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Sample records for bacterial transcription initiation

  1. Initial Events in Bacterial Transcription Initiation

    PubMed Central

    Ruff, Emily F.; Record, M. Thomas; Artsimovitch, Irina

    2015-01-01

    Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the −10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis. PMID:26023916

  2. The interaction between bacterial transcription factors and RNA polymerase during the transition from initiation to elongation.

    PubMed

    Yang, Xiao; Lewis, Peter J

    2010-01-01

    There are three stages of transcription: initiation, elongation and termination, and traditionally there has been a clear distinction between the stages. The specificity factor sigma is completely released from bacterial RNA polymerase after initiation, and then recycled for another round of transcription. Elongation factors then associate with the polymerase followed by termination factors (where necessary). These factors dissociate prior to initiation of a new round of transcription. However, there is growing evidence suggesting that sigma factors can be retained in the elongation complex. The structure of bacterial RNAP in complex with an essential elongation factor NusA has recently been published, which suggested rather than competing for the major σ binding site, NusA binds to a discrete region on RNAP. A model was proposed to help explain the way in which both factors could be associated with RNAP during the transition from transcription initiation to elongation.

  3. A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism.

    PubMed

    Sheppard, Carol; James, Ellen; Barton, Geraint; Matthews, Stephen; Severinov, Konstantin; Wigneshweraraj, Sivaramesh

    2013-04-01

    The process of transcription initiation is the major target for regulation of gene expression in bacteria and is performed by a multi-subunit RNA polymerase enzyme (RNAp). A complex network of regulatory elements controls the activity of the RNAp to fine-tune transcriptional output. Thus, RNAp is a nexus for controlling bacterial gene expression at the transcription level. Many bacteriophages, viruses that infect bacteria, encode transcription factors that specifically target and modulate the activity of the host RNAp and, thereby, facilitate the acquisition of the host bacteria by the phage. Here, we describe the modus operandi of a T7 bacteriophage-encoded small protein called Gp2 and define Gp2 as a non-bacterial regulator of bacterial transcription.

  4. CoSMoS unravels mysteries of transcription initiation.

    PubMed

    Gourse, Richard L; Landick, Robert

    2012-02-17

    Using a fluorescence method called colocalization single-molecule spectroscopy (CoSMoS), Friedman and Gelles dissect the kinetics of transcription initiation at a bacterial promoter. Ultimately, CoSMoS could greatly aid the study of the effects of DNA sequence and transcription factors on both prokaryotic and eukaryotic promoters.

  5. Topography of the euryarchaeal transcription initiation complex.

    PubMed

    Bartlett, Michael S; Thomm, Michael; Geiduschek, E Peter

    2004-02-13

    Transcription in the Archaea is carried out by RNA polymerases and transcription factors that are highly homologous to their eukaryotic counterparts, but little is known about the structural organization of the archaeal transcription complex. To address this, transcription initiation complexes have been formed with Pyrococcus furiosus transcription factors (TBP and TFB1), RNA polymerase, and a linear DNA fragment containing a strong promoter. The arrangement of proteins from base pair -35 to +20 (relative to the transcriptional start site) has been analyzed by photochemical protein-DNA cross-linking. TBP cross-links to the TATA box and TFB1 cross-links both upstream and downstream of the TATA box, as expected, but the sites of most prominent TFB1 cross-linking are located well downstream of the TATA box, reaching as far as the start site of transcription, suggesting a role for TFB1 in initiation of transcription that extends beyond polymerase recruitment. These cross-links indicate the transcription factor orientation in the initiation complex. The pattern of cross-linking of four RNA polymerase subunits (B, A', A", and H) to the promoter suggests a path for promoter DNA relative to the RNA polymerase surface in this archaeal transcription initiation complex. In addition, an unidentified protein approximately the size of TBP cross-links to the non-transcribed DNA strand near the upstream edge of the transcription bubble. Cross-linking is specific to the polymerase-containing initiation complex and requires the gdh promoter TATA box. The location of this protein suggests that it, like TFB1, could also have a role in transcription initiation following RNA polymerase recruitment.

  6. DNA dynamically directs its own transcription initiation

    SciTech Connect

    Rasmussen, K. O.; Kalosakas, G.; Bishop, A. R.; Choi, C. H.; Usheva, A.

    2004-01-01

    Initiation of DNA gene transcription requires a transient opening in the double helix at the transcriptional start site. It is generally assumed that the location of this 'transcriptional bubble' is determined by sequence-specific protein binding, and that the energy required for unwinding the double helix comes from torsional strain. Physical twisting should cause DNA to open consistently in weakly bonded A/T rich stretches, however, simple base-pairing energetics alone can not account for the variety of observed transcriptional start sites. Applying the Peyrard-Bishop nonlinear cooperativity model to DNA, we are able to predict that thermally-induced DNA bubbles, similar in size to transcription bubbles, form at specific locations on DNA promoters. These predicted openings agree remarkably well with experiment, and that they correlate exactly with known transcription start sites and important regulatory sites on three different promoters. We propose that the sequence-specific location of the transcriptional start site is predetermined by the inherent opening patterns of specific DNA sequences. As DNA bubble formation is independent of protein binding, it appears that DNA is not only a passive carrier of information, but its dynamics plays an important role in directing the transcription and regulation of the genes it contains.

  7. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

    PubMed Central

    Martínez, Luary C.; Vadyvaloo, Viveka

    2014-01-01

    Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation, and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins, and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches, and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria. PMID:24724055

  8. Bacterial Transcription as a Target for Antibacterial Drug Development.

    PubMed

    Ma, Cong; Yang, Xiao; Lewis, Peter J

    2016-03-01

    Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

  9. Bacterial Transcription as a Target for Antibacterial Drug Development

    PubMed Central

    Ma, Cong; Yang, Xiao

    2016-01-01

    SUMMARY Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design. PMID:26764017

  10. Bacterial antisense RNAs are mainly the product of transcriptional noise

    PubMed Central

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  11. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

    PubMed

    Tillett, D; Burns, B P; Neilan, B A

    2000-03-01

    A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

  12. Non-canonical transcription initiation: the expanding universe of transcription initiating substrates.

    PubMed

    Barvík, Ivan; Rejman, Dominik; Panova, Natalya; Šanderová, Hana; Krásný, Libor

    2017-03-01

    RNA polymerase (RNAP) is the central enzyme of transcription of the genetic information from DNA into RNA. RNAP recognizes four main substrates: ATP, CTP, GTP and UTP. Experimental evidence from the past several years suggests that, besides these four NTPs, other molecules can be used to initiate transcription: (i) ribooligonucleotides (nanoRNAs) and (ii) coenzymes such as NAD+, NADH, dephospho-CoA and FAD. The presence of these molecules at the 5΄ ends of RNAs affects the properties of the RNA. Here, we discuss the expanding portfolio of molecules that can initiate transcription, their mechanism of incorporation, effects on RNA and cellular processes, and we present an outlook toward other possible initiation substrates.

  13. Molecular basis of transcription initiation in Archaea.

    PubMed

    De Carlo, Sacha; Lin, Shih-Chieh; Taatjes, Dylan J; Hoenger, Andreas

    2010-01-01

    Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.

  14. Transcript profiling of early lateral root initiation.

    PubMed

    Himanen, Kristiina; Vuylsteke, Marnik; Vanneste, Steffen; Vercruysse, Steven; Boucheron, Elodie; Alard, Philippe; Chriqui, Dominique; Van Montagu, Marc; Inzé, Dirk; Beeckman, Tom

    2004-04-06

    At the onset of lateral root initiation in Arabidopsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation in Arabidopsis. The reproducible expression profiles during a time course allowed us to define four stages that precede the cell division in the pericycle. These early stages were characterized by G1 cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took place within 6 h after transfer from N-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.

  15. The TRTGn motif stabilizes the transcription initiation open complex.

    PubMed

    Voskuil, Martin I; Chambliss, Glenn H

    2002-09-20

    The effect on transcription initiation by the extended -10 motif (5'-TRTG(n)-3'), positioned upstream of the -10 region, was investigated using a series of base substitution mutations in the alpha-amylase promoter (amyP). The extended -10 motif, previously referred to as the -16 region, is found frequently in Gram-positive bacterial promoters and several extended -10 promoters from Escherichia coli. The inhibitory effects of the non-productive promoter site (amyP2), which overlaps the upstream region of amyP, were eliminated by mutagenesis of the -35 region and the TRTG motif of amyP2. Removal by mutagenesis of the competitive effects of amyP2 resulted in a reduced dependence of amyP on the TRTG motif. In the absence of the second promoter, mutations in the TRTG motif of amyP destabilized the open complex and prevented the maintenance of open complexes at low temperatures. The open complex half-life was up to 26-fold shorter in the mutant TRTG motif promoters than in the wild-type promoter. We demonstrate that the amyP TRTG motif dramatically stabilizes the open complex intermediate during transcription initiation. Even though the open complex is less stable in the mutant promoters, the region of melted DNA is the same in the wild-type and mutant promoters. However, upon addition of the first three nucleotides, which trap RNAP (RNA polymerase) in a stable initiating complex, the melted DNA region contracts at the 5'-end in a TRTG motif promoter mutant but not at the wild-type promoter, indicating that the motif contributes to maintaining DNA-strand separation.

  16. Bacterial RNA polymerase can retain σ70 throughout transcription.

    PubMed

    Harden, Timothy T; Wells, Christopher D; Friedman, Larry J; Landick, Robert; Hochschild, Ann; Kondev, Jane; Gelles, Jeff

    2016-01-19

    Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription.

  17. Collective Properties of a Transcription Initiation Model Under Varying Environment.

    PubMed

    Hu, Yucheng; Lowengrub, John S

    2016-01-01

    The dynamics of gene transcription is tightly regulated in eukaryotes. Recent experiments have revealed various kinds of transcriptional dynamics, such as RNA polymerase II pausing, that involves regulation at the transcription initiation stage, and the choice of different regulation pattern is closely related to the physiological functions of the target gene. Here we consider a simplified model of transcription initiation, a process including the assembly of transcription complex and the pausing and releasing of the RNA polymerase II. Focusing on the collective behaviors of a population level, we explore the potential regulatory functions this model can offer. These functions include fast and synchronized response to environmental change, or long-term memory about the transcriptional status. As a proof of concept we also show that, by selecting different control mechanisms cells can adapt to different environments. These findings may help us better understand the design principles of transcriptional regulation.

  18. Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

    PubMed Central

    Deshpande, Aishwarya P.; Sultana, Shemaila

    2015-01-01

    The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples. PMID:25095993

  19. Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase.

    PubMed

    Deshpande, Aishwarya P; Patel, Smita S

    2012-01-01

    Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP "sensor" to regulate gene expression. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

  20. Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

    PubMed Central

    Wu, Zhe; Ietswaart, Robert; Liu, Fuquan; Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-01-01

    The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context. PMID:26699513

  1. DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

    PubMed

    Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

    2016-04-01

    RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells.

  2. INITIATION AND REGULATION OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION

    PubMed Central

    Noton, Sarah L.; Fearns, Rachel

    2015-01-01

    The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. PMID:25683441

  3. Two independent transcription initiation codes overlap on vertebrate core promoters.

    PubMed

    Haberle, Vanja; Li, Nan; Hadzhiev, Yavor; Plessy, Charles; Previti, Christopher; Nepal, Chirag; Gehrig, Jochen; Dong, Xianjun; Akalin, Altuna; Suzuki, Ana Maria; van IJcken, Wilfred F J; Armant, Olivier; Ferg, Marco; Strähle, Uwe; Carninci, Piero; Müller, Ferenc; Lenhard, Boris

    2014-03-20

    A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable

  4. Two independent transcription initiation codes overlap on vertebrate core promoters

    NASA Astrophysics Data System (ADS)

    Haberle, Vanja; Li, Nan; Hadzhiev, Yavor; Plessy, Charles; Previti, Christopher; Nepal, Chirag; Gehrig, Jochen; Dong, Xianjun; Akalin, Altuna; Suzuki, Ana Maria; van Ijcken, Wilfred F. J.; Armant, Olivier; Ferg, Marco; Strähle, Uwe; Carninci, Piero; Müller, Ferenc; Lenhard, Boris

    2014-03-01

    A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable

  5. Dissecting the stochastic transcription initiation process in live Escherichia coli.

    PubMed

    Lloyd-Price, Jason; Startceva, Sofia; Kandavalli, Vinodh; Chandraseelan, Jerome G; Goncalves, Nadia; Oliveira, Samuel M D; Häkkinen, Antti; Ribeiro, Andre S

    2016-06-01

    We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

  6. Dissecting the stochastic transcription initiation process in live Escherichia coli

    PubMed Central

    Lloyd-Price, Jason; Startceva, Sofia; Kandavalli, Vinodh; Chandraseelan, Jerome G.; Goncalves, Nadia; Oliveira, Samuel M. D.; Häkkinen, Antti; Ribeiro, Andre S.

    2016-01-01

    We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics. PMID:27026687

  7. Structural Basis of RNA Polymerase I Transcription Initiation.

    PubMed

    Engel, Christoph; Gubbey, Tobias; Neyer, Simon; Sainsbury, Sarah; Oberthuer, Christiane; Baejen, Carlo; Bernecky, Carrie; Cramer, Patrick

    2017-03-23

    Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

  8. Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates

    PubMed Central

    Melaugh, Gavin; Hutchison, Jaime; Kragh, Kasper Nørskov; Irie, Yasuhiko; Roberts, Aled; Bjarnsholt, Thomas; Diggle, Stephen P.; Gordon, Vernita D.; Allen, Rosalind J.

    2016-01-01

    Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities. PMID:26934187

  9. Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates.

    PubMed

    Melaugh, Gavin; Hutchison, Jaime; Kragh, Kasper Nørskov; Irie, Yasuhiko; Roberts, Aled; Bjarnsholt, Thomas; Diggle, Stephen P; Gordon, Vernita D; Allen, Rosalind J

    2016-01-01

    Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.

  10. R-loops in bacterial transcription: their causes and consequences.

    PubMed

    Gowrishankar, J; Leela, J Krishna; Anupama, K

    2013-01-01

    Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

  11. Upstream Binding of Idling RNA Polymerase Modulates Transcription Initiation from a Nearby Promoter*

    PubMed Central

    Gerganova, Veneta; Maurer, Sebastian; Stoliar, Liubov; Japaridze, Aleksandre; Dietler, Giovanni; Nasser, William; Kutateladze, Tamara; Travers, Andrew; Muskhelishvili, Georgi

    2015-01-01

    The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter. PMID:25648898

  12. The RPB2 Flap Loop of Human RNA Polymerase II Is Dispensable for Transcription Initiation and Elongation▿†

    PubMed Central

    Palangat, Murali; Grass, Jeffrey A.; Langelier, Marie-France; Coulombe, Benoit; Landick, Robert

    2011-01-01

    The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential. PMID:21670157

  13. Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells.

    PubMed

    Jin, Ding Jun; Mata Martin, Carmen; Sun, Zhe; Cagliero, Cedric; Zhou, Yan Ning

    2017-02-01

    We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is three-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis, and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

  14. Transcription initiation factor DksA has diverse effects on RNA chain elongation

    PubMed Central

    Furman, Ran; Sevostyanova, Anastasiya; Artsimovitch, Irina

    2012-01-01

    Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex. PMID:22210857

  15. The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling.

    PubMed

    Shandilya, Jayasha; Roberts, Stefan G E

    2012-05-01

    The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.

  16. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    SciTech Connect

    Volkman, Brian Finley

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  17. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

    PubMed Central

    Tropel, David; van der Meer, Jan Roelof

    2004-01-01

    Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways. PMID:15353566

  18. An iron detection system determines bacterial swarming initiation and biofilm formation

    PubMed Central

    Lin, Chuan-Sheng; Tsai, Yu-Huan; Chang, Chih-Jung; Tseng, Shun-Fu; Wu, Tsung-Ru; Lu, Chia-Chen; Wu, Ting-Shu; Lu, Jang-Jih; Horng, Jim-Tong; Martel, Jan; Ojcius, David M.; Lai, Hsin-Chih; Young, John D.

    2016-01-01

    Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising a two-component system (TCS) and the TCS-regulated iron chelator 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) that directly senses and modulates environmental ferric iron (Fe3+) availability to determine swarming initiation and biofilm formation. We demonstrate that the two-component system RssA-RssB (RssAB) directly senses environmental ferric iron (Fe3+) and transcriptionally modulates biosynthesis of flagella and the iron chelator ICDH-Coumarin whose production requires the pvc cluster. Addition of Fe3+, or loss of ICDH-Coumarin due to pvc deletion results in prolonged RssAB signaling activation, leading to delayed swarming initiation and increased biofilm formation. We further show that ICDH-Coumarin is able to chelate Fe3+ to switch off RssAB signaling, triggering swarming initiation and biofilm reduction. Our findings reveal a novel cellular system that senses iron levels to regulate bacterial surface lifestyle. PMID:27845335

  19. Ocean microbes. Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages.

    PubMed

    Ottesen, Elizabeth A; Young, Curtis R; Gifford, Scott M; Eppley, John M; Marin, Roman; Schuster, Stephan C; Scholin, Christopher A; DeLong, Edward F

    2014-07-11

    Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.

  20. Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

    PubMed

    Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

    2017-03-09

    Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

  1. A new way to start: nanoRNA-mediated priming of transcription initiation.

    PubMed

    Nickels, Bryce E

    2012-01-01

    A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.

  2. Initial stages of bacterial fouling during dead-end microfiltration.

    PubMed

    Xu, Wendong; Chellam, Shankararaman

    2005-09-01

    Constant pressure experiments were performed using track-etched polycarbonate membranes and rod-shaped bacteria (viz., Brevundimonas diminuta and Serratia marcescens) to study flux decline and backwashing during the early stages of microfiltration. The intermediate blocking law originally derived for spherical particles was modified to account for the approximate cylindrical shape of the selected bacteria. A deposition factor was introduced to empirically account for the morphology of bacterial deposits. The initial stages of flux decline prior to the secretion of new extracellular polymeric substances (EPS) was quantitatively described by the intermediate blocking law before transitioning to cake filtration at later times. Scanning electron microscopy (SEM) provided additional visual evidence that bacteria simultaneously deposited directly on the membrane and on each other during early stages of filtration as assumed bythe intermediate blocking law. Empirical deposition factors decreased with initial permeate flux indicating its effect on bacteria deposition patterns, which was also confirmed by SEM. Bacteria were easily removed following short filtration times before significant secretion of new EPS by simply rinsing with ultrapure water, thereby completely restoring the clean membrane permeability. In contrast, this rinsing procedure did not completely recover the membrane permeability following longer durations when significant amounts of new EPS proteins and polysaccharides were secreted. Consequently, backwashing effectiveness during water and wastewater microfiltration will be high prior to EPS production whereas flux recovery may not be possible solely by hydrodynamic means once EPS are secreted.

  3. Initial bacterial adhesion on resin, titanium and zirconia in vitro

    PubMed Central

    Lee, Byung-Chul; Jung, Gil-Yong; Kim, Dae-Joon

    2011-01-01

    PURPOSE The aim of this in vitro study was to investigate the adhesion of initial colonizer, Streptococcus sanguis, on resin, titanium and zirconia under the same surface polishing condition. MATERIALS AND METHODS Specimens were prepared from Z-250, cp-Ti and 3Y-TZP and polished with 1 µm diamond paste. After coating with saliva, each specimen was incubated with Streptococcus sanguis. Scanning electron microscope, crystal violet staining and measurement of fluorescence intensity resulting from resazurin reduction were performed for quantifying the bacterial adhesion. RESULTS Surface of resin composite was significantly rougher than that of titanium and zirconia, although all tested specimens are classified as smooth. The resin specimens showed lower value of contact angle compared with titanium and zirconia specimens, and had hydrophilic surfaces. The result of scanning electron microscopy demonstrated that bound bacteria were more abundant on resin in comparison with titanium and zirconia. When total biofilm mass determined by crystal violet, absorbance value of resin was significantly higher than that of titanium or zirconia. The result of relative fluorescence intensities also demonstrated that the highest fluorescence intensity was found on the surface of resin. Absorbance value and fluorescence intensity on titanium was not significantly different from those on zirconia. CONCLUSION Resin specimens showed the roughest surface and have a significantly higher susceptibility to adhere Streptococcus sanguis than titanium and zirconia when surfaces of each specimen were polished under same condition. There was no significant difference in bacteria adhesion between titanium and zirconia in vitro. PMID:21814616

  4. Transcriptional Responses of Treponema denticola to Other Oral Bacterial Species

    PubMed Central

    Simanian, Emil J.; Shi, Wenyuan; Lux, Renate

    2014-01-01

    an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket. PMID:24505483

  5. The dynamic nature and territory of transcriptional machinery in the bacterial chromosome

    PubMed Central

    Jin, Ding J.; Cagliero, Cedric; Martin, Carmen M.; Izard, Jerome; Zhou, Yan N.

    2015-01-01

    Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment. PMID:26052320

  6. Initial insights into bacterial succession during human decomposition.

    PubMed

    Hyde, Embriette R; Haarmann, Daniel P; Petrosino, Joseph F; Lynne, Aaron M; Bucheli, Sibyl R

    2015-05-01

    Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession.

  7. Binding motifs in bacterial gene promoters modulate transcriptional effect of global regulators

    SciTech Connect

    Leuze, Michael Rex; Karpinets, Tatiana V; Syed, Mustafa H; Beliaev, Alexander S; Uberbacher, Edward C

    2012-01-01

    Bacterial gene regulation involves transcription factors (TFs) that influence the expression of many genes. Global regulators, including CRP (cAMP Receptor Protein), ArcA, and FNR, can modulate the transcriptional activity of multiple operons. The similarity of a regulatory element s sequence to a TF s consensus binding site (BS) and the position of the regulatory element in an operon promoter are considered the most important determinants of this TF s regulatory influence. In this study we explore the hypothesis that the number of TFBS half-sites (where a half-site is one half of the palindromic BS consensus sequence, which we shall refer to as a binding motif or a BM) of a global regulator in an operon s promoter plays an important role in the operon s transcriptional regulation. We examine empirical data from transcriptional profiling of the CRP regulon in Shewanella oneidenses MR 1 and Escherichia coli, and of the ArcA regulon in S. oneidenses MR 1. We compare the power of CRP BM counts and of full, symmetrical CRP TFBS characteristics, namely similarity to consensus and location, to predict CRP-induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full-length TFBS quality or location. Regression analysis indicates that IHF (Integration Host Factor) and ArcA have synergistic effects on CRP-induced gene transcription, positive and negative, respectively. Based on these results, we propose that the fine-tuning of bacterial transcriptional activity by CRP may involves not only the bending of the operon promoter, facilitated by CRP in cooperation with the histone-like protein IHF, but also the cumulative binding affinity of multiple weak BMs.

  8. Human Mitochondrial Transcription Initiation Complexes Have Similar Topology on the Light and Heavy Strand Promoters.

    PubMed

    Morozov, Yaroslav I; Temiakov, Dmitry

    2016-06-24

    Transcription is a highly regulated process in all domains of life. In human mitochondria, transcription of the circular genome involves only two promoters, called light strand promoter (LSP) and heavy strand promoter (HSP), located in the opposite DNA strands. Initiation of transcription occurs upon sequential assembly of an initiation complex that includes mitochondrial RNA polymerase (mtRNAP) and the initiation factors mitochondrial transcription factor A (TFAM) and TFB2M. It has been recently suggested that the transcription initiation factor TFAM binds to HSP and LSP in opposite directions, implying that the mechanisms of transcription initiation are drastically dissimilar at these promoters. In contrast, we found that binding of TFAM to HSP and the subsequent recruitment of mtRNAP results in a pre-initiation complex that is remarkably similar in topology and properties to that formed at the LSP promoter. Our data suggest that assembly of the pre-initiation complexes on LSP and HSP brings these transcription units in close proximity, providing an opportunity for regulatory proteins to simultaneously control transcription initiation in both mtDNA strands.

  9. Possible interaction between the bacterial transcription factor ArtA and the eukaryotic RNA polymerase III promoter.

    PubMed

    Matsutani, Sachiko

    2016-06-01

    Eukaryotic RNA polymerase III (RNAP III) transcribes tRNA genes and short interspersed elements that have internal promoters consisting of A- and B-blocks. The B-block binding subunit of the transcription initiation factor TFIIIC binds to the B-block. The mobile bacterial insertion sequence (IS) 1 contains a RNAP III promoter-like sequence, which stimulates bacterial transcription along with the bacterial ArtA protein. Here, the DNA-binding ability of ArtA was examined in vitro using a simple, newly developed method. Various DNA fragments, including RNAP III promoter fragments, were separately incubated with purified ArtA, and then loaded onto a polyacrylamide gel. Since DNAs bound by ArtA remain in the gel wells during electrophoresis, SDS was added into the wells at the electrophoresis halfway point. It was hypothesized that SDS would dissociate the DNA-ArtA complexes in the wells, and then the DNAs would begin to migrate. In fact, new bands appeared in all of the lanes at similar intensities, indicating that ArtA binds nonspecifically to DNA. Therefore, labeled wild-type RNAP III promoter fragments were incubated with either the unlabeled wild-type or mutant fragments and ArtA, and electrophoresed. The B-block(-like) sequences of IS1, a human Alu element, and an anuran tRNA gene were important for binding to ArtA. Additionally, in silico analyses revealed the presence of the RNAP III promoter-like structures in the IS1 isoforms and the IS3 family elements. These results suggest the presence of parts of the RNAP III transcription machinery in bacteria, and might imply that its prototype existed in the common ancestor.

  10. Specific Inhibition of HER-2/NEU Transcription Initiation

    DTIC Science & Technology

    2007-07-01

    quadruplex-forming region in the c-myb promoter is a critical cis-acting element that may repress c-myb promoter activity through MAZ 6 W81XWH-04-1-0560...oligonucleotides representing the c-MYB promoter. o Identification of MAZ as a transcription factor that can recognize alternate DNA conformations in

  11. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    PubMed Central

    Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Morrison, John M.; Dunman, Paul M.

    2009-01-01

    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript. PMID:18992285

  12. Specific Inhibition of HER-2/neu Transcription Initiation

    DTIC Science & Technology

    2006-07-01

    disproportionate to gene copy number with or without gene amplification as a result of transcriptional activation. A polypurine tract (PPT) containing...DNA polymerase (Taq Pol) arrest assay (5;6). We evaluated two commercially available compounds, the cationic porphyrins TmPyP2 and TmPyP4 (7-10...225)-luc/FRT. These vectors are useful for creating stable, integrated genes in host cells bearing a flp recombinase target sequence (FRT) using the

  13. Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

    PubMed

    Fishburn, James; Galburt, Eric; Hahn, Steven

    2016-06-17

    Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

  14. Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters.

    PubMed Central

    Du, H; Roy, A L; Roeder, R G

    1993-01-01

    Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad-ML) promoter by binding to an E-box motif located at position -60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix-loop-helix protein. In this report, we demonstrate a 25-to 30-fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine-rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV-1 promoter, as well as those in the Ad-ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co-transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43-dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV-1 and Ad-ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear. Images PMID:8440240

  15. The interaction surface of a bacterial transcription elongation factor required for complex formation with an antiterminator during transcription antitermination.

    PubMed

    Mishra, Saurabh; Mohan, Shalini; Godavarthi, Sapna; Sen, Ranjan

    2013-09-27

    The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription elongation complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA N terminus (NTD) specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53 (S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C) of the NusA binding region of the N protein. We conclude that when bound to the EC, the N interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator.

  16. The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

    PubMed Central

    2012-01-01

    Summary: Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ54 for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ54-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ54 to the bacterial cell and its unique role in regulating transcription. PMID:22933558

  17. Transcriptional activation domains stimulate initiation and elongation at different times and via different residues.

    PubMed Central

    Brown, S A; Weirich, C S; Newton, E M; Kingston, R E

    1998-01-01

    Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation. PMID:9606196

  18. Snapshots of a viral RNA polymerase switching gears from transcription initiation to elongation.

    PubMed

    Theis, Karsten

    2013-12-01

    During transcription initiation, RNA polymerase binds tightly to the promoter DNA defining the start of transcription, transcribes comparatively slowly, and frequently releases short transcripts (3-8 nucleotides) in a process called abortive cycling. Transitioning to elongation, the second phase of transcription, the polymerase dissociates from the promoter while RNA synthesis continues. Elongation is characterized by higher rates of transcription and tight binding to the RNA transcript. The RNA polymerase from enterophage T7 (T7 RNAP) has been used as a model to understand the mechanism of transcription in general, and the transition from initiation to elongation specifically. This single-subunit enzyme undergoes dramatic conformational changes during this transition to support the changing requirements of nucleic acid interactions while continuously maintaining polymerase function. Crystal structures, available of multiple stages of the initiation complex and of the elongation complex, combined with biochemical and biophysical data, offer molecular detail of the transition. Some of the crystal structures contain a variant of T7 RNAP where proline 266 is substituted by leucine. This variant shows less abortive products and altered timing of transition, and is a valuable tool to study these processes. The structural transitions from early to late initiation are well understood and are consistent with solution data. The timing of events and the structural intermediates in the transition from late initiation to elongation are less well understood, but the available data allows one to formulate testable models of the transition to guide further research.

  19. Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

    PubMed Central

    Tang, Wei; Wu, Yufeng; Li, Moran; Wang, Jian; Mei, Sha

    2016-01-01

    ABSTRACT Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5′ untranslated region (5′ UTR) of <10 nucleotides (nt) and leadered transcripts with a 5′ UTR of ≥10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG1 and AUG2) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG1 is more efficient than AUG2 for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG1 but did not affect initiation at AUG2, indicating that AUG2-initiated translation does not involve ribosome scanning from the 5′ end of the transcript. Furthermore, the efficiency of AUG2-initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG1 and AUG2 contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. IMPORTANCE In eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for

  20. Integrated Circuits: How Transcriptional Silencing and Counter-Silencing Facilitate Bacterial Evolution

    PubMed Central

    Will, W. Ryan; Navarre, William W.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally- and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks. PMID:25461567

  1. Ribavirin suppresses bacterial virulence by targeting LysR-type transcriptional regulators

    PubMed Central

    Mandal, Rahul Shubhra; Ta, Atri; Sinha, Ritam; Theeya, Nagaraja; Ghosh, Anirban; Tasneem, Mohsina; Bhunia, Anirban; Koley, Hemanta; Das, Santasabuj

    2016-01-01

    Targeting bacterial virulence mechanisms without compromising bacterial growth is a promising strategy to prevent drug resistance. LysR-type transcriptional regulators (LTTRs) possess structural conservation across bacterial species and regulate virulence in numerous pathogens, making them attractive targets for antimicrobial agents. We targeted AphB, a Vibrio cholerae LTTR, which regulates the expression of genes encoding cholera toxin and toxin-co-regulated pilus for inhibitor designing. Since AphB ligand is unknown, we followed a molecular fragment-based approach for ligand designing using FDA-approved drugs and subsequent screen to identify molecules that exhibited high-affinity binding to AphB ligand-binding pocket. Among the identified compounds, ribavirin, an anti-viral drug, antagonized AphB functions. Ribavirin perturbed Vibrio cholerae pathogenesis in animal models. The inhibitory effects of the drug was limited to the bacteria expressing wild type AphB, but not its constitutively active mutant (AphBN100E), which represents the ligand-bound state, suggesting that ribavirin binds to the active site of AphB to exert its inhibitory role and there exists no AphB-independent mechanism of its action. Similarly, ribavirin suppressed the functions of Salmonella Typhi LTTR Hrg, indicating its broad spectrum efficacy. Moreover, ribavirin did not affect the bacterial viability in culture. This study cites an example of drug repurposing for anti-infective therapy. PMID:27991578

  2. Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors

    PubMed Central

    Garbeva, Paolina; Silby, Mark W; Raaijmakers, Jos M; Levy, Stuart B; Boer, Wietse de

    2011-01-01

    The ability of soil bacteria to successfully compete with a range of other microbial species is crucial for their growth and survival in the nutrient-limited soil environment. In the present work, we studied the behavior and transcriptional responses of soil-inhabiting Pseudomonas fluorescens strain Pf0-1 on nutrient-poor agar to confrontation with strains of three phylogenetically different bacterial genera, that is, Bacillus, Brevundimonas and Pedobacter. Competition for nutrients was apparent as all three bacterial genera had a negative effect on the density of P. fluorescens Pf0-1; this effect was most strong during the interaction with Bacillus. Microarray-based analyses indicated strong differences in the transcriptional responses of Pf0-1 to the different competitors. There was higher similarity in the gene expression response of P. fluorescens Pf0-1 to the Gram-negative bacteria as compared with the Gram-positive strain. The Gram-negative strains did also trigger the production of an unknown broad-spectrum antibiotic in Pf0-1. More detailed analysis indicated that expression of specific Pf0-1 genes involved in signal transduction and secondary metabolite production was strongly affected by the competitors' identity, suggesting that Pf0-1 can distinguish among different competitors and fine-tune its competitive strategies. The results presented here demonstrate that P. fluorescens Pf0-1 shows a species-specific transcriptional and metabolic response to bacterial competitors and provide new leads in the identification of specific cues in bacteria–bacteria interactions and of novel competitive strategies, antimicrobial traits and genes. PMID:21228890

  3. The Chinese hamster dihydrofolate reductase replication origin decision point follows activation of transcription and suppresses initiation of replication within transcription units.

    PubMed

    Sasaki, Takayo; Ramanathan, Sunita; Okuno, Yukiko; Kumagai, Chiharu; Shaikh, Seemab S; Gilbert, David M

    2006-02-01

    Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.

  4. The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

    PubMed Central

    Sasaki, Takayo; Ramanathan, Sunita; Okuno, Yukiko; Kumagai, Chiharu; Shaikh, Seemab S.; Gilbert, David M.

    2006-01-01

    Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP. PMID:16428457

  5. Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

    PubMed Central

    Chen, Yun; Pai, Athma A.; Herudek, Jan; Lubas, Michal; Meola, Nicola; Järvelin, Aino I.; Andersson, Robin; Pelechano, Vicent; Steinmetz, Lars M.; Heick Jensen, Torben; Sandelin, Albin

    2016-01-01

    Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes. PMID:27455346

  6. Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

    PubMed Central

    Green, Jeffrey; Rolfe, Matthew D; Smith, Laura J

    2014-01-01

    Molecular oxygen (O2) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O2 for their production. Furthermore, the ability to adapt to changes in O2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron–sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O2 and/or NO. Indirect sensors perceive the physiological effects of O2 starvation. Thus, O2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O2- and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis. PMID:25603427

  7. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  8. The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo.

    PubMed Central

    Kennell, J C; Wang, H; Lambowitz, A M

    1994-01-01

    The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript. Images PMID:8164665

  9. Environmental regulation operating at the promoter clearance step of bacterial transcription

    PubMed Central

    Laishram, Rakesh S.; Gowrishankar, Jayaraman

    2007-01-01

    In vivo transcription of the Escherichia coli argO gene, which encodes an arginine (Arg) exporter, requires the LysR-family regulator protein ArgP (previously called IciA) and is induced in the presence of Arg or its naturally occurring antimetabolite analog canavanine. Lysine (Lys) addition, on the other hand, phenocopies an argP mutation to result in the shutoff of argO expression. We now report that the ArgP dimer by itself is able to bind the argO promoter-operator region to form a binary complex, but that the formation of a ternary complex with RNA polymerase is greatly stimulated only in presence of a coeffector. Both Arg and Lys were proficient as coeffectors for ArgP-mediated recruitment of RNA polymerase to, and open complex formation at, the argO promoter, although only Arg (but not Lys) was competent to activate transcription. The two coeffectors competed for binding to ArgP, and the ternary complex that had been assembled on the argO template in the presence of Lys could be chased into a transcriptionally active state upon Arg addition. Our results support a novel mechanism of argO regulation in which Lys-bound ArgP reversibly restrains RNA polymerase at the promoter, at a step (following open complex formation) that precedes, and is common to, both abortive and productive transcription. This represents, therefore, the first example of an environmental signal regulating the final step of promoter clearance by RNA polymerase in bacterial transcription. We propose that, in E. coli cells, the ternary complex remains assembled and poised at the argO promoter at all times to respond, positively or negatively, to instantaneous changes in the ratio of intracellular Arg to Lys concentrations. PMID:17504942

  10. Developmental regulation of transcription initiation: more than just changing the actors.

    PubMed

    Müller, Ferenc; Zaucker, Andreas; Tora, Làszlò

    2010-10-01

    The traditional model of transcription initiation nucleated by the TFIID complex has suffered significant erosion in the last decade. The discovery of cell-specific paralogs of TFIID subunits and a variety of complexes that replace TFIID in transcription initiation of protein coding genes have been paralleled by the description of diverse core promoter sequences. These observations suggest an additional level of regulation of developmental and tissue-specific gene expression at the core promoter level. Recent work suggests that this regulation may function through specific roles of distinct TBP-type factors and TBP-associated factors (TAFs), however the picture emerging is still far from complete. Here we summarize the proposed models of transcription initiation by alternative initiation complexes in distinct stages of developmental specialization during vertebrate ontogeny.

  11. Effects of rate-limiting steps in transcription initiation on genetic filter motifs.

    PubMed

    Häkkinen, Antti; Tran, Huy; Yli-Harja, Olli; Ribeiro, Andre S

    2013-01-01

    The behavior of genetic motifs is determined not only by the gene-gene interactions, but also by the expression patterns of the constituent genes. Live single-molecule measurements have provided evidence that transcription initiation is a sequential process, whose kinetics plays a key role in the dynamics of mRNA and protein numbers. The extent to which it affects the behavior of cellular motifs is unknown. Here, we examine how the kinetics of transcription initiation affects the behavior of motifs performing filtering in amplitude and frequency domain. We find that the performance of each filter is degraded as transcript levels are lowered. This effect can be reduced by having a transcription process with more steps. In addition, we show that the kinetics of the stepwise transcription initiation process affects features such as filter cutoffs. These results constitute an assessment of the range of behaviors of genetic motifs as a function of the kinetics of transcription initiation, and thus will aid in tuning of synthetic motifs to attain specific characteristics without affecting their protein products.

  12. Directional transition from initiation to elongation in bacterial translation

    PubMed Central

    Goyal, Akanksha; Belardinelli, Riccardo; Maracci, Cristina; Milón, Pohl; Rodnina, Marina V.

    2015-01-01

    The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNAfMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNAfMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation. PMID:26338773

  13. Initiator-dependent transcription in vitro by a wheat germ chromatin extract.

    PubMed

    Schweizer, P; Mösinger, E

    1994-04-01

    The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.

  14. Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Adjalley, Sophie H; Chabbert, Christophe D; Klaus, Bernd; Pelechano, Vicent; Steinmetz, Lars M

    2016-03-15

    A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

  15. oriC-encoded instructions for the initiation of bacterial chromosome replication

    PubMed Central

    Wolański, Marcin; Donczew, Rafał; Zawilak-Pawlik, Anna; Zakrzewska-Czerwińska, Jolanta

    2014-01-01

    Replication of the bacterial chromosome initiates at a single origin of replication that is called oriC. This occurs via the concerted action of numerous proteins, including DnaA, which acts as an initiator. The origin sequences vary across species, but all bacterial oriCs contain the information necessary to guide assembly of the DnaA protein complex at oriC, triggering the unwinding of DNA and the beginning of replication. The requisite information is encoded in the unique arrangement of specific sequences called DnaA boxes, which form a framework for DnaA binding and assembly. Other crucial sequences of bacterial origin include DNA unwinding element (DUE, which designates the site at which oriC melts under the influence of DnaA) and binding sites for additional proteins that positively or negatively regulate the initiation process. In this review, we summarize our current knowledge and understanding of the information encoded in bacterial origins of chromosomal replication, particularly in the context of replication initiation and its regulation. We show that oriC encoded instructions allow not only for initiation but also for precise regulation of replication initiation and coordination of chromosomal replication with the cell cycle (also in response to environmental signals). We focus on Escherichia coli, and then expand our discussion to include several other microorganisms in which additional regulatory proteins have been recently shown to be involved in coordinating replication initiation to other cellular processes (e.g., Bacillus, Caulobacter, Helicobacter, Mycobacterium, and Streptomyces). We discuss diversity of bacterial oriC regions with the main focus on roles of individual DNA recognition sequences at oriC in binding the initiator and regulatory proteins as well as the overall impact of these proteins on the formation of initiation complex. PMID:25610430

  16. Events during eucaryotic rRNA transcription initiation and elongation: Conversion from the closed to the open promoter complex requires nucleotide substrates

    SciTech Connect

    Bateman, E.; Paule, M.R.

    1988-05-01

    Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyro-carbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

  17. Real-time observation of the initiation of RNA polymerase II transcription.

    PubMed

    Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

    2015-09-10

    Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

  18. Bacterial Shape and ActA Distribution Affect Initiation of Listeria monocytogenes Actin-Based Motility

    PubMed Central

    Rafelski, Susanne M.; Theriot, Julie A.

    2005-01-01

    We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed. PMID:15980176

  19. Binding motifs in bacterial gene promoters modulate transcriptional effects of global regulators CRP and ArcA

    SciTech Connect

    Leuze, Mike; Karpinets, Tatiana V.; Syed, Mustafa H.; Beliaev, Alex S.; Uberbacher, Edward

    2012-05-30

    Bacterial gene regulation involves transcription factors (TF) that bind to DNA recognition sequences in operon promoters. These recognition sequences, many of which are palindromic, are known as regulatory elements or transcription factor binding sites (TFBS). Some TFs are global regulators that can modulate the expression of hundreds of genes. In this study we examine global regulator half-sites, where a half-site, which we shall call a binding motif (BM), is one half of a palindromic TFBS. We explore the hypothesis that the number of BMs plays an important role in transcriptional regulation, examining empirical data from transcriptional profiling of the CRP and ArcA regulons. We compare the power of BM counts and of full TFBS characteristics to predict induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full TFBS quality or location.

  20. Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation

    PubMed Central

    Ramachandran, Aparna; Basu, Urmimala; Sultana, Shemaila; Nandakumar, Divya; Patel, Smita S.

    2017-01-01

    Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has ‘post-recruitment’ roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from −4 to +1 in the open complex with all three proteins and from −4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery. PMID:27903899

  1. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

    PubMed Central

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

    2016-01-01

    DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. PMID:27001521

  2. A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

    PubMed Central

    Sogayar, Mari Cleide; Camargo, Anamaria A.

    2004-01-01

    We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms. PMID:15197164

  3. SeqTU: A Web Server for Identification of Bacterial Transcription Units

    PubMed Central

    Chen, Xin; Chou, Wen-Chi; Ma, Qin; Xu, Ying

    2017-01-01

    A transcription unit (TU) consists of K ≥ 1consecutive genes on the same strand of a bacterial genome that are transcribed into a single mRNA molecule under certain conditions. Their identification is an essential step in elucidation of transcriptional regulatory networks. We have recently developed a machine-learning method to accurately identify TUs from RNA-seq data, based on two features of the assembled RNA reads: the continuity and stability of RNA-seq coverage across a genomic region. While good performance was achieved by the method on Escherichia coli and Clostridium thermocellum, substantial work is needed to make the program generally applicable to all bacteria, knowing that the program requires organism specific information. A web server, named SeqTU, was developed to automatically identify TUs with given RNA-seq data of any bacterium using a machine-learning approach. The server consists of a number of utility tools, in addition to TU identification, such as data preparation, data quality check and RNA-read mapping. SeqTU provides a user-friendly interface and automated prediction of TUs from given RNA-seq data. The predicted TUs are displayed intuitively using HTML format along with a graphic visualization of the prediction. PMID:28266571

  4. Short term memory of Caenorhabditis elegans against bacterial pathogens involves CREB transcription factor.

    PubMed

    Prithika, Udayakumar; Vikneswari, Ramaraj; Balamurugan, Krishnaswamy

    2017-04-01

    One of the key issues pertaining to the control of memory is to respond to a consistently changing environment or microbial niche present in it. Human cyclic AMP response element binding protein (CREB) transcription factor which plays a crucial role in memory has a homolog in C. elegans, crh-1. crh-1 appears to influence memory processes to certain extent by habituation of the host to a particular environment. The discrimination between the pathogen and a non-pathogen is essential for C. elegans in a microbial niche which determines its survival. Training the nematodes in the presence of a virulent pathogen (S. aureus) and an opportunistic pathogen (P. mirabilis) separately exhibits a different behavioural paradigm. This appears to be dependent on the CREB transcription factor. Here we show that C. elegans homolog crh-1 helps in memory response for a short term against the interacting pathogens. Following conditioning of the nematodes to S. aureus and P. mirabilis, the wild type nematodes exhibited a positive response towards the respective pathogens which diminished slowly after 2h. By contrast, the crh-1 deficient nematodes had a defective memory post conditioning. The molecular data reinforces the importance of crh-1 gene in retaining the memory of nematode. Our results also suggest that involvement of neurotransmitters play a crucial role in modulating the memory of the nematode with the assistance of CREB. Therefore, we elucidate that CREB is responsible for the short term memory response in C. elegans against bacterial pathogens.

  5. SeqTU: A Web Server for Identification of Bacterial Transcription Units.

    PubMed

    Chen, Xin; Chou, Wen-Chi; Ma, Qin; Xu, Ying

    2017-03-07

    A transcription unit (TU) consists of K ≥ 1consecutive genes on the same strand of a bacterial genome that are transcribed into a single mRNA molecule under certain conditions. Their identification is an essential step in elucidation of transcriptional regulatory networks. We have recently developed a machine-learning method to accurately identify TUs from RNA-seq data, based on two features of the assembled RNA reads: the continuity and stability of RNA-seq coverage across a genomic region. While good performance was achieved by the method on Escherichia coli and Clostridium thermocellum, substantial work is needed to make the program generally applicable to all bacteria, knowing that the program requires organism specific information. A web server, named SeqTU, was developed to automatically identify TUs with given RNA-seq data of any bacterium using a machine-learning approach. The server consists of a number of utility tools, in addition to TU identification, such as data preparation, data quality check and RNA-read mapping. SeqTU provides a user-friendly interface and automated prediction of TUs from given RNA-seq data. The predicted TUs are displayed intuitively using HTML format along with a graphic visualization of the prediction.

  6. Neuronal Transcriptional Repressor REST Suppresses an Atoh7-Independent Program for Initiating Retinal Ganglion Cell Development

    PubMed Central

    Mao, Chai-An; Tsai, Wen-Wei; Cho, Jang-Hyeon; Pan, Ping; Barton, Michelle Craig; Klein, William H.

    2010-01-01

    As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs. PMID:20969844

  7. Conservation of Transcription Start Sites within Genes across a Bacterial Genus

    SciTech Connect

    Shao, Wenjun; Price, Morgan N.; Deutschbauer, Adam M.; Romine, Margaret F.; Arkin, Adam P.

    2014-07-01

    Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function.

  8. TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation.

    PubMed

    Papai, Gabor; Tripathi, Manish K; Ruhlmann, Christine; Layer, Justin H; Weil, P Anthony; Schultz, Patrick

    2010-06-17

    Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.

  9. High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

    PubMed Central

    Labhart, P; Reeder, R H

    1989-01-01

    We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters. Images PMID:2470092

  10. Transcription activator structure reveals redox control of a replication initiation reaction†

    PubMed Central

    Sanders, Cyril M.; Sizov, Dmytro; Seavers, Philippa R.; Ortiz-Lombardía, Miguel; Antson, Alfred A.

    2007-01-01

    Redox changes are one of the factors that influence cell-cycle progression and that control the processes of cellular proliferation, differentiation, senescence and apoptosis. Proteins regulated through redox-sensitive cysteines have been characterized but specific ‘sulphydryl switches’ in replication proteins remain to be identified. In bovine papillomavirus type-1, DNA replication begins when the viral transcription factor E2 recruits the viral initiator protein E1 to the origin of DNA replication (ori). Here we show that a novel dimerization interface in the E2 transcription activation domain is stabilized by a disulphide bond. Oxidative cross-linking via Cys57 sequesters the interaction surface between E1 and E2, preventing pre-initiation and replication initiation complex formation. Our data demonstrate that as well as a mechanism for regulating DNA binding, redox reactions can control replication by modulating the tertiary structure of critical protein factors using a specific redox sensor. PMID:17478495

  11. X-ray Crystal Structures Elucidate the Nucleotidyl Transfer Reaction of Transcript Initiation Using Two Nucleotides

    SciTech Connect

    M Gleghorn; E Davydova; R Basu; L Rothman-Denes; K Murakami

    2011-12-31

    We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

  12. A model for regulation of mammalian ribosomal DNA transcription. Co-ordination of initiation and termination.

    PubMed Central

    Nashimoto, M; Mishima, Y

    1988-01-01

    Based on recent experimental data about transcription initiation and termination, a model for regulation of mammalian ribosomal DNA transcription is developed using a simple kinetic scheme. In this model, the existence of the transition pathway from the terminator to the promoter increases the rate of ribosomal RNA precursor synthesis. In addition to this 'non-transcribed spacer' traverse of RNA polymerase I, the co-ordination of initiation and termination allows a rapid on/off switch transition from the minimum to the maximum rate of ribosomal RNA precursor synthesis. Furthermore, taking account of the participation of two factors in the termination event, we propose a plausible molecular mechanism for the co-ordination of initiation and termination. This co-ordination is emphasized by repetition of the terminator unit. PMID:3223915

  13. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure.

    PubMed

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG.

  14. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure

    PubMed Central

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG. PMID:26779399

  15. Anti-Biofilm Performance of Three Natural Products against Initial Bacterial Attachment

    PubMed Central

    Salta, Maria; Wharton, Julian A.; Dennington, Simon P.; Stoodley, Paul; Stokes, Keith R.

    2013-01-01

    Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent “slime” attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5–20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria. PMID:24192819

  16. Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

    PubMed Central

    Vassena, Rita; Boué, Stéphanie; González-Roca, Eva; Aran, Begoña; Auer, Herbert; Veiga, Anna; Belmonte, Juan Carlos Izpisua

    2011-01-01

    The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming. PMID:21775417

  17. Plant and Bacterial Symbiotic Mutants Define Three Transcriptionally Distinct Stages in the Development of the Medicago truncatula/Sinorhizobium meliloti Symbiosis1

    PubMed Central

    Mitra, Raka Mustaphi; Long, Sharon Rugel

    2004-01-01

    In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation. Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants. We have identified three symbiotically regulated plant genes encoding a β,1–3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor. The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes. The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M. truncatula by S. meliloti. PMID:14739349

  18. Developmental trajectories of amphibian microbiota: response to bacterial therapy depends on initial community structure.

    PubMed

    Davis, Leyla R; Bigler, Laurent; Woodhams, Douglas C

    2017-02-22

    Improving host health through microbial manipulation requires untangling factors that shape the microbiome. There is currently little understanding of how initial community structure may drive the microbiota trajectory across host development or influence bacterial therapy outcomes. Probiotic baths of surface symbionts, Pseudomonas fluorescens and Flavobacterium johnsoniae were administered to 240 tadpoles of the midwife toad, Alytes obstetricans in semi-natural outdoor mesocosms originating from geographically and genetically distinct populations in Switzerland. Host bacterial and fungal assemblages were compared in tadpoles from the pond of origin, across metamorphosis, and in toadlets via microbial fingerprinting. Bacterial and fungal community structures differed significantly among populations and a microbial population signature persisted from the tadpole stage, through metamorphosis, and following probiotic treatment. A minimal core surface microbiota is described by persistence through development and by shared membership across populations. The impact of F. johnsoniae on the tadpole surface microbiome was assessed with shotgun metagenomics. Bacterial therapy reduced abundance, diversity, and functional repertoire compared to untreated controls. A correlation between host skin peptides and microbiota suggests a mechanism of host-directed symbiosis throughout development. Early developmental stages are ideal targets for amphibian bacterial therapy that can govern a microbiome trajectory at critical timepoints and may impact susceptibility to disease.

  19. Structure of an RNA Polymerase II-TFIIB Complex and the Transcription Initiation Mechanism

    SciTech Connect

    Liu, Xin; Bushnell, David A; Wang, Dong; Calero, Guillermo; Kornberg, Roger D

    2010-01-14

    Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the 'B finger,' reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

  20. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA.

    PubMed

    Hubin, Elizabeth A; Fay, Allison; Xu, Catherine; Bean, James M; Saecker, Ruth M; Glickman, Michael S; Darst, Seth A; Campbell, Elizabeth A

    2017-01-09

    RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the -10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD.

  1. The elongation factor Spt5 facilitates transcription initiation for rapid induction of inflammatory-response genes

    PubMed Central

    Diamant, Gil; Bahat, Anat; Dikstein, Rivka

    2016-01-01

    A subset of inflammatory-response NF-κB target genes is activated immediately following pro-inflammatory signal. Here we followed the kinetics of primary transcript accumulation after NF-κB activation when the elongation factor Spt5 is knocked down. While elongation rate is unchanged, the transcript synthesis at the 5′-end and at the earliest time points is delayed and reduced, suggesting an unexpected role in early transcription. Investigating the underlying mechanism reveals that the induced TFIID–promoter association is practically abolished by Spt5 depletion. This effect is associated with a decrease in promoter-proximal H3K4me3 and H4K5Ac histone modifications that are differentially required for rapid transcriptional induction. In contrast, the displacement of TFIIE and Mediator, which occurs during promoter escape, is attenuated in the absence of Spt5. Our findings are consistent with a central role of Spt5 in maintenance of TFIID–promoter association and promoter escape to support rapid transcriptional induction and re-initiation of inflammatory-response genes. PMID:27180651

  2. A cellular repressor regulates transcription initiation from the minute virus of mice P38 promoter.

    PubMed Central

    Krauskopf, A; Aloni, Y

    1994-01-01

    We previously reported that the P38 promoter of minute virus of mice (MVM) is trans activated by the viral nonstructural protein, NS1, through an interaction with a downstream promoter element designated DPE. In this communication we report the identification of a distinct downstream promoter element which inhibits transcription from the P38 promoter in vitro, in the absence of the DPE. Removal of 34 bp from the region between +95 and +129 downstream from the P38 initiation start site relieved inhibition of transcription in whole-cell extract. Inhibition was also relieved by the addition, to the transcription reaction, of excess DNA fragments which span the putative inhibiting element. This indicated the involvement of a trans-acting factor, in inhibition of transcription from the P38. Gel retardation experiments demonstrated the specific binding of a cellular protein to the inhibitory element. This P38 inhibitory element shows spacing and orientation dependence as well as promoter specificity. The regulation of viral transcription by a cellular repressor may play an important role in obtaining a fine temporal order of viral gene expression during the course of infection. Images PMID:8139925

  3. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA

    PubMed Central

    Hubin, Elizabeth A; Fay, Allison; Xu, Catherine; Bean, James M; Saecker, Ruth M; Glickman, Michael S; Darst, Seth A; Campbell, Elizabeth A

    2017-01-01

    RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001 PMID:28067618

  4. DciA is an ancestral replicative helicase operator essential for bacterial replication initiation

    PubMed Central

    Brézellec, Pierre; Vallet-Gely, Isabelle; Possoz, Christophe; Quevillon-Cheruel, Sophie; Ferat, Jean-Luc

    2016-01-01

    Delivery of the replicative helicase onto DNA is an essential step in the initiation of replication. In bacteria, DnaC (in Escherichia coli) and DnaI (in Bacillus subtilis) are representative of the two known mechanisms that assist the replicative helicase at this stage. Here, we establish that these two strategies cannot be regarded as prototypical of the bacterial domain since dnaC and dnaI (dna[CI]) are present in only a few bacterial phyla. We show that dna[CI] was domesticated at least seven times through evolution in bacteria and at the expense of one gene, which we rename dciA (dna[CI] antecedent), suggesting that DciA and Dna[CI] share a common function. We validate this hypothesis by establishing in Pseudomonas aeruginosa that DciA possesses the attributes of the replicative helicase-operating proteins associated with replication initiation. PMID:27830752

  5. The elongation factor RfaH and the initiation factor σ bind to the same site on the transcription elongation complex

    PubMed Central

    Sevostyanova, Anastasiya; Svetlov, Vladimir; Vassylyev, Dmitry G.; Artsimovitch, Irina

    2008-01-01

    RNA polymerase is a target for numerous regulatory events in all living cells. Recent studies identified a few “hot spots” on the surface of bacterial RNA polymerase that mediate its interactions with diverse accessory proteins. Prominent among these hot spots, the β′ subunit clamp helices serve as a major binding site for the initiation factor σ and for the elongation factor RfaH. Furthermore, the two proteins interact with the nontemplate DNA strand in transcription complexes and thus may interfere with each other's activity. We show that RfaH does not inhibit transcription initiation but, once recruited to RNA polymerase, abolishes σ-dependent pausing. We argue that this apparent competition is due to a steric exclusion of σ by RfaH that is stably bound to the nontemplate DNA and clamp helices, both of which are necessary for the σ recruitment to the transcription complex. Our findings highlight the key regulatory role played by the clamp helices during both initiation and elongation stages of transcription. PMID:18195372

  6. Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis.

    PubMed

    Nepal, Chirag; Hadzhiev, Yavor; Previti, Christopher; Haberle, Vanja; Li, Nan; Takahashi, Hazuki; Suzuki, Ana Maria M; Sheng, Ying; Abdelhamid, Rehab F; Anand, Santosh; Gehrig, Jochen; Akalin, Altuna; Kockx, Christel E M; van der Sloot, Antoine A J; van Ijcken, Wilfred F J; Armant, Olivier; Rastegar, Sepand; Watson, Craig; Strähle, Uwe; Stupka, Elia; Carninci, Piero; Lenhard, Boris; Müller, Ferenc

    2013-11-01

    Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.

  7. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    PubMed Central

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  8. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    NASA Astrophysics Data System (ADS)

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-07-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

  9. An unmethylated 3' promoter-proximal region is required for efficient transcription initiation.

    PubMed

    Appanah, Ruth; Dickerson, David R; Goyal, Preeti; Groudine, Mark; Lorincz, Matthew C

    2007-02-16

    The promoter regions of approximately 40% of genes in the human genome are embedded in CpG islands, CpG-rich regions that frequently extend on the order of one kb 3' of the transcription start site (TSS) region. CpGs 3' of the TSS of actively transcribed CpG island promoters typically remain methylation-free, indicating that maintaining promoter-proximal CpGs in an unmethylated state may be important for efficient transcription. Here we utilize recombinase-mediated cassette exchange to introduce a Moloney Murine Leukemia Virus (MoMuLV)-based reporter, in vitro methylated 1 kb downstream of the TSS, into a defined genomic site. In a subset of clones, methylation spreads to within approximately 320 bp of the TSS, yielding a dramatic decrease in transcript level, even though the promoter/TSS region remains unmethylated. Chromatin immunoprecipitation analyses reveal that such promoter-proximal methylation results in loss of RNA polymerase II and TATA-box-binding protein (TBP) binding in the promoter region, suggesting that repression occurs at the level of transcription initiation. While DNA methylation-dependent trimethylation of H3 lysine (K)9 is confined to the intragenic methylated region, the promoter and downstream regions are hypo-acetylated on H3K9/K14. Furthermore, DNase I hypersensitivity and methylase-based single promoter analysis (M-SPA) experiments reveal that a nucleosome is positioned over the unmethylated TATA-box in these clones, indicating that dense DNA methylation downstream of the promoter region is sufficient to alter the chromatin structure of an unmethylated promoter. Based on these observations, we propose that a DNA methylation-free region extending several hundred bases downstream of the TSS may be a prerequisite for efficient transcription initiation. This model provides a biochemical explanation for the typical positioning of TSSs well upstream of the 3' end of the CpG islands in which they are embedded.

  10. A Critical, Nonlinear Threshold Dictates Bacterial Invasion and Initial Kinetics During Influenza

    PubMed Central

    Smith, Amber M.; Smith, Amanda P.

    2016-01-01

    Secondary bacterial infections increase morbidity and mortality of influenza A virus (IAV) infections. Bacteria are able to invade due to virus-induced depletion of alveolar macrophages (AMs), but this is not the only contributing factor. By analyzing a kinetic model, we uncovered a nonlinear initial dose threshold that is dependent on the amount of virus-induced AM depletion. The threshold separates the growth and clearance phenotypes such that bacteria decline for dose-AM depletion combinations below the threshold, stay constant near the threshold, and increase above the threshold. In addition, the distance from the threshold correlates to the growth rate. Because AM depletion changes throughout an IAV infection, the dose requirement for bacterial invasion also changes accordingly. Using the threshold, we found that the dose requirement drops dramatically during the first 7d of IAV infection. We then validated these analytical predictions by infecting mice with doses below or above the predicted threshold over the course of IAV infection. These results identify the nonlinear way in which two independent factors work together to support successful post-influenza bacterial invasion. They provide insight into coinfection timing, the heterogeneity in outcome, the probability of acquiring a coinfection, and the use of new therapeutic strategies to combat viral-bacterial coinfections. PMID:27974820

  11. A Critical, Nonlinear Threshold Dictates Bacterial Invasion and Initial Kinetics During Influenza

    NASA Astrophysics Data System (ADS)

    Smith, Amber M.; Smith, Amanda P.

    2016-12-01

    Secondary bacterial infections increase morbidity and mortality of influenza A virus (IAV) infections. Bacteria are able to invade due to virus-induced depletion of alveolar macrophages (AMs), but this is not the only contributing factor. By analyzing a kinetic model, we uncovered a nonlinear initial dose threshold that is dependent on the amount of virus-induced AM depletion. The threshold separates the growth and clearance phenotypes such that bacteria decline for dose-AM depletion combinations below the threshold, stay constant near the threshold, and increase above the threshold. In addition, the distance from the threshold correlates to the growth rate. Because AM depletion changes throughout an IAV infection, the dose requirement for bacterial invasion also changes accordingly. Using the threshold, we found that the dose requirement drops dramatically during the first 7d of IAV infection. We then validated these analytical predictions by infecting mice with doses below or above the predicted threshold over the course of IAV infection. These results identify the nonlinear way in which two independent factors work together to support successful post-influenza bacterial invasion. They provide insight into coinfection timing, the heterogeneity in outcome, the probability of acquiring a coinfection, and the use of new therapeutic strategies to combat viral-bacterial coinfections.

  12. Convergent Transcription At Intragenic Super-Enhancers Targets AID-initiated Genomic Instability

    PubMed Central

    Meng, Fei-Long; Du, Zhou; Federation, Alexander; Hu, Jiazhi; Wang, Qiao; Kieffer-Kwon, Kyong-Rim; Meyers, Robin M.; Amor, Corina; Wasserman, Caitlyn R.; Neuberg, Donna; Casellas, Rafael; Nussenzweig, Michel C.; Bradner, James E.; Liu, X. Shirley; Alt, Frederick W.

    2015-01-01

    Summary Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single stranded DNA targets. While largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-Seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from Super-Enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells. PMID:25483776

  13. RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

    PubMed Central

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E.; Liau, Brian B.; Mack, Stephen C.; Yang, Kailin; Factor, Daniel C.; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E.; Bao, Shideng; Rich, Jeremy N.

    2016-01-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH. PMID:27322055

  14. RBPJ maintains brain tumor-initiating cells through CDK9-mediated transcriptional elongation.

    PubMed

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E; Liau, Brian B; Mack, Stephen C; Yang, Kailin; Factor, Daniel C; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E; Bao, Shideng; Rich, Jeremy N

    2016-07-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

  15. Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

    PubMed Central

    Weake, Vikki M.; Dyer, Jamie O.; Seidel, Christopher; Box, Andrew; Swanson, Selene K.; Peak, Allison; Florens, Laurence; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2011-01-01

    The Spt–Ada–Gcn5–acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5′ end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression. PMID:21764853

  16. Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation

    PubMed Central

    Zhang, Zhengjian; Boskovic, Zarko; Hussain, Mahmud M; Hu, Wenxin; Inouye, Carla; Kim, Han-Je; Abole, A Katherine; Doud, Mary K; Lewis, Timothy A; Koehler, Angela N; Schreiber, Stuart L; Tjian, Robert

    2015-01-01

    Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions. DOI: http://dx.doi.org/10.7554/eLife.07777.001 PMID:26314865

  17. Transcriptional Response of Honey Bee Larvae Infected with the Bacterial Pathogen Paenibacillus larvae

    PubMed Central

    Cornman, Robert Scott; Lopez, Dawn; Evans, Jay D.

    2013-01-01

    American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and a general immunity is achieved by larvae as they age, the basis of which has not been identified. To quickly identify a pool of candidate genes responsive to P. larvae infection, we sequenced transcripts from larvae inoculated with P. larvae at 12 hours post-emergence and incubated for 72 hours, and compared expression levels to a control cohort. We identified 75 genes with significantly higher expression and six genes with significantly lower expression. In addition to several antimicrobial peptides, two genes encoding peritrophic-matrix domains were also up-regulated. Extracellular matrix proteins, proteases/protease inhibitors, and members of the Osiris gene family were prevalent among differentially regulated genes. However, analysis of Drosophila homologs of differentially expressed genes revealed spatial and temporal patterns consistent with developmental asynchrony as a likely confounder of our results. We therefore used qPCR to measure the consistency of gene expression changes for a subset of differentially expressed genes. A replicate experiment sampled at both 48 and 72 hours post infection allowed further discrimination of genes likely to be involved in host response. The consistently responsive genes in our test set included a hymenopteran-specific protein tyrosine kinase, a hymenopteran specific serine endopeptidase, a cytochrome P450 (CYP9Q1), and a homolog of trynity, a zona pellucida domain protein. Of the known honey bee antimicrobial peptides, apidaecin was responsive at both time-points studied whereas hymenoptaecin was more consistent in its level of change between biological replicates and had the greatest increase in expression by RNA-seq analysis

  18. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

    PubMed

    Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

    2012-09-01

    Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

  19. ITPI: Initial Transcription Process-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula

    PubMed Central

    Zhang, Baixia; Li, Yanwen; Zhang, Yanling; Li, Zhiyong; Bi, Tian; He, Yusu; Song, Kuokui; Wang, Yun

    2016-01-01

    Identification of bioactive components is an important area of research in traditional Chinese medicine (TCM) formula. The reported identification methods only consider the interaction between the components and the target proteins, which is not sufficient to explain the influence of TCM on the gene expression. Here, we propose the Initial Transcription Process-based Identification (ITPI) method for the discovery of bioactive components that influence transcription factors (TFs). In this method, genome-wide chip detection technology was used to identify differentially expressed genes (DEGs). The TFs of DEGs were derived from GeneCards. The components influencing the TFs were derived from STITCH. The bioactive components in the formula were identified by evaluating the molecular similarity between the components in formula and the components that influence the TF of DEGs. Using the formula of Tian-Zhu-San (TZS) as an example, the reliability and limitation of ITPI were examined and 16 bioactive components that influence TFs were identified. PMID:27034696

  20. SigmoID: a user-friendly tool for improving bacterial genome annotation through analysis of transcription control signals

    PubMed Central

    Damienikan, Aliaksandr U.

    2016-01-01

    The majority of bacterial genome annotations are currently automated and based on a ‘gene by gene’ approach. Regulatory signals and operon structures are rarely taken into account which often results in incomplete and even incorrect gene function assignments. Here we present SigmoID, a cross-platform (OS X, Linux and Windows) open-source application aiming at simplifying the identification of transcription regulatory sites (promoters, transcription factor binding sites and terminators) in bacterial genomes and providing assistance in correcting annotations in accordance with regulatory information. SigmoID combines a user-friendly graphical interface to well known command line tools with a genome browser for visualising regulatory elements in genomic context. Integrated access to online databases with regulatory information (RegPrecise and RegulonDB) and web-based search engines speeds up genome analysis and simplifies correction of genome annotation. We demonstrate some features of SigmoID by constructing a series of regulatory protein binding site profiles for two groups of bacteria: Soft Rot Enterobacteriaceae (Pectobacterium and Dickeya spp.) and Pseudomonas spp. Furthermore, we inferred over 900 transcription factor binding sites and alternative sigma factor promoters in the annotated genome of Pectobacterium atrosepticum. These regulatory signals control putative transcription units covering about 40% of the P. atrosepticum chromosome. Reviewing the annotation in cases where it didn’t fit with regulatory information allowed us to correct product and gene names for over 300 loci. PMID:27257541

  1. SigmoID: a user-friendly tool for improving bacterial genome annotation through analysis of transcription control signals.

    PubMed

    Nikolaichik, Yevgeny; Damienikan, Aliaksandr U

    2016-01-01

    The majority of bacterial genome annotations are currently automated and based on a 'gene by gene' approach. Regulatory signals and operon structures are rarely taken into account which often results in incomplete and even incorrect gene function assignments. Here we present SigmoID, a cross-platform (OS X, Linux and Windows) open-source application aiming at simplifying the identification of transcription regulatory sites (promoters, transcription factor binding sites and terminators) in bacterial genomes and providing assistance in correcting annotations in accordance with regulatory information. SigmoID combines a user-friendly graphical interface to well known command line tools with a genome browser for visualising regulatory elements in genomic context. Integrated access to online databases with regulatory information (RegPrecise and RegulonDB) and web-based search engines speeds up genome analysis and simplifies correction of genome annotation. We demonstrate some features of SigmoID by constructing a series of regulatory protein binding site profiles for two groups of bacteria: Soft Rot Enterobacteriaceae (Pectobacterium and Dickeya spp.) and Pseudomonas spp. Furthermore, we inferred over 900 transcription factor binding sites and alternative sigma factor promoters in the annotated genome of Pectobacterium atrosepticum. These regulatory signals control putative transcription units covering about 40% of the P. atrosepticum chromosome. Reviewing the annotation in cases where it didn't fit with regulatory information allowed us to correct product and gene names for over 300 loci.

  2. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed Central

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-01-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994). PMID:7651413

  3. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-09-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

  4. Transcriptional Regulation in Saccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

    PubMed Central

    Hahn, Steven; Young, Elton T.

    2011-01-01

    Here we review recent advances in understanding the regulation of mRNA synthesis in Saccharomyces cerevisiae. Many fundamental gene regulatory mechanisms have been conserved in all eukaryotes, and budding yeast has been at the forefront in the discovery and dissection of these conserved mechanisms. Topics covered include upstream activation sequence and promoter structure, transcription factor classification, and examples of regulated transcription factor activity. We also examine advances in understanding the RNA polymerase II transcription machinery, conserved coactivator complexes, transcription activation domains, and the cooperation of these factors in gene regulatory mechanisms. PMID:22084422

  5. Key Roles of the Downstream Mobile Jaw of Escherichia coli RNA Polymerase in Transcription Initiation

    PubMed Central

    Drennan, Amanda; Kraemer, Mark; Capp, Michael; Gries, Theodore; Ruff, Emily; Sheppard, Carol; Wigneshweraraj, Sivaramesh; Artsimovitch, Irina; Record, M. Thomas

    2012-01-01

    Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λPR promoter DNA to form the unstable open intermediate I2, which rapidly converts to much more stable open complexes (I3, RPo). Conversion of I2 to RPo involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β’ jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ΔJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5–10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ΔJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally-uncharacterized intermediate I3. Overall rate constants (ka) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of ka for WT RNAP with DT+12 and full-length λPR are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RPo formation, while downstream DNA functions primarily as the assembly platform after DNA opening. PMID:23116321

  6. Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase.

    PubMed

    Henderson, Kate L; Felth, Lindsey C; Molzahn, Cristen M; Shkel, Irina; Wang, Si; Chhabra, Munish; Ruff, Emily F; Bieter, Lauren; Kraft, Joseph E; Record, M Thomas

    2017-04-11

    To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ββ'ωσ(70)), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

  7. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  8. Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization.

    PubMed

    Kremer, Natacha; Philipp, Eva E R; Carpentier, Marie-Christine; Brennan, Caitlin A; Kraemer, Lars; Altura, Melissa A; Augustin, René; Häsler, Robert; Heath-Heckman, Elizabeth A C; Peyer, Suzanne M; Schwartzman, Julia; Rader, Bethany A; Ruby, Edward G; Rosenstiel, Philip; McFall-Ngai, Margaret J

    2013-08-14

    Upon transit to colonization sites, bacteria often experience critical priming that prepares them for subsequent, specific interactions with the host; however, the underlying mechanisms are poorly described. During initiation of the symbiosis between the bacterium Vibrio fischeri and its squid host, which can be observed directly and in real time, approximately five V. fischeri cells aggregate along the mucociliary membranes of a superficial epithelium prior to entering host tissues. Here, we show that these few early host-associated symbionts specifically induce robust changes in host gene expression that are critical to subsequent colonization steps. This exquisitely sensitive response to the host's specific symbiotic partner includes the upregulation of a host endochitinase, whose activity hydrolyzes polymeric chitin in the mucus into chitobiose, thereby priming the symbiont and also producing a chemoattractant gradient that promotes V. fischeri migration into host tissues. Thus, the host responds transcriptionally upon initial symbiont contact, which facilitates subsequent colonization.

  9. Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

    PubMed Central

    Haycocks, James R. J.; Grainger, David C.

    2016-01-01

    A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality. PMID:27258043

  10. Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

    PubMed Central

    Wright, David P; Ulijasz, Andrew T

    2014-01-01

    Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

  11. Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.

    PubMed

    Angermayr, Michaela; Oechsner, Ulrich; Gregor, Kerstin; Schroth, Gary P; Bandlow, Wolfhard

    2002-10-01

    The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6). Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.

  12. Transcription in archaea

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Ouzounis, C. A.; Woese, C. R. (Principal Investigator)

    1999-01-01

    Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

  13. The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

    PubMed Central

    Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

    1980-01-01

    Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

  14. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    SciTech Connect

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  15. Transcription initiation factor IID-interactive histone chaperone CIA-II implicated in mammalian spermatogenesis.

    PubMed

    Umehara, Takashi; Horikoshi, Masami

    2003-09-12

    Histones are thought to have specific roles in mammalian spermatogenesis, because several subtypes of histones emerge that are post-translationally modified during spermatogenesis. Though regular assembly of nucleosome is guaranteed by histone chaperones, their involvement in spermatogenesis is yet to be characterized. Here we identified a histone chaperone-related factor, which we designated as CCG1-interacting factor A-II (CIA-II), through interaction with bromodomains of TAFII250/CCG1, which is the largest subunit of human transcription initiation factor IID (TFIID). We found that human CIA-II (hCIA-II) localizes in HeLa nuclei and is highly expressed in testis and other proliferating cell-containing tissues. Expression of mouse CIA-II (mCIA-II) does not occur in the germ cell-lacking testes of adult WBB6F1-W/Wv mutant mice, indicating its expression in testis to be specific to germ cells. Fractionation of testicular germ cells revealed that mCIA-II transcripts accumulate in pachytene spermatocytes but not in spermatids. In addition, the mCIA-II transcripts in testis were present as early as 4 days after birth and decreased at 56 days after birth. These findings indicate that mCIA-II expression in testis is restricted to premeiotic to meiotic stages during spermatogenesis. Also, we found that hCIA-II interacts with histone H3 in vivo and with histones H3/H4 in vitro and that it facilitates supercoiling of circular DNA when it is incubated with core histones and topoisomerase I in vitro. These data suggest that CIA-II is a histone chaperone and is implicated in the regulation of mammalian spermatogenesis.

  16. Three-dimensional EM Structure of an Intact Activator-dependent Transcription Initiation Complex

    SciTech Connect

    Hudson, B.; Quispe, J; Lara-González, S; Kim, Y; Berman, H; Arnold, E; Ebright, R; Lawson, C

    2009-01-01

    We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-{angstrom} electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP {alpha} subunit C-terminal domain ({alpha}CTD), interactions of {alpha}CTD with {sigma}70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP {beta}{prime}, {beta}, and {sigma}70 subunits.

  17. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    NASA Astrophysics Data System (ADS)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  18. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    PubMed Central

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

  19. Defective transcription initiation causes postnatal growth failure in a mouse model of nucleotide excision repair (NER) progeria.

    PubMed

    Kamileri, Irene; Karakasilioti, Ismene; Sideri, Aria; Kosteas, Theodoros; Tatarakis, Antonis; Talianidis, Iannis; Garinis, George A

    2012-02-21

    Nucleotide excision repair (NER) defects are associated with cancer, developmental disorders and neurodegeneration. However, with the exception of cancer, the links between defects in NER and developmental abnormalities are not well understood. Here, we show that the ERCC1-XPF NER endonuclease assembles on active promoters in vivo and facilitates chromatin modifications for transcription during mammalian development. We find that Ercc1(-/-) mice demonstrate striking physiological, metabolic and gene expression parallels with Taf10(-/-) animals carrying a liver-specific transcription factor II D (TFIID) defect in transcription initiation. Promoter occupancy studies combined with expression profiling in the liver and in vitro differentiation cell assays reveal that ERCC1-XPF interacts with TFIID and assembles with POL II and the basal transcription machinery on promoters in vivo. Whereas ERCC1-XPF is required for the initial activation of genes associated with growth, it is dispensable for ongoing transcription. Recruitment of ERCC1-XPF on promoters is accompanied by promoter-proximal DNA demethylation and histone marks associated with active hepatic transcription. Collectively, the data unveil a role of ERCC1/XPF endonuclease in transcription initiation establishing its causal contribution to NER developmental disorders.

  20. Kinetics of transcription initiation directed by multiple cis-regulatory elements on the glnAp2 promoter

    PubMed Central

    Wang, Yaolai; Liu, Feng; Wang, Wei

    2016-01-01

    Transcription initiation is orchestrated by dynamic molecular interactions, with kinetic steps difficult to detect. Utilizing a hybrid method, we aim to unravel essential kinetic steps of transcriptional regulation on the glnAp2 promoter, whose regulatory region includes two enhancers (sites I and II) and three low-affinity sequences (sites III-V), to which the transcriptional activator NtrC binds. By structure reconstruction, we analyze all possible organization architectures of the transcription apparatus (TA). The main regulatory mode involves two NtrC hexamers: one at enhancer II transiently associates with site V such that the other at enhancer I can rapidly approach and catalyze the σ54-RNA polymerase holoenzyme. We build a kinetic model characterizing essential steps of the TA operation; with the known kinetics of the holoenzyme interacting with DNA, this model enables the kinetics beyond technical detection to be determined by fitting the input-output function of the wild-type promoter. The model further quantitatively reproduces transcriptional activities of various mutated promoters. These results reveal different roles played by two enhancers and interpret why the low-affinity elements conditionally enhance or repress transcription. This work presents an integrated dynamic picture of regulated transcription initiation and suggests an evolutionarily conserved characteristic guaranteeing reliable transcriptional response to regulatory signals. PMID:27899598

  1. Initial bacterial deposition on bare and zeolite-coated aluminum alloy and stainless steel.

    PubMed

    Chen, Gexin; Beving, Derek E; Bedi, Rajwant S; Yan, Yushan S; Walker, Sharon L

    2009-02-03

    In this study, the impact of zeolite thin film coatings on bacterial deposition and "biofouling" of surfaces has been investigated in an aqueous environment. The synthesis of two types of zeolite coatings, ZSM-5 coated on aluminum alloy and zeolite A coated on stainless steel, and the characterization of the coated and bare metal surfaces are described. The extent of cell deposition onto the bare and zeolite-coated aluminum alloy and stainless steel surfaces is investigated in a parallel plate flow chamber system under a laminar flow conditions. The initial rates of bacterial transfer to the various surfaces are compared by utilizing a marine bacterium, Halomonas pacifica g, under a range of ionic strength conditions. H. pacifica g deposited onto bare metal surfaces to a greater extent as compared with cells deposited onto the zeolite coatings. The surface properties found to have the most notable effect on attachment are the electrokinetic and hydrophobicity properties of the metal and zeolite-coated surfaces. These results suggest that a combination of two chemical mechanisms-hydrophobic and electrostatic interactions-contribute to the antifouling nature of the zeolite surface. Additional observations on the relative role of the hydrodynamic and physical phenomena are also discussed.

  2. Pituitary adenoma apoplexy with initial presentation mimicking bacterial meningoencephalitis: a case report.

    PubMed

    Huang, Wen-Yi; Chien, Yu-Yi; Wu, Chia-Lun; Weng, Wei-Chieh; Peng, Tsung-I; Chen, Hsien-Chih

    2009-05-01

    Pituitary apoplexy is a rare but life-threatening disorder. Clinical presentation of this condition includes severe headache, impaired consciousness, fever, visual disturbance, and variable ocular paresis. Signs of meningeal irritation are very rare. However, if present and associated with headache, fever, and pleocytosis, meningeal irritation may lead to misinterpretation as infectious meningoencephalitis. To the best of our knowledge, pituitary apoplexy with an initial presentation mimicking infectious meningoencephalitis had rarely been reported in the literature. Here, we report a 57-year-old man who had acute severe headache, high fever, neck stiffness, disturbance in consciousness, and left ocular paresis. Laboratory data showed leukocytosis, an elevated C-reactive protein level, and neutrophilic pleocytosis in the cerebrospinal fluid. Because bacterial meningoencephalitis was suspected, empiric antibiotic therapy was administered but in vain. Further examinations indicated a diagnosis of pituitary adenoma with apoplexy. After the immediate administration of intravenous corticosteroid supplement and surgical decompression, the patient recovered.

  3. Novel functions of thyroid hormone receptor mutants: Beyond nucleus-initiated transcription

    PubMed Central

    Furuya, Fumihiko; Ying, Hao; Zhao, Li; Cheng, Sheue-yann

    2009-01-01

    Study of molecular actions of thyroid hormone receptor β (TRβ) mutants in vivo has been facilitated by creation of a mouse model (TRβPV mouse) that harbors a knockin mutant of TRβ (denoted PV). PV, which was identified in a patient with resistance to thyroid hormone, has lost T3 binding activity and transcription capacity. The striking phenotype of thyroid cancer exhibited by TRβPV/PV mice has allowed the elucidation of novel oncogenic activity of a TRβ mutant (PV) [PAS1]beyond nucleus-initiated transcription. PV was found to physically interact with the regulatory p85α subunit of phosphatidylinositol 3-kinase (PI3K) in both the nuclear and cytoplasmic compartments. This protein-protein interaction activates the PI3K signaling by increasing phosphorylation of AKT, mammalian target of rapamycin (mTOR), and p70S6K. PV, via interaction with p85α, also activates the PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathway in the extra-nuclear compartment. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. In addition to affecting these membrane-initiated signaling events, PV affects [PAS2]the stability of the pituitary tumor-transforming gene (PTTG) product. PTTG (also known as securin), a critical mitotic checkpoint protein, is physically associated with TRβ or PV in vivo. Concomitant with T3-induced degradation of TRβ, PTTG is degraded by the proteasome machinery, but no such degradation occurs when PTTG is associated with PV. The degradation of PTTG/TRβ is activated by the direct interaction of the T3-bound TRβ with the steroid receptor coactivator-3 (SRC-3) that recruits a proteasome activator (PA28γ). PV that does not bind T3 cannot interact directly with SRC-3/PA28γ to activate proteasome degradation, and the absence of degradation results in an aberrant accumulation of PTTG. The PV-induced failure of timely degradation of PTTG results in mitotic abnormalities. PV, via novel

  4. Early Canine Plaque Biofilms: Characterization of Key Bacterial Interactions Involved in Initial Colonization of Enamel

    PubMed Central

    Holcombe, Lucy J.; Patel, Niran; Colyer, Alison; Deusch, Oliver; O’Flynn, Ciaran; Harris, Stephen

    2014-01-01

    Periodontal disease (PD) is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops. PMID:25463050

  5. Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

    PubMed Central

    Aregger, Michael; Cowling, Victoria H.

    2013-01-01

    Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation. PMID:23863084

  6. [SWI/SNF Protein Complexes Participate in the Initiation and Elongation Stages of Drosophila hsp70 Gene Transcription].

    PubMed

    Mazina, M Yu; Nikolenko, Yu V; Krasnov, A N; Vorobyeva, N E

    2016-02-01

    The participation of the SWI/SNF chromatin remodeling complex in the stimulation of the RNA polymerase II binding to gene promotors was demonstrated in all model eukaryotic organisms. It was shown eight years ago that the SWI/SNF complex influence on transcription is not limited to its role in initiation but also includes participation in elongation and alternative splicing. In the current work, we describe the subunit composition of the SWI/SNF complexes participating in initiation, preparing for the elongation and elongation of hsp70 gene transcription in Drosophila melanogaster. The data reveal the high mobility of the SWI/SNF complex composition during the hsp 70 gene transcription process. We suggest a model describing the process of sequential SWI/SNF complex formation during heat-shock induced transcription of the hsp 70 gene.

  7. Overexpression of the Eggplant (Solanum melongena) NAC Family Transcription Factor SmNAC Suppresses Resistance to Bacterial Wilt

    PubMed Central

    Na, Chen; Shuanghua, Wu; Jinglong, Fu; Bihao, Cao; Jianjun, Lei; Changming, Chen; Jin, Jiang

    2016-01-01

    Bacterial wilt (BW) is a serious disease that affects eggplant (Solanum melongena) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (SmNAC) from eggplant and characterized its expression, its localization at the tissue and subcellular levels, and its role in BW resistance. To this end, transgenic eggplant lines were generated in which the expression of SmNAC was constitutively up regulated or suppressed using RNAi. The results indicated that overexpression of SmNAC decreases resistance to BW. Moreover, SmNAC overexpression resulted in the reduced accumulation of the plant immune signaling molecule salicylic acid (SA) and reduced expression of ICS1 (a gene that encode isochorismate synthase 1, which is involved in SA biosynthesis). We propose that reduced SA content results in increased bacterial wilt susceptibility in the transgenic lines. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in eggplant. PMID:27528282

  8. Overexpression of the Eggplant (Solanum melongena) NAC Family Transcription Factor SmNAC Suppresses Resistance to Bacterial Wilt.

    PubMed

    Na, Chen; Shuanghua, Wu; Jinglong, Fu; Bihao, Cao; Jianjun, Lei; Changming, Chen; Jin, Jiang

    2016-08-16

    Bacterial wilt (BW) is a serious disease that affects eggplant (Solanum melongena) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (SmNAC) from eggplant and characterized its expression, its localization at the tissue and subcellular levels, and its role in BW resistance. To this end, transgenic eggplant lines were generated in which the expression of SmNAC was constitutively up regulated or suppressed using RNAi. The results indicated that overexpression of SmNAC decreases resistance to BW. Moreover, SmNAC overexpression resulted in the reduced accumulation of the plant immune signaling molecule salicylic acid (SA) and reduced expression of ICS1 (a gene that encode isochorismate synthase 1, which is involved in SA biosynthesis). We propose that reduced SA content results in increased bacterial wilt susceptibility in the transgenic lines. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in eggplant.

  9. Transcription factor Interplay between LEAFY and APETALA1/ CAULIFLOWER during Floral Initiation.

    PubMed

    Goslin, Kevin; Zheng, Beibei; Serrano-Mislata, Antonio; Rae, Liina; Ryan, Patrick T; Kwaśniewska, Kamila; Thomson, Bennett; O'Maoileidigh, Diarmuid; Madueno, Francisco; Wellmer, Frank; Graciet, Emmanuelle

    2017-04-06

    The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL, is currently unknown. To better understand the regulatory relationship between LFY and AP1/CAL during floral initiation, we monitored the activity of LFY in the absence of AP1/CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL may act as part of an incoherent feed-forward loop to control the establishment of a stable developmental program for the formation of flowers.

  10. Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and...

  11. Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensis Live Vaccine Strain (LVS) Supports a General Host Suppression and Bacterial Uptake by Macropinocytosis*

    PubMed Central

    Bradburne, Christopher E.; Verhoeven, Anne B.; Manyam, Ganiraju C.; Chaudhry, Saira A.; Chang, Eddie L.; Thach, Dzung C.; Bailey, Charles L.; van Hoek, Monique L.

    2013-01-01

    Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2–6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. PMID:23322778

  12. AF4 uses the SL1 components of RNAP1 machinery to initiate MLL fusion- and AEP-dependent transcription

    PubMed Central

    Okuda, Hiroshi; Kanai, Akinori; Ito, Shinji; Matsui, Hirotaka; Yokoyama, Akihiko

    2015-01-01

    Gene rearrangements generate MLL fusion genes, which can lead to aggressive leukemia. In most cases, MLL fuses with a gene encoding a component of the AEP (AF4 family/ENL family/P-TEFb) coactivator complex. MLL–AEP fusion proteins constitutively activate their target genes to immortalize haematopoietic progenitors. Here we show that AEP and MLL–AEP fusion proteins activate transcription through selectivity factor 1 (SL1), a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). The pSER domain of AF4 family proteins associates with SL1 on chromatin and loads TATA-binding protein (TBP) onto the promoter to initiate RNA polymerase II (RNAP2)-dependent transcription. These results reveal a previously unknown transcription initiation mechanism involving AEP and a role for SL1 as a TBP-loading factor in RNAP2-dependent gene activation. PMID:26593443

  13. Sophisticated Regulation of Transcriptional Factors by the Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

    PubMed

    Galinier, Anne; Deutscher, Josef

    2017-03-24

    The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a carbohydrate transport and phosphorylation system present in bacteria of all different phyla and in archaea. It is usually composed of three proteins or protein complexes, enzyme I, HPr, and enzyme II, which are phosphorylated at histidine or cysteine residues. However, in many bacteria, HPr can also be phosphorylated at a serine residue. The PTS not only functions as a carbohydrate transporter but also regulates numerous cellular processes either by phosphorylating its target proteins or by interacting with them in a phosphorylation-dependent manner. The target proteins can be catabolic enzymes, transporters, and signal transduction proteins but are most frequently transcriptional regulators. In this review, we will describe how PTS components interact with or phosphorylate proteins to regulate directly or indirectly the activity of transcriptional repressors, activators, or antiterminators. We will briefly summarize the well-studied mechanism of carbon catabolite repression in firmicutes, where the transcriptional regulator catabolite control protein A needs to interact with seryl-phosphorylated HPr in order to be functional. We will present new results related to transcriptional activators and antiterminators containing specific PTS regulation domains, which are the phosphorylation targets for three different types of PTS components. Moreover, we will discuss how the phosphorylation level of the PTS components precisely regulates the activity of target transcriptional regulators or antiterminators, with or without PTS regulation domain, and how the availability of PTS substrates and thus the metabolic status of the cell are connected with various cellular processes, such as biofilm formation or virulence of certain pathogens.

  14. Bacterial rRNA-targeted reverse transcription-PCR used to identify pathogens responsible for fever with neutropenia.

    PubMed

    Sakaguchi, Sachi; Saito, Masahiro; Tsuji, Hirokazu; Asahara, Takashi; Takata, Oto; Fujimura, Junya; Nagata, Satoru; Nomoto, Koji; Shimizu, Toshiaki

    2010-05-01

    The purpose of this study was to evaluate the clinical utility of bacterial rRNA-targeted reverse transcription-quantitative PCR (BrRNA RT-qPCR) assays for identifying the bacterial pathogens that cause fever with neutropenia in pediatric cancer patients, by comparing the bacterial detection rate of this technique with that of blood culture. One milliliter of blood was collected from pediatric patients who developed fever with neutropenia following cancer chemotherapy. BrRNA RT-qPCR was performed using 16 primer sets, each designed for a specific type of bacteria. The entire BrRNA RT-qPCR procedure took less than 5 h. Blood culture was performed at the same time, following the standard institutional procedure. Blood from 13 patients was collected during 23 febrile neutropenic episodes. Of these samples, bacteria were identified in 16 by BrRNA RT-qPCR (69.6%) and in 4 by blood culture (17.4%, P<0.001). In all 4 blood culture-positive samples, BrRNA RT-qPCR detected the same type of bacteria as that identified by culture. In 9 samples, more than 4 types of bacteria were identified simultaneously by BrRNA RT-qPCR, most of which were anaerobic bacteria known to be part of the gut flora. We conclude that BrRNA RT-qPCR could be useful in the diagnosis of fever with neutropenia, given its high bacterial detection rate, short turnaround time, and the small blood sample required compared with the standard blood culture techniques. Our findings also indicate that anaerobic intestinal bacteria, which are difficult to detect by standard culture techniques, may be responsible for some cases of febrile neutropenia.

  15. Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates

    SciTech Connect

    Knezetic, J.A.; Jacob, G.A.; Luse, D.S.

    1988-08-01

    The authors have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, they demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that the observations are not the result of slow displacemnt of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

  16. A model of membrane contraction predicting initiation and completion of bacterial cell division.

    PubMed

    Dow, Claire E; Rodger, Alison; Roper, David I; van den Berg, Hugo A

    2013-05-01

    Bacterial cell division involves a complex and dynamic sequence of events whereby polymers of the protein FtsZ assemble at the division plane and rearrange to achieve the goal of contracting the cell membrane at the site of cell division, thus dividing the parent cell into two daughter cells. We present a mathematical model (which we refer to as CAM-FF: Critical Accumulation of Membrane-bound FtsZ Fibres) of the assembly of the contractile ring in terms of the accumulation of short linear polymers of FtsZ that associate and dissociate from the cell membrane. In prokaryotes, the biochemical function of FtsZ is thought to underpin the assembly and at least the initial kinetic force of ring contraction. Our model extends earlier work of Surovtsev et al. [PLoS Comput. Biol., 2008, 4, e1000102] by adding (i) the kinetics of FtsZ accumulation on cell membrane anchor proteins and (ii) the physical forces required to deform the cell against its surface tension. Moreover, we provide a more rigorous treatment of intracellular diffusion and we revise some of the model parameter values in light of the experimental evidence now available. We derive a critical contraction parameter which links the chemical population dynamics of membrane-bound FtsZ molecules to the force of contraction. Using this parameter as a tool to predict the ability of the cell to initiate division, we are able to predict the division outcome in cells depleted of key FtsZ-binding proteins.

  17. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader.

    PubMed

    Kriner, Michelle A; Groisman, Eduardo A

    2017-01-25

    Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5' leader mRNA of the Salmonella corA gene can adopt two mutually exclusive conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals.

  18. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader

    PubMed Central

    Kriner, Michelle A.; Groisman, Eduardo A.

    2017-01-01

    Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5′ leader mRNA of the Salmonella corA gene can adopt two mutually exclusive conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals. PMID:28123036

  19. Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

    PubMed Central

    Hamon, Véronique; Erales, Jenny; Bignon, Christophe; Roche, Philippe

    2016-01-01

    Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region. PMID:27936158

  20. From Peptide Aptamers to Inhibitors of FUR, Bacterial Transcriptional Regulator of Iron Homeostasis and Virulence.

    PubMed

    Mathieu, Sophie; Cissé, Cheickna; Vitale, Sylvia; Ahmadova, Aynur; Degardin, Mélissa; Pérard, Julien; Colas, Pierre; Miras, Roger; Boturyn, Didier; Covès, Jacques; Crouzy, Serge; Michaud-Soret, Isabelle

    2016-09-16

    FUR (Ferric Uptake Regulator) protein is a global transcriptional regulator that senses iron status and controls the expression of genes involved in iron homeostasis, virulence, and oxidative stress. Ubiquitous in Gram-negative bacteria and absent in eukaryotes, FUR is an attractive antivirulence target since the inactivation of the fur gene in various pathogens attenuates their virulence. The characterization of 13-aa-long anti-FUR linear peptides derived from the variable part of the anti-FUR peptide aptamers, that were previously shown to decrease pathogenic E. coli strain virulence in a fly infection model, is described herein. Modeling, docking, and experimental approaches in vitro (activity and interaction assays, mutations) and in cells (yeast two-hybrid assays) were combined to characterize the interactions of the peptides with FUR, and to understand their mechanism of inhibition. As a result, reliable structure models of two peptide-FUR complexes are given. Inhibition sites are mapped in the groove between the two FUR subunits where DNA should also bind. Another peptide behaves differently and interferes with the dimerization itself. These results define these novel small peptide inhibitors as lead compounds for inhibition of the FUR transcription factor.

  1. Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

    PubMed

    Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

    2012-01-01

    The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

  2. Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

    PubMed

    Mookherjee, Neeloffer; Wilson, Heather L; Doria, Silvana; Popowych, Yurij; Falsafi, Reza; Yu, Jie Jessie; Li, Yuexin; Veatch, Sarah; Roche, Fiona M; Brown, Kelly L; Brinkman, Fiona S L; Hokamp, Karsten; Potter, Andy; Babiuk, Lorne A; Griebel, Philip J; Hancock, Robert E W

    2006-12-01

    Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host

  3. Influence of membrane surface properties on the behavior of initial bacterial adhesion and biofilm development onto nanofiltration membranes.

    PubMed

    Myint, Aye Aye; Lee, Wonil; Mun, Sungmin; Ahn, Chang Hoon; Lee, Seockheon; Yoon, Jeyong

    2010-01-01

    In order to investigate biofouling problems, the fundamental behaviors of initial bacterial adhesion and biofilm development on four different nanofiltration (NF) membranes were evaluated using Pseudomonas aeruginosa PAO1 as a model bacterial strain. Initial cell adhesion was considerably higher on an aromatic polyamide-based NF membrane with a hydrophobic and rough surface, whereas cell aggregation on a polypiperazine-based NF membrane with a relatively hydrophilic and smooth surface was lower. Moreover, significant differences in the structural heterogeneity of the biofilms were observed among the four NF membranes. This study shows that the surface roughness and hydrophobicity of a membrane play an important role in determining initial cell adhesion, aggregation and favorable localization sites for colony formation. In addition, it was found that biofilm development was strongly influenced by the surface morphology of a membrane.

  4. The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

    PubMed Central

    García-López, M. Carmen; Pelechano, Vicent; Mirón-García, M. Carmen; Garrido-Godino, Ana I.; García, Alicia; Calvo, Olga; Werner, Michel; Pérez-Ortín, José E.; Navarro, Francisco

    2011-01-01

    RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. PMID:21954159

  5. Inhibition of transcription by the Caenorhabditis elegans germline protein PIE-1: genetic evidence for distinct mechanisms targeting initiation and elongation.

    PubMed

    Ghosh, Dolan; Seydoux, Geraldine

    2008-01-01

    In Caenorhabditis elegans embryos, specification of the germ lineage depends on PIE-1, a maternal protein that blocks mRNA transcription in germline blastomeres. Studies in mammalian cell culture have suggested that PIE-1 inhibits P-TEFb, a kinase that phosphorylates serine 2 in the carboxyl-terminal domain (CTD) repeats of RNA polymerase II during transcriptional elongation. We have tested this hypothesis using an in vivo complementation assay for PIE-1 function. Our results support the view that PIE-1 inhibits P-TEFb using the CTD-like motif YAPMAPT. This activity is required to block serine 2 phosphorylation in germline blastomeres, but unexpectedly is not essential for transcriptional repression or specification of the germline. We find that sequences outside of the YAPMAPT are required to inhibit serine 5 phosphorylation, and that this second inhibitory mechanism is essential for transcriptional repression and specification of the germ lineage. Our results suggest that PIE-1 uses partially redundant mechanisms to block transcription by targeting both the initiation and elongation phases of the transcription cycle.

  6. Transcription-coupled DNA supercoiling dictates the chromosomal arrangement of bacterial genes

    PubMed Central

    Sobetzko, Patrick

    2016-01-01

    Over the recent decade, the central importance of DNA supercoiling in chromosome organization and global gene regulation of bacteria became more and more visible. With a regulon comprising more than 2000 genes in Escherichia coli, DNA supercoiling is among the most influential regulators of gene expression found in bacteria so far. However, the mechanism creating thousands of diverse temporal gene expression patterns coordinated by DNA supercoiling remains unclear. In this study we show that a specific chromosomal arrangement of genes modulates the local levels of DNA supercoiling at gene promoters via transcription-coupled DNA supercoiling (TCDS) in the model organism E. coli. Our findings provide a consistent explanation for the strong positive coupling of temporal gene expression patterns of neighboring genes. Using comparative genomics we are furthermore able to provide evidence that TCDS is a driving force for the evolution of chromosomal gene arrangement patterns in other Enterobacteriaceae. With the currently available data of promoter supercoiling sensitivity we prove that the same principle is applicable also for the evolutionary distant gram-positive pathogenic bacterium Streptococcus pneumoniae. Moreover, our findings are fully consistent with recent investigations concerning the regulatory impact of TCDS on gene pairs in eukaryots underpinning the broad applicability of our analysis. PMID:26783203

  7. SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

    PubMed Central

    Babin, Brett M.; Bergkessel, Megan; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Newman, Dianne K.; Tirrell, David A.

    2016-01-01

    Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A). PMID:26787849

  8. Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA.

    SciTech Connect

    Zhang, R.; Pappas, T.; Brace, J.; Miller, P.; Oulmassov, T.; Molyneaux, J.; Anderson, J.; Bashkin, J.; Winans, S.; Joachimiak, A.; Biosciences Division; Cornell Univ.; Monsanto Co.

    2002-06-27

    Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis1. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-l-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an {alpha}/{beta}/{alpha} sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix-turn-helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

  9. Effect of Soil Clay Content on RNA Isolation and on Detection and Quantification of Bacterial Gene Transcripts in Soil by Quantitative Reverse Transcription-PCR ▿†

    PubMed Central

    Novinscak, A.; Filion, M.

    2011-01-01

    In this study, we evaluated the effect of soil clay content on RNA isolation and on quantitative reverse transcription-PCR (qRT-PCR) quantification of microbial gene transcripts. The amount of clay significantly altered RNA isolation yields and qRT-PCR analyses. Recommendations are made for quantifying microbial gene transcripts in soil samples varying in clay content. PMID:21724880

  10. Genetic and biochemical interactions between the bacterial replication initiator DnaA and the nucleoid-associated protein Rok in Bacillus subtilis.

    PubMed

    Seid, Charlotte A; Smith, Janet L; Grossman, Alan D

    2017-03-01

    We identified interactions between the conserved bacterial replication initiator and transcription factor DnaA and the nucleoid-associated protein Rok of Bacillus subtilis. DnaA binds directly to clusters of DnaA boxes at the origin of replication and elsewhere, including the promoters of several DnaA-regulated genes. Rok, an analog of H-NS from gamma-proteobacteria that affects chromosome architecture and of Lsr2 from Mycobacteria, binds A+T-rich sequences throughout the genome and represses expression of many genes. Using crosslinking and immunoprecipitation followed by deep sequencing (ChIP-seq), we found that DnaA was associated with eight previously identified regions containing clusters of DnaA boxes, plus 36 additional regions that were also bound by Rok. Association of DnaA with these additional regions appeared to be indirect as it was dependent on Rok and independent of the DNA-binding domain of DnaA. Gene expression and mutant analyses support a model in which DnaA and Rok cooperate to repress transcription of yxaJ, the yybNM operon and the sunA-bdbB operon. Our results indicate that DnaA modulates the activity of Rok. We postulate that this interaction might affect nucleoid architecture. Furthermore, DnaA might interact similarly with Rok analogues in other organisms.

  11. Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination

    PubMed Central

    Eriksen, Mette; Sneppen, Kim; Pedersen, Steen; Mitarai, Namiko

    2017-01-01

    Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be “hit” by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination. PMID:28382022

  12. Impact of wall shear stress on initial bacterial adhesion in rotating annular reactor.

    PubMed

    Saur, Thibaut; Morin, Emilie; Habouzit, Frédéric; Bernet, Nicolas; Escudié, Renaud

    2017-01-01

    The objective of this study was to investigate the bacterial adhesion under different wall shear stresses in turbulent flow and using a diverse bacterial consortium. A better understanding of the mechanisms governing microbial adhesion can be useful in diverse domains such as industrial processes, medical fields or environmental biotechnologies. The impact of wall shear stress-four values ranging from 0.09 to 7.3 Pa on polypropylene (PP) and polyvinyl chloride (PVC)-was carried out in rotating annular reactors to evaluate the adhesion in terms of morphological and microbiological structures. A diverse inoculum consisting of activated sludge was used. Epifluorescence microscopy was used to quantitatively and qualitatively characterize the adhesion. Attached bacterial communities were assessed by molecular fingerprinting profiles (CE-SSCP). It has been demonstrated that wall shear stress had a strong impact on both quantitative and qualitative aspects of the bacterial adhesion. ANOVA tests also demonstrated the significant impact of wall shear stress on all three tested morphological parameters (surface coverage, number of objects and size of objects) (p-values < 2.10-16). High wall shear stresses increased the quantity of attached bacteria but also altered their spatial distribution on the substratum surface. As the shear increased, aggregates or clusters appeared and their size grew when increasing the shears. Concerning the microbiological composition, the adhered bacterial communities changed gradually with the applied shear.

  13. Impact of wall shear stress on initial bacterial adhesion in rotating annular reactor

    PubMed Central

    Saur, Thibaut; Morin, Emilie; Habouzit, Frédéric; Bernet, Nicolas

    2017-01-01

    The objective of this study was to investigate the bacterial adhesion under different wall shear stresses in turbulent flow and using a diverse bacterial consortium. A better understanding of the mechanisms governing microbial adhesion can be useful in diverse domains such as industrial processes, medical fields or environmental biotechnologies. The impact of wall shear stress—four values ranging from 0.09 to 7.3 Pa on polypropylene (PP) and polyvinyl chloride (PVC)—was carried out in rotating annular reactors to evaluate the adhesion in terms of morphological and microbiological structures. A diverse inoculum consisting of activated sludge was used. Epifluorescence microscopy was used to quantitatively and qualitatively characterize the adhesion. Attached bacterial communities were assessed by molecular fingerprinting profiles (CE-SSCP). It has been demonstrated that wall shear stress had a strong impact on both quantitative and qualitative aspects of the bacterial adhesion. ANOVA tests also demonstrated the significant impact of wall shear stress on all three tested morphological parameters (surface coverage, number of objects and size of objects) (p-values < 2.10−16). High wall shear stresses increased the quantity of attached bacteria but also altered their spatial distribution on the substratum surface. As the shear increased, aggregates or clusters appeared and their size grew when increasing the shears. Concerning the microbiological composition, the adhered bacterial communities changed gradually with the applied shear. PMID:28207869

  14. Initial community and environment determine the response of bacterial communities to dispersant and oil contamination.

    PubMed

    Ortmann, Alice C; Lu, YueHan

    2015-01-15

    Bioremediation of seawater by natural bacterial communities is one potential response to coastal oil spills, but the success of the approach may vary, depending on geographical location, oil composition and the timing of spill. The short term response of coastal bacteria to dispersant, oil and dispersed oil was characterized using 16S rRNA gene tags in two mesocosm experiments conducted two months apart. Despite differences in the amount of oil-derived alkanes across the treatments and experiments, increases in the contributions of hydrocarbon degrading taxa and decreases in common estuarine bacteria were observed in response to dispersant and/or oil. Between the two experiments, the direction and rates of changes in particulate alkane concentrations differed, as did the magnitude of the bacterial response to oil and/or dispersant. Together, our data underscore large variability in bacterial responses to hydrocarbon pollutants, implying that bioremediation success varies with starting biological and environmental conditions.

  15. Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes.

    PubMed Central

    Financsek, I; Mizumoto, K; Mishima, Y; Muramatsu, M

    1982-01-01

    The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The sequenced region was found to consist of 75% G and C and to contain a number of short direct and inverted repeats and palindromes. By comparison of the corresponding initiation regions of three mammalian species, several conserved sequences were found upstream and downstream from the transcription starting point. Two short A + T-rich sequences are present on human, mouse, and rat ribosomal RNA genes between the initiation site and 40 nucleotides upstream, and a C + T cluster is located at a position around -60. At and downstream from the initiation site, a common sequence, T-AG-C-T-G-A-C-A-C-G-C-T-G-T-C-C-T-CT-T, was found in the three genes from position -1 through +18. The strong conservation of these sequences suggests their functional significance in rDNA. The S1 nuclease protection experiments with cloned rDNA fragments indicated the presence in human 45S RNA of molecules several hundred nucleotides shorter than the supposed primary transcript. The first 19 nucleotides of these molecules appear identical--except for one mismatch--to the nucleotide sequence of the 5' end of a supposed early processing product of the mouse 45S RNA. Images PMID:6954460

  16. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

    PubMed Central

    Schwartz, Allison R.; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J.

    2017-01-01

    AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions. PMID:28100489

  17. HFR1 sequesters PIF1 to govern the transcriptional network underlying light-initiated seed germination in Arabidopsis.

    PubMed

    Shi, Hui; Zhong, Shangwei; Mo, Xiaorong; Liu, Na; Nezames, Cynthia D; Deng, Xing Wang

    2013-10-01

    Seed germination is the first step for seed plants to initiate a new life cycle. Light plays a predominant role in promoting seed germination, where the initial phase is mediated by photoreceptor phytochrome B (phyB). Previous studies showed that phytochrome-interacting factor1 (PIF1) represses seed germination downstream of phyB. Here, we identify a positive regulator of phyB-dependent seed germination, long hypocotyl in far-red1 (HFR1). HFR1 blocks PIF1 transcriptional activity by forming a heterodimer with PIF1 that prevents PIF1 from binding to DNA. Our whole-genomic analysis shows that HFR1 and PIF1 oppositely mediate the light-regulated transcriptome in imbibed seeds. Through the HFR1-PIF1 module, light regulates expression of numerous genes involved in cell wall loosening, cell division, and hormone pathways to initiate seed germination. The functionally antagonistic HFR1-PIF1 pair constructs a fail-safe mechanism for fine-tuning seed germination during low-level illumination, ensuring a rapid response to favorable environmental changes. This study identifies the HFR1-PIF1 pair as a central module directing the whole genomic transcriptional network to rapidly initiate light-induced seed germination.

  18. Temperature-Dependent Model of Multi-step Transcription Initiation in Escherichia coli Based on Live Single-Cell Measurements

    PubMed Central

    Lloyd-Price, Jason; Tran, Huy; Ribeiro, Andre S.

    2016-01-01

    Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme. For this, we compared detailed stochastic models fit to the empirical data in maximum likelihood sense using statistical methods. Using this analysis, we found that temperature affects the rate limiting steps unequally, as nonlinear changes in the closed complex formation suffice to explain the differences in transcription dynamics between conditions. Meanwhile, a similar analysis of the PtetA promoter revealed that it has a different rate limiting step configuration, with temperature regulating different steps. Finally, we used the derived models to explore a possible cause for why the identified steps are preferred as the main cause for behavior modifications with temperature: we find that transcription dynamics is either insensitive or responds reciprocally to changes in the other steps. Our results suggests that different promoters employ different rate limiting step patterns that control not only their rate and variability, but also their sensitivity to environmental changes. PMID:27792724

  19. Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

    PubMed Central

    Wippel, Kathrin

    2016-01-01

    ABSTRACT The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa. The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469 (designated dksA), SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCE The stringent response is a bacterial transcription regulation process triggered upon nutritional stress. Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot

  20. Effect of malathion on the initiation and elongation steps of transcription.

    PubMed

    Walter, Z; Wiszkowska, H

    1990-01-01

    In isolated cell nuclei of pig thymus malathion, S-1,2-bis(ethoxycarbonyl)-ethyl-O,O-dimethyldithiophosphate inhibited both initiation and elongation of all three classes of nuclear RNA polymerases; proflavin was used as an inhibitor of initiation, and actinomycin, as an inhibitor of elongation.

  1. Alternative transcription initiation sites and polyadenylation sites are recruited during Mu suppression at the rf2a locus of maize.

    PubMed Central

    Cui, Xiangqin; Hsia, An-Ping; Liu, Feng; Ashlock, Daniel A; Wise, Roger P; Schnable, Patrick S

    2003-01-01

    Even in the absence of excisional loss of the associated Mu transposons, some Mu-induced mutant alleles of maize can lose their capacity to condition a mutant phenotype. Three of five Mu-derived rf2a alleles are susceptible to such Mu suppression. The suppressible rf2a-m9437 allele has a novel Mu transposon insertion (Mu10) in its 5' untranslated region (UTR). The suppressible rf2a-m9390 allele has a Mu1 insertion in its 5' UTR. During suppression, alternative transcription initiation sites flanking the Mu1 transposon yield functional transcripts. The suppressible rf2a-m8110 allele has an rcy/Mu7 insertion in its 3' UTR. Suppression of this allele occurs via a previously unreported mechanism; sequences in the terminal inverted repeats of rcy/Mu7 function as alternative polyadenylation sites such that the suppressed rf2a-m8110 allele yields functional rf2a transcripts. No significant differences were observed in the nucleotide compositions of these alternative polyadenylation sites as compared with 94 other polyadenylation sites from maize genes. PMID:12618406

  2. Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

    PubMed Central

    Wilder, Hannah K.; Raffel, Sandra J.; Barbour, Alan G.; Porcella, Stephen F.; Sturdevant, Daniel E.; Vaisvil, Benjamin; Kapatral, Vinayak; Schmitt, Daniel P.; Schwan, Tom G.; Lopez, Job E.

    2016-01-01

    Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle. PMID:26845332

  3. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling

    PubMed Central

    Quin, Jaclyn; Chan, Keefe T.; Devlin, Jennifer R.; Cameron, Donald P.; Diesch, Jeannine; Cullinane, Carleen; Ahern, Jessica; Khot, Amit; Hein, Nadine; George, Amee J.; Hannan, Katherine M; Poortinga, Gretchen; Sheppard, Karen E.; Khanna, Kum Kum; Johnstone, Ricky W.; Drygin, Denis; McArthur, Grant A.; Pearson, Richard B.

    2016-01-01

    RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy. PMID:27391441

  4. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling.

    PubMed

    Quin, Jaclyn; Chan, Keefe T; Devlin, Jennifer R; Cameron, Donald P; Diesch, Jeannine; Cullinane, Carleen; Ahern, Jessica; Khot, Amit; Hein, Nadine; George, Amee J; Hannan, Katherine M; Poortinga, Gretchen; Sheppard, Karen E; Khanna, Kum Kum; Johnstone, Ricky W; Drygin, Denis; McArthur, Grant A; Pearson, Richard B; Sanij, Elaine; Hannan, Ross D

    2016-08-02

    RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

  5. Protein Mis-Termination Initiates Genetic Diseases, Cancers, and Restricts Bacterial Genome Expansion.

    PubMed

    Wong, Tit-Yee; Schwartzbach, Steve D

    2015-01-01

    Protein termination is an important cellular process. Protein termination relies on the stop-codons in the mRNA interacting properly with the releasing factors on the ribosome. One third of inherited diseases, including cancers, are associated with the mutation of the stop-codons. Many pathogens and viruses are able to manipulate their stop-codons to express their virulence. The influence of stop-codons is not limited to the primary reading frame of the genes. Stop-codons in the second and third reading frames are referred as premature stop signals (PSC). Stop-codons and PSCs together are collectively referred as stop-signals. The ratios of the stop-signals (referred as translation stop-signals ratio or TSSR) of genetically related bacteria, despite their great differences in gene contents, are much alike. This nearly identical Genomic-TSSR value of genetically related bacteria may suggest that bacterial genome expansion is limited by their unique stop-signals bias. We review the protein termination process and the different types of stop-codon mutation in plants, animals, microbes, and viruses, with special emphasis on the role of PSCs in directing bacterial evolution in their natural environments. Knowing the limit of genomic boundary could facilitate the formulation of new strategies in controlling the spread of diseases and combat antibiotic-resistant bacteria.

  6. Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

    PubMed Central

    Geisler, Christoph; Jarvis, Donald L.

    2012-01-01

    The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems. PMID:23022569

  7. Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor.

    PubMed Central

    Mishima, Y; Financsek, I; Kominami, R; Muramatsu, M

    1982-01-01

    Mouse and human cell extracts (S100) can support an accurate and efficient transcription initiation on homologous ribosomal RNA gene (rDNA) templates. The cell extracts were fractionated with the aid of a phosphocellulose column into four fractions (termed A, B, C and D), including one containing a major part of the RNA polymerase I activity. Various reconstitution experiments indicate that fraction D is an absolute requirement for the correct and efficient transcription initiation by RNA polymerase I on both mouse and human genes. Fraction B effectively suppresses random initiation on these templates. Fraction A appears to further enhance the transcription which takes place with fractions C and D. Although fractions A, B and C are interchangeable between mouse and human extracts, fraction D is not; i.e. initiation of transcription required the presence of a homologous fraction D for both templates. The factor(s) in fraction D, however, is not literally species-specific, since mouse D fraction is capable of supporting accurate transcription initiation on a rat rDNA template in the presence of all the other fractions from human cell extract under the conditions where human D fraction is unable to support it. We conclude from these experiments that a species-dependent factor in fraction D plays an important role in the initiation of rDNA transcription in each animal species. Images PMID:7177852

  8. Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor.

    PubMed

    Goh, Tatsuaki; Toyokura, Koichi; Wells, Darren M; Swarup, Kamal; Yamamoto, Mayuko; Mimura, Tetsuro; Weijers, Dolf; Fukaki, Hidehiro; Laplaze, Laurent; Bennett, Malcolm J; Guyomarc'h, Soazig

    2016-09-15

    Lateral root formation is an important determinant of root system architecture. In Arabidopsis, lateral roots originate from pericycle cells, which undergo a program of morphogenesis to generate a new lateral root meristem. Despite its importance for root meristem organization, the onset of quiescent center (QC) formation during lateral root morphogenesis remains unclear. Here, we used live 3D confocal imaging to monitor cell organization and identity acquisition during lateral root development. Our dynamic observations revealed an early morphogenesis phase and a late meristem formation phase as proposed in the bi-phasic growth model. Establishment of lateral root QCs coincided with this developmental phase transition. QC precursor cells originated from the outer layer of stage II lateral root primordia, within which the SCARECROW (SCR) transcription factor was specifically expressed. Disrupting SCR function abolished periclinal divisions in this lateral root primordia cell layer and perturbed the formation of QC precursor cells. We conclude that de novo QC establishment in lateral root primordia operates via SCR-mediated formative cell division and coincides with the developmental phase transition.

  9. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.).

    PubMed

    Wang, I-C; Ustiyan, V; Zhang, Y; Cai, Y; Kalin, T V; Kalinichenko, V V

    2014-11-13

    Lung cancer is the leading cause of deaths in cancer patients in the United States. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small-cell lung cancer. Transgenic expression of activated Kras(G12D) in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas; however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. Foxm1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing Kras(G12D) under control of Sftpc promoter to demonstrate that Foxm1 was induced in type II epithelial cells before the formation of lung tumors. Conditional deletion of Foxm1 from Kras(G12D)-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.

  10. Transcription regulation mechanisms of bacteriophages

    PubMed Central

    Yang, Haiquan; Ma, Yingfang; Wang, Yitian; Yang, Haixia; Shen, Wei; Chen, Xianzhong

    2014-01-01

    Phage diversity significantly contributes to ecology and evolution of new bacterial species through horizontal gene transfer. Therefore, it is essential to understand the mechanisms underlying phage-host interactions. After initial infection, the phage utilizes the transcriptional machinery of the host to direct the expression of its own genes. This review presents a view on the transcriptional regulation mechanisms of bacteriophages, and its contribution to phage diversity and classification. Through this review, we aim to broaden the understanding of phage-host interactions while providing a reference source for researchers studying the regulation of phage transcription. PMID:25482231

  11. A retroviral RNA secondary structure required for efficient initiation of reverse transcription.

    PubMed Central

    Cobrinik, D; Soskey, L; Leis, J

    1988-01-01

    Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication. Images PMID:2458484

  12. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    PubMed

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  13. Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator

    PubMed Central

    Severinov, Konstantin; Minakhin, Leonid; Sekine, Shun-ichi; Lopatina, Anna; Yokoyama, Shigeyuki

    2014-01-01

    Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue. PMID:25105059

  14. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    PubMed

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  15. Bound to Succeed: transcription factor binding-site prediction and its contribution to understanding virulence and environmental adaptation in bacterial plant pathogens.

    PubMed

    Saha, Surya; Lindeberg, Magdalen

    2013-10-01

    Bacterial plant pathogens rely on a battalion of transcription factors to fine-tune their response to changing environmental conditions and to marshal the genetic resources required for successful pathogenesis. Prediction of transcription factor binding sites (TFBS) represents an important tool for elucidating regulatory networks and has been conducted in multiple genera of plant-pathogenic bacteria for the purpose of better understanding mechanisms of survival and pathogenesis. The major categories of TFBS that have been characterized are reviewed here, with emphasis on in silico methods used for site identification and challenges therein, their applicability to different types of sequence datasets, and insights into mechanisms of virulence and survival that have been gained through binding-site mapping. An improved strategy for establishing E-value cutoffs when using existing models to screen uncharacterized genomes is also discussed.

  16. Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.

    PubMed

    Sasano, Yu; Watanabe, Daisuke; Ukibe, Ken; Inai, Tomomi; Ohtsu, Iwao; Shimoi, Hitoshi; Takagi, Hiroshi

    2012-04-01

    Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.

  17. Salvage microbiology: opportunities and challenges in the detection of bacterial pathogens following initiation of antimicrobial treatment

    PubMed Central

    Farrell, John J.; Hujer, Andrea M.; Sampath, Rangarajan; Bonomo, Robert A.

    2015-01-01

    Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with implementation of these strategies in clinical practice. PMID:25523281

  18. Influence of cAMP receptor protein (CRP) on bacterial virulence and transcriptional regulation of allS by CRP in Klebsiella pneumoniae.

    PubMed

    Xue, Jian; Tan, Bin; Yang, Shiya; Luo, Mei; Xia, Huiming; Zhang, Xian; Zhou, Xipeng; Yang, Xianxian; Yang, Ruifu; Li, Yingli; Qiu, Jingfu

    2016-11-15

    cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. The gene allS was well-known as allantoin-utilizing capability and involving in bacterial virulence in Klebsiella pneumoniae (K. pneumoniae). The specific DNA recognition motif of transcription regulator CRP was found in allS promoter region. Therefore, this study is aimed to investigate the function of CRP on virulence and its transcriptional regulation mechanism to gene allS in K. pneumoniae. The wild-type (WT) K. pneumoniae NTUH-2044, crp knockout (Kp-Δcrp) and the complemented knockout (KpC-Δcrp) strains were used to determine the function of crp gene. The lacZ fusion, qRT-PCR, electrophoretic mobility shift and DNase I footprinting assays were performed to study the transcriptional regulation of CRP on allS. The result showed a decreased virulence in crp knockout strain. Complement through supplementing crp fragment in expression plasmid partially restore virulence of knockout bacteria. The CRP could bind to the allS promoter-proximal region and the binding site was further refined to be located from 60bp to 94bp upstream of the allS promoter. Based on these results, we proposed that CRP is an essential virulence regulator and knock out of crp gene will result in reduced virulence in K. pneumoniae. In the meantime, the transcription of gene allS is positively regulated by CRP via directly binding to upstream of allS promoter.

  19. Identification and transcriptional profile of multiple genes in the posterior kidney of Nile tilapia at 6h post bacterial infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophi...

  20. An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

    PubMed Central

    Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

    2016-01-01

    The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

  1. An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

    PubMed Central

    Iguchi-Ariga, S M; Okazaki, T; Itani, T; Ogata, M; Sato, Y; Ariga, H

    1988-01-01

    We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression. Images PMID:3053161

  2. Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance.

    PubMed

    Sohn, Kee Hoon; Lee, Sung Chul; Jung, Ho Won; Hong, Jeum Kyu; Hwang, Byung Kook

    2006-08-01

    A novel pathogen-induced gene encoding the RAV (Related to ABI3/VP1) transcription factor, CARAV1, was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. CARAV1 contains two distinct DNA-binding domains AP2 and B3 uniquely found in higher plants. Transient expression analysis of the smGFP:CARAV1 fusion construct in Arabidopsis protoplasts and pepper epidermal cells revealed the CARAV1 protein to be localized in the nucleus. The N-terminal region of CARAV1 fused to the GAL4 DNA-binding domain was required to activate transcription of reporter genes in yeast. In yeast one-hybrid, the recognition of CAACA and CACCTG motifs also were essential for the CARAV1 protein to bind to a specific target gene and activate the reporter gene. The expression of the CARAV1 gene was strongly induced early in pepper leaves during the pathogen infection, abiotic elicitors and environmental stresses. CARAV1 transcripts were localized in the phloem cells of leaf tissues during pathogen infection and ethylene treatment. Ectopic expression of the CARAV1 gene in transgenic Arabidopsis plants induced some PR genes and enhanced resistance against infection by Pseudomonas syringae pv. tomato DC3000 and osmotic stresses by high salinity and dehydration. The CARAV1 promoter activation was induced by P. syringae pv. tabaci, salicylic acid and abscisic acid. These data suggest that pathogen- and abiotic stress-inducible CARAV1 functions as a transcriptional activator triggering resistance to bacterial infection and tolerance to osmotic stresses.

  3. Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

    PubMed

    Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

    2016-03-01

    Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

  4. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    PubMed

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  5. The Phytohormone Ethylene Enhances Cellulose Production, Regulates CRP/FNRKx Transcription and Causes Differential Gene Expression within the Bacterial Cellulose Synthesis Operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582

    PubMed Central

    Augimeri, Richard V.; Strap, Janice L.

    2015-01-01

    Komagataeibacter (formerly Gluconacetobacter) xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC) biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid) to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx). Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx, and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA) induced differential expression of genes within the bacterial cellulose synthesis (bcs) operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature. PMID:26733991

  6. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel transcription activator-like (TAL) effector of Xanthomonas oryzae pv. oryzae.

    PubMed

    Wang, Chun-Lian; Qin, Teng-Fei; Yu, Hong-Man; Zhang, Xiao-Ping; Che, Jin-Ying; Gao, Ying; Zheng, Chong-Ke; Yang, Bing; Zhao, Kai-Jun

    2014-05-01

    Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is not only a disease devastating rice production worldwide, but also an ideal model system for the study of the interaction between plants and their bacterial pathogens. The rice near-isogenic line (NIL) CBB23, derived from a cross between a wild rice Oryza rufipogon accession (RBB16) and a susceptible indica rice variety (Jingang 30), is highly resistant to all field Xoo strains tested so far. Although the BB resistance of CBB23 has been widely used in rice breeding programmes, the mechanism of its extremely broad-spectrum resistance remains unknown. Here, we report the molecular cloning of an avirulence gene, designated as avrXa23, from Xoo strain PXO99(A) . We validate that AvrXa23, a novel transcription activator-like effector, specifically triggers the broad-spectrum BB resistance in CBB23. The prevalence of avrXa23 in all 38 Xoo strains surveyed may explain the broad-spectrum feature of BB resistance in CBB23. The results will significantly facilitate the molecular cloning of the corresponding resistance (R) gene in the host, and provide new insights into our understanding of the molecular mechanism for broad-spectrum disease resistance in plants.

  7. Bacterially-Associated Transcriptional Remodelling in a Distinct Genomic Subtype of Colorectal Cancer Provides a Plausible Molecular Basis for Disease Development

    PubMed Central

    Goosen, Ryan W.

    2016-01-01

    The relevance of specific microbial colonisation to colorectal cancer (CRC) disease pathogenesis is increasingly recognised, but our understanding of possible underlying molecular mechanisms that may link colonisation to disease in vivo remains limited. Here, we investigate the relationships between the most commonly studied CRC-associated bacteria (Enterotoxigenic Bacteroides fragilis, pks+ Escherichia coli, Fusobacterium spp., afaC+ E. coli, Enterococcus faecalis & Enteropathogenic E. coli) and altered transcriptomic and methylation profiles of CRC patients, in order to gain insight into the potential contribution of these bacteria in the aetiopathogenesis of CRC. We show that colonisation by E. faecalis and high levels of Fusobacterium is associated with a specific transcriptomic subtype of CRC that is characterised by CpG island methylation, microsatellite instability and a significant increase in inflammatory and DNA damage pathways. Analysis of the significant, bacterially-associated changes in host gene expression, both at the level of individual genes as well as pathways, revealed a transcriptional remodeling that provides a plausible mechanistic link between specific bacterial colonisation and colorectal cancer disease development and progression in this subtype; these included upregulation of REG3A, REG1A and REG1P in the case of high-level colonization by Fusobacterium, and CXCL10 and BMI1 in the case of colonisation by E. faecalis. The enrichment of both E. faecalis and Fusobacterium in this CRC subtype suggests that polymicrobial colonisation of the colonic epithelium may well be an important aspect of colonic tumourigenesis. PMID:27846243

  8. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    PubMed Central

    Milano, Teresa

    2016-01-01

    The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups. PMID:27446613

  9. Isolation of flowering genes and seasonal changes in their transcript levels related to flower induction and initiation in apple (Malus domestica).

    PubMed

    Hättasch, Conny; Flachowsky, Henryk; Kapturska, Danuta; Hanke, Magda-Viola

    2008-10-01

    Flower development in apple (Malus domestica Borkh.) extends over two consecutive seasons. During the first season, most shoot apical meristems change to reproductive growth and initiate flowers. After winter dormancy, flower development continues during the second season and ends with anthesis in the spring. To determine the beginning of the transition to reproductive growth at the molecular level and to identify genes involved in this critical phase of flower development, we examined transcript levels of the putative flowering genes MdCOL1, MdCOL2, MdFT, MdSOC1, MdMADS2, MdMADS5, MdTFL1-1 and MdTFL1-2 in vegetative terminal buds of the apple cultivar Pinova during the first season by quantitative real-time PCR. Transcript levels of these genes peaked at the end of April during blooming of coexisting floral buds. Subsequently, there was a large increase in transcription, which started on May 22 for AFL2 and MdMADS2, followed by MdFT and AFL1 one week later. We propose that the increased transcription at the end of May marks the beginning of flower induction. Transcript levels of MdSOC1, MdTFL1-1 and MdTFL1-2 increased at the end of June, suggesting that these genes are involved in flower initiation, which follows flower induction. In contrast, MdMADS5 transcription was too weak to be quantified, and the transcript levels of MdCOL1 and MdCOL2 showed no detectable trends during the study.

  10. Impact of Alternative Initiation, Splicing, and Termination on the Diversity of the mRNA Transcripts Encoded by the Mouse Transcriptome

    PubMed Central

    Zavolan, Mihaela; Kondo, Shinji; Schönbach, Christian; Adachi, Jun; Hume, David A.; Hayashizaki, Yoshihide; Gaasterland, Terry

    2003-01-01

    We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleotide distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs. PMID:12819126

  11. A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription.

    PubMed Central

    Milkereit, P; Tschochner, H

    1998-01-01

    Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells. The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation. PMID:9649439

  12. Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs

    SciTech Connect

    Korner, Heinz; Sofia, Heidi J. ); Zumft, Walter G.

    2003-12-30

    The Crp-Fnr regulators, named after the first two identified members, are DNA-binding proteins which predominantly function as positive transcription factors, though roles of repressors are also important. Among over 1200 proteins with an N-terminally-located nucleotide-binding domain similar to the cAMP receptor protein, the distinctive additional trait of the Crp-Fnr superfamily is a C-terminally-located helix-turn-helix motif for DNA binding. From a curated database of 369 family members exhibiting both features, we provide a protein tree of Crp-Fnr proteins according to their phylogenetic relationships. This results in the assembly of the regulators ArcR, CooA, CprK, Crp, Dnr, FixK, Flp, Fnr, FnrBac, FnrN, MalR, NnrR, NtcA, PrfA, and YeiL and their homologues in distinct clusters. Lead members and representatives of these groups are described, placing emphasis on the less well known regulators and target processes. Several more groups consist of sequence-derived proteins of unknown physiological role; some of them are tight clusters of highly similar members. The Crp-Fnr regulators stand out in responding to a broad spectrum of intracellular and exogenous signals such as cyclic AMP, anoxia, the redox state, oxidative and nitrosative stress, nitric oxide (NO), carbon monoxide (CO), 2-oxoglutarate, or temperature. To accomplish their roles Crp-Fnr members have intrinsic sensory modules allowing the binding of allosteric effector molecules, or have prosthetic groups for the interaction with the signal. The regulatory adaptability and structural flexibility represented in the Crp-Fnr scaffold has led to the evolution of an important group of physiologically versatile transcription factors.

  13. The Bacillus subtilis Late Competence Operon comE Is Transcriptionally Regulated by yutB and under Post-Transcription Initiation Control by comN (yrzD) ▿ †

    PubMed Central

    Ogura, Mitsuo; Tanaka, Teruo

    2009-01-01

    The Bacillus subtilis genome has been sequenced, and disruptants with disruptions in genes that were not characterized previously were systematically generated. We screened these gene disruptants for decreased transformation frequency and identified two genes, yrzD and yutB, whose disruption resulted in severely reduced transformation frequency and modestly reduced transformation frequency, respectively. In the regulation of competence development, various signals affect the expression of comK, which encodes a master regulator of genetic competence that drives late competence gene transcription. Epistatic analyses of both the yrzD and yutB genes revealed no significant differences in the expression of comK. Further analysis of the expression of late competence genes in the yrzD disruptant revealed that yrzD is specifically required for regulation of the comE operon, which is one of the late competence operons, and thus was renamed comN. An analysis of various comE-lacZ fusions revealed that the target cis element for comN action is in the large (approximately 1-kb) 5′ untranslated region of comE, while the activity of the comE promoter was not affected by disruption of comN. These results suggested that there is post-transcription initiation control of comE by comN. A sequential deletion analysis of this region revealed the 35-bp region required for comN action. The yutB gene encodes a putative lipoic acid synthetase and yet is specifically required for transcription of comE, based on the results of lacZ fusion analyses. Therefore, yutB and comN regulate comE at the transcription and post-transcription initiation levels, respectively. These results demonstrate that a comE-specific regulatory mechanism is involved in development of genetic competence. PMID:19028902

  14. Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis.

    PubMed

    Norais, N; Nogarotto, R; Lacobini, E T; Garaguso, I; Grifantini, R; Gauli, G; Grandi, G

    2001-11-01

    The most popular approach for proteomics analysis is based on the combination of two-dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two-dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.

  15. A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

    PubMed Central

    Al-Khouri, Anna Maria; Paule, Marvin R.

    2002-01-01

    In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

  16. Model-based Characterization of the Parameters of Dissimilatory Sulfate Reduction Under the Effect of Different Initial Density of Desulfovibrio piger Vib-7 Bacterial Cells.

    PubMed

    Kushkevych, Ivan; Bolis, Marco; Bartos, Milan

    2015-01-01

    The objective of this study was to design a model of dissimilatory sulfate reduction process using the Verhulst function, with a particular focus on the kinetics of bacterial growth, sulfate and lactate consumption, and accumulation of hydrogen sulfide and acetate. The effect of the initial density (0.12±0.011, 0.25±0.024, 0.5±0.048 and 1.0±0.096 mg cells/ml of medium) of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 on the growth and dissimilatory sulfate reduction was studied. The exponential growth phase of the D. piger Vib-7 was observed for 72 hours of cultivation at the (0.12 and 0.25 mg/ml) initial concentration of bacterial cells. Sulfate and lactate were consumed incompletely during this time. The increase in the initial concentration of cells to 0.5 and 1 mg/ml led to a shortening of the exponential bacterial growth phase and a shift to the stationary phase of the growth. In the case of 0.5 mg/ml seeding, the stationary growth phase was observed in the 36(th) hour of cultivation. The increase in the initial concentration of cells to 1 mg/ml led to the beginning of the stationary growth phase in 24th hours of cultivation. Under these conditions, sulfate and lactate were consumed completely in the 48th hour of cultivation. The kinetic analysis of the curves of bacterial growth and the process of dissimilatory sulfate reduction by D. piger Vib-7 was carried out.

  17. Structural basis of the role of the NikA ribbon-helix-helix domain in initiating bacterial conjugation.

    PubMed

    Yoshida, Hitoshi; Furuya, Nobuhisa; Lin, Yi-Jan; Güntert, Peter; Komano, Teruya; Kainosho, Masatsune

    2008-12-19

    Conjugation is a fundamental process for the rapid evolution of bacteria, enabling them, for example, to adapt to various environmental conditions or to acquire multi-drug resistance. NikA is one of the relaxosomal proteins that initiate the intercellular transfer of the R64 conjugative plasmid with the P-type origin of transfer, oriT. The three-dimensional structure of the N-terminal 51 residue fragment of NikA, NikA(1-51), which binds to the 17-bp repeat A sequence in R64 oriT, was determined by NMR to be a homodimer composed of two identical ribbon-helix-helix (RHH) domains, which are commonly found in transcriptional repressors. The structure determination of NikA(1-51) was achieved using automated NOE assignment with CYANA, without measuring filtered NOESY experiments to distinguish between the intra- and intermolecular NOEs, and without any a priori assumption on the tertiary or quaternary structure of the protein. Mutational experiments revealed that the DNA-binding region of the NikA(1-51) dimer is an anti-parallel beta-sheet composed of one beta-strand from each of the N-terminal ends of the two domains. Various biochemical experiments have indicated that the full length NikA(1-109) exists as a homotetramer formed through an alpha-helical domain at the C-terminus, and that the anti-parallel beta-sheets of both dimeric domains bind to two homologous 5 bp internal repeats within repeat A. As a tetramer, the full length NikA(1-109) showed higher affinity to repeat A and bent the oriT duplex more strongly than NikA(1-51) did. Many RHH proteins are involved in specific DNA recognition and in protein-protein interactions. The discovery of the RHH fold in NikA suggests that NikA binds to oriT and interacts with the relaxase, NikB, which is unable to bind to the nick region in oriT without NikA.

  18. Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

    PubMed Central

    Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

    2016-01-01

    All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

  19. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    PubMed Central

    Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.; Yu, Minmin; Hung, Li-Wei; Cieslik, Marcin; Derewenda, Urszula; Lesley, Scott A.; Wilson, Ian A.; Giedroc, David P.; Derewenda, Zygmunt S.

    2009-01-01

    The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR_C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni2+ ions but that it is able to bind Zn2+ with K d < 70 nM. It is concluded that Zn2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors. PMID:19307717

  20. Crystal structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    SciTech Connect

    Zheng, Meiying; Cooper, David; Grossoehmerb, Nickolas; Yu, Minmin; Hung, Li-Wei; Cieslik, Murcin; Derewendaro, Urszula; Lesley, Scott; Wilson, Ian; Giedrocb, David; Derewenda, Zygmunt

    2009-06-06

    The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged helix (WH) DNA-binding domains and diverse C-terminal, regulatory domains, which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all a-helical regulatory domains classified into two related Pfam families: FadR{_}C and FCD. Only two crystal structures of the FadR family members, i.e. the E. coli FadR protein and the LldR from C. glutamicum, have been described to date in literature. Here we describe the crystal structure of TM0439, a GntR regulator with an FCD domain, found in the Thermotoga maritima genome. The FCD domain is similar to that of the LldR regulator, and contains a buried metal binding site. Using atomic absorption spectroscopy and Trp fluorescence, we show that the recombinant protein contains bound Ni{sup 2+} ions, but it is able to bind Zn{sup 2+} with K{sub D} < 70 nM . We conclude that Zn{sup 2+} is the likely physiological metal, where it may perform either or both structural and regulatory roles. Finally, we compare the TM0439 structure to two other FadR family structures recently deposited by Structural Genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

  1. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn{sup 2+}-binding FCD domains

    SciTech Connect

    Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.; Yu, Minmin; Hung, Li-Wei; Cieslik, Marcin; Derewenda, Urszula; Lesley, Scott A.; Wilson, Ian A.; Giedroc, David P.; Derewenda, Zygmunt S.

    2009-04-01

    Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni{sup 2+} ions but that it is able to bind Zn{sup 2+} with K{sub d} < 70 nM. It is concluded that Zn{sup 2+} is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

  2. Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription.

    PubMed

    Xu, Muyu; Gonzalez-Hurtado, Elsie; Martinez, Ernest

    2016-04-01

    Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

  3. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

    PubMed Central

    Hossain, Manzar; Stillman, Bruce

    2016-01-01

    Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment. DOI: http://dx.doi.org/10.7554/eLife.12785.001 PMID:27458800

  4. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts.

    PubMed

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-09-29

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

  5. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts

    PubMed Central

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-01-01

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil. PMID:27319579

  6. Toll-like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar typhimurium infection, but not for the initiation of bacterial clearance.

    PubMed

    Talbot, Suzanne; Tötemeyer, Sabine; Yamamoto, Masahiro; Akira, Shizuo; Hughes, Katherine; Gray, David; Barr, Tom; Mastroeni, Pietro; Maskell, Duncan J; Bryant, Clare E

    2009-12-01

    Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

  7. Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP-dependent transcription initiation mechanism in E. coli lac operon.

    PubMed

    Tutar, Yusuf

    2008-06-01

    The cyclic AMP receptor protein (CRP) of Escherichia coli regulates the activity of more than 150 genes. Allosteric changes in CRP structure accompanied by cAMP binding, initiate transcription through protein binding to specific DNA sequences. Initially, researchers proposed a two-site cAMP-binding model for CRP-dependent transcription activation since biophysical methods showed two transitions during titration experiments. Three conformational states were considered; apo-CRP, CRP:(cAMP)(1) and CRP:(cAMP)(2), and CRP:(cAMP)(1) was proposed as the active form in this initial model. X-ray data indicated an anti conformation and in contrast NMR experiments suggested a syn conformation for bound cAMPs. For years this paradigm about ligand conformation has been ambiguous. When CRP was crystallized with four bound cAMP in the last decade, two cAMPs were assigned to syn and the other two to anti conformations. Again three conformational states were suggested; apo-CRP, CRP:(cAMP)(2), and CRP:(cAMP)(4). This new structure changed the view of CRP allosteric activation from a two-site model to a four-site model in the literature and the new model has been supported by biochemical and genetic data so far. According to the accepted model, binding of the first two cAMP molecules displays positive cooperativity, however, binding of the last two cAMP molecules shows negative cooperativity. This resolved the conflict between dynamic and static experimental observations. However, this new model cannot explain the initiation mechanism as previously proposed because functionally active CRP has only one cAMP equivalent. Gene regulation and transcription factors are involved in regulating both prokaryotic and eukaryotic metabolism. Although gene regulation and expression are much more complex in eukaryotes, CRP-mediated transcription initiation is a model of general interest to life sciences and medicine. Therefore, the aim of this review is to summarize recent works and developments on

  8. UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

    PubMed Central

    Gyenis, Ákos; Umlauf, David; Újfaludi, Zsuzsanna; Boros, Imre; Ye, Tao; Tora, Làszlò

    2014-01-01

    Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation. PMID:25058334

  9. Transcriptional Response in Mouse Thyroid Tissue after 211At Administration: Effects of Absorbed Dose, Initial Dose-Rate and Time after Administration

    PubMed Central

    Rudqvist, Nils; Spetz, Johan; Schüler, Emil; Parris, Toshima Z.; Langen, Britta; Helou, Khalil; Forssell-Aronsson, Eva

    2015-01-01

    Background 211At-labeled radiopharmaceuticals are potentially useful for tumor therapy. However, a limitation has been the preferential accumulation of released 211At in the thyroid gland, which is a critical organ for such therapy. The aim of this study was to determine the effect of absorbed dose, dose-rate, and time after 211At exposure on genome-wide transcriptional expression in mouse thyroid gland. Methods BALB/c mice were i.v. injected with 1.7, 7.5 or 100 kBq 211At. Animals injected with 1.7 kBq were killed after 1, 6, or 168 h with mean thyroid absorbed doses of 0.023, 0.32, and 1.8 Gy, respectively. Animals injected with 7.5 and 100 kBq were killed after 6 and 1 h, respectively; mean thyroid absorbed dose was 1.4 Gy. Total RNA was extracted from pooled thyroids and the Illumina RNA microarray platform was used to determine mRNA levels. Differentially expressed transcripts and enriched GO terms were determined with adjusted p-value <0.01 and fold change >1.5, and p-value <0.05, respectively. Results In total, 1232 differentially expressed transcripts were detected after 211At administration, demonstrating a profound effect on gene regulation. The number of regulated transcripts increased with higher initial dose-rate/absorbed dose at 1 or 6 h. However, the number of regulated transcripts decreased with mean absorbed dose/time after 1.7 kBq 211At administration. Furthermore, similar regulation profiles were seen for groups administered 1.7 kBq. Interestingly, few previously proposed radiation responsive genes were detected in the present study. Regulation of immunological processes were prevalent at 1, 6, and 168 h after 1.7 kBq administration (0.023, 0.32, 1.8 Gy). PMID:26177204

  10. Barley MLA Immune Receptors Directly Interfere with Antagonistically Acting Transcription Factors to Initiate Disease Resistance Signaling[C][W

    PubMed Central

    Chang, Cheng; Yu, Deshui; Jiao, Jian; Jing, Shaojuan; Schulze-Lefert, Paul; Shen, Qian-Hua

    2013-01-01

    The nucleotide binding domain and Leucine-rich repeat (NLR)–containing proteins in plants and animals mediate pathogen sensing inside host cells and mount innate immune responses against microbial pathogens. The barley (Hordeum vulgare) mildew A (MLA) locus encodes coiled-coil (CC)–type NLRs mediating disease resistance against the powdery mildew pathogen Blumeria graminis. Here, we report direct interactions between MLA and two antagonistically acting transcription factors, MYB6 and WRKY1. The N-terminal CC signaling domain of MLA interacts with MYB6 to stimulate its DNA binding activity. MYB6 functions as a positive regulator of basal and MLA-mediated immunity responses to B. graminis. MYB6 DNA binding is antagonized by direct association with WRKY1 repressor, which in turn also interacts with the MLA CC domain. The activated form of full-length MLA10 receptor is needed to release MYB6 activator from WRKY1 repression and to stimulate MYB6-dependent gene expression. This implies that, while sequestered by the WRKY1 repressor in the presence of the resting immune receptor, MYB6 acts as an immediate and positive postactivation signaling component of the active state of MLA during transcriptional reprogramming for innate immune responses. PMID:23532068

  11. Medium-dependent regulation of proteinase gene expression in Lactococcus lactis: control of transcription initiation by specific dipeptides.

    PubMed Central

    Marugg, J D; Meijer, W; van Kranenburg, R; Laverman, P; Bruinenberg, P G; de Vos, W M

    1995-01-01

    Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene (gusA) were used to study the medium- and growth-dependent expression of the divergently transcribed genes involved in proteinase production (prtP and prtM) of Lactococcus lactis SK11. The results show that both the prtP and prtM genes are controlled at the transcriptional level by the peptide content of the medium and, to a lesser extent, by the growth rate. A more than 10-fold regulation in beta-glucuronidase activity was observed for both prtP and prtM promoters in batch and continuous cultures. The level of expression of the prtP and prtM promoters was high in whey permeate medium with relatively low concentrations of peptides, whereas at increased concentrations the expression of the promoters was repressed. The lowest level of expression was observed in peptide- and amino acid-rich laboratory media, such as glucose-M17 and MRS. The addition of specific dipeptides, such as leucylproline and prolylleucine, to the growth medium negatively affected the expression of the prtP-gusA fusions. The repression by dipeptides was not observed in mutants defective in the uptake of di-tripeptides, indicating that the internal concentration of dipeptides or derivatives is important in the regulation of proteinase production. PMID:7768792

  12. Transcriptional and metabolic signatures of Arabidopsis responses to chewing damage by an insect herbivore and bacterial infection and the consequences of their interaction

    PubMed Central

    Appel, Heidi M.; Maqbool, Shahina B.; Raina, Surabhi; Jagadeeswaran, Guru; Acharya, Biswa R.; Hanley, John C.; Miller, Kathryn P.; Hearnes, Leonard; Jones, A. Daniel; Raina, Ramesh; Schultz, Jack C.

    2014-01-01

    Plants use multiple interacting signaling systems to identify and respond to biotic stresses. Although it is often assumed that there is specificity in signaling responses to specific pests, this is rarely examined outside of the gene-for-gene relationships of plant-pathogen interactions. In this study, we first compared early events in gene expression and later events in metabolite profiles of Arabidopsis thaliana following attack by either the caterpillar Spodoptera exigua or avirulent (DC3000 avrRpm1) Pseudomonas syringae pv. tomato at three time points. Transcriptional responses of the plant to caterpillar feeding were rapid, occurring within 1 h of feeding, and then decreased at 6 and 24 h. In contrast, plant response to the pathogen was undetectable at 1 h but grew larger and more significant at 6 and 24 h. There was a surprisingly large amount of overlap in jasmonate and salicylate signaling in responses to the insect and pathogen, including levels of gene expression and individual hormones. The caterpillar and pathogen treatments induced different patterns of expression of glucosinolate biosynthesis genes and levels of glucosinolates. This suggests that when specific responses develop, their regulation is complex and best understood by characterizing expression of many genes and metabolites. We then examined the effect of feeding by the caterpillar Spodoptera exigua on Arabidopsis susceptibility to virulent (DC3000) and avirulent (DC3000 avrRpm1) P. syringae pv. tomato, and found that caterpillar feeding enhanced Arabidopsis resistance to the avirulent pathogen and lowered resistance to the virulent strain. We conclude that efforts to improve plant resistance to bacterial pathogens are likely to influence resistance to insects and vice versa. Studies explicitly comparing plant responses to multiple stresses, including the role of elicitors at early time points, are critical to understanding how plants organize responses in natural settings. PMID:25278943

  13. Pseudoalteromonas spp. serve as initial bacterial attractants in mesocosms of coastal waters but have subsequent antifouling capacity in mesocosms and when embedded in paint.

    PubMed

    Bernbom, Nete; Ng, Yoke Yin; Olsen, Stefan Møller; Gram, Lone

    2013-11-01

    The purpose of the present study was to determine if the monoculture antifouling effect of several pigmented pseudoalteromonads was retained in in vitro mesocosm systems using natural coastal seawater and when the bacteria were embedded in paint used on surfaces submerged in coastal waters. Pseudoalteromonas piscicida survived on a steel surface and retained antifouling activity for at least 53 days in sterile seawater, whereas P. tunicata survived and had antifouling activity for only 1 week. However, during the first week, all Pseudoalteromonas strains facilitated rather than prevented bacterial attachment when used to coat stainless steel surfaces and submerged in mesocosms with natural seawater. The bacterial density on surfaces coated with sterile growth medium was 10(5) cells/cm(2) after 7 days, whereas counts on surfaces precoated with Pseudoalteromonas were significantly higher, at 10(6) to 10(8) cells/cm(2). However, after 53 days, seven of eight Pseudoalteromonas strains had reduced total bacterial adhesion compared to the control. P. piscicida, P. antarctica, and P. ulvae remained on the surface, at levels similar to those in the initial coating, whereas P. tunicata could not be detected. Larger fouling organisms were observed on all plates precoated with Pseudoalteromonas; however, plates coated only with sterile growth medium were dominated by a bacterial biofilm. Suspensions of a P. piscicida strain and a P. tunicata strain were incorporated into ship paints (Hempasil x3 87500 and Hempasil 77500) used on plates that were placed at the Hempel A/S test site in Jyllinge Harbor. For the first 4 months, no differences were observed between control plates and treated plates, but after 5 to 6 months, the control plates were more fouled than the plates with pseudoalteromonad-based paint. Our study demonstrates that no single laboratory assay can predict antifouling effects and that a combination of laboratory and real-life methods must be used to determine

  14. Rad3-Cds1 mediates coupling of initiation of meiotic recombination with DNA replication. Mei4-dependent transcription as a potential target of meiotic checkpoint.

    PubMed

    Ogino, Keiko; Masai, Hisao

    2006-01-20

    Premeiotic S-phase and meiotic recombination are known to be strictly coupled in Saccharomyces cerevisiae. However, the checkpoint pathway regulating this coupling has been largely unknown. In fission yeast, Rad3 is known to play an essential role in coordination of DNA replication and cell division during both mitotic growth and meiosis. Here we have examined whether the Rad3 pathway also regulates the coupling of DNA synthesis and recombination. Inhibition of premeiotic S-phase with hydroxyurea completely abrogates the progression of meiosis, including the formation of DNA double-strand breaks (DSBs). DSB formation is restored in rad3 mutant even in the presence of hydroxyurea, although repair of DSBs does not take place or is significantly delayed, indicating that the subsequent recombination steps may be still inhibited. Examination of the roles of downstream checkpoint kinases reveals that Cds1, but not Chk1 or Mek1, is required for suppression of DSB in the presence of hydroxyurea. Transcriptional induction of some rec+ genes essential for DSB occurs at a normal timing and to a normal level in the absence of DNA synthesis in both the wild-type and cds1delta cells. On the other hand, the transcriptional induction of the mei4+ transcription factor and cdc25+ phosphatase, which is significantly suppressed by hydroxyurea in the wild-type cells, occurs almost to a normal level in cds1delta cells even in the presence of hydroxyurea. These results show that the Rad3-Cds1 checkpoint pathway coordinates initiation of meiotic recombination and meiotic cell divisions with premeiotic DNA synthesis. Because mei4+ is known to be required for DSB formation and cdc25+ is required for activation of meiotic cell divisions, we propose an intriguing possibility that the Rad3-Cds1 meiotic checkpoint pathway may target transcription of these factors.

  15. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development.

    PubMed

    Danzer, John; Mellott, Eric; Bui, Anhthu Q; Le, Brandon H; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M; Somers, David A; Goldberg, Robert B; Harada, John J

    2015-07-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and inducer of C-repeat/dehydration responsive element-binding factor expression1/scream2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development.

  16. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development1[OPEN

    PubMed Central

    Danzer, John; Mellott, Eric; Bui, Anhthu Q.; Le, Brandon H.; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M.; Somers, David A.; Goldberg, Robert B.; Harada, John J.

    2015-01-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and INDUCER OF C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT-BINDING FACTOR EXPRESSION1/SCREAM2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development. PMID:25963149

  17. The bacterial enhancer-dependent RNA polymerase

    PubMed Central

    Zhang, Nan; Darbari, Vidya C.; Glyde, Robert; Zhang, Xiaodong; Buck, Martin

    2016-01-01

    Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript. PMID:27789741

  18. Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

    PubMed Central

    Gaillard, Coline; Tokuyasu, Taku A.; Rosen, Galit; Sotzen, Jason; Vitaliano-Prunier, Adeline; Roy, Ritu; Passegué, Emmanuelle; de Thé, Hugues; Figueroa, Maria E.; Kogan, Scott C.

    2015-01-01

    Acute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment. PMID:26088929

  19. Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected

    PubMed Central

    Gómez-Cuadrado, A; Martín, M; Noël, M; Ruiz-Carrillo, A

    1995-01-01

    Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism. PMID:8524232

  20. Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor.

    PubMed

    Hwang, C; Wu, W-M; Gentry, T J; Carley, J; Carroll, S L; Schadt, C; Watson, D; Jardine, P M; Zhou, J; Hickey, R F; Criddle, C S; Fields, M W

    2006-08-01

    High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n = 500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.

  1. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome

    PubMed Central

    Zhang, Jingji; Ieong, Ka-Weng; Johansson, Magnus; Ehrenberg, Måns

    2015-01-01

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200–80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNAHis and, as also seen in vivo, Glu-tRNAGlu. We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here. PMID:26195797

  2. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome.

    PubMed

    Zhang, Jingji; Ieong, Ka-Weng; Johansson, Magnus; Ehrenberg, Måns

    2015-08-04

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

  3. Transcription regulation mechanisms of bacteriophages: recent advances and future prospects.

    PubMed

    Yang, Haiquan; Ma, Yingfang; Wang, Yitian; Yang, Haixia; Shen, Wei; Chen, Xianzhong

    2014-01-01

    Phage diversity significantly contributes to ecology and evolution of new bacterial species through horizontal gene transfer. Therefore, it is essential to understand the mechanisms underlying phage-host interactions. After initial infection, the phage utilizes the transcriptional machinery of the host to direct the expression of its own genes. This review presents a view on the transcriptional regulation mechanisms of bacteriophages, and its contribution to phage diversity and classification. Through this review, we aim to broaden the understanding of phage-host interactions while providing a reference source for researchers studying the regulation of phage transcription.

  4. A novel transcription initiation factor (TIF), TIF-IE, is required for homogeneous Acanthamoeba castellanii TIF-IB (SL1) to form a committed complex.

    PubMed

    Radebaugh, C A; Kubaska, W M; Hoffman, L H; Stiffler, K; Paule, M R

    1998-10-16

    The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460, 000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 +/- 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

  5. The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

    PubMed Central

    Gu, Tao; Zhou, Chaoyang; Sørensen, Sebastian R.; Zhang, Ji; He, Jian; Yu, Peiwen; Li, Shunpeng

    2013-01-01

    The environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase. PMID:24123738

  6. Why is initial bacterial colonization of the intestine important to the infant’s and child’s health?

    PubMed Central

    Houghteling, Pearl D.; Walker, W. Allan

    2014-01-01

    Microbial colonization of the infant occurs during a critical time window for immune and gastrointestinal development. Infant colonization sets the stage for the adult microbiome. This review is a broad survey of the factors affecting infant colonization and the downstream effects on gastrointestinal health and disease. Major topics affecting colonization include initial inoculation dependent on birth mode, the impact of breastfeeding, and inside-out modulation of the developing microbiome by the immune system. Major outcomes of colonization include the timing-dependent education of the neonatal immune system, which is interconnected with barrier function and metabolism. These all engage in further continuing cross-talk with the microbiome, genetics and nutrition. This review will also briefly discuss mechanisms of disease resulting from disrupted colonization as well as nutritional and microbial therapies. PMID:25313849

  7. Rethinking transcription coupled DNA repair.

    PubMed

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity.

  8. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    PubMed Central

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  9. Non-transcriptional regulatory processes shape transcriptional network dynamics.

    PubMed

    Ray, J Christian J; Tabor, Jeffrey J; Igoshin, Oleg A

    2011-10-11

    Information about the extra- or intracellular environment is often captured as biochemical signals that propagate through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programmes in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. Cellular response dynamics are ultimately determined by interactions between transcriptional and non-transcriptional networks, with dramatic implications for physiology and evolution. Here, we provide an overview of non-transcriptional interactions that can affect the performance of natural and synthetic bacterial regulatory networks.

  10. Proteome changes in the initial bacterial colonist during ecological succession in an acid mine drainage biofilm community

    SciTech Connect

    Mueller, Ryan; Dill, Brian; Pan, Chongle; Belnap, Christopher P.; Thomas, Brian; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.

    2011-01-01

    Proteomes of acid mine drainage biofilms at different stages of ecological succession were examined to understand microbial responses to changing community membership. We evaluated the degree of reproducibility of the community proteomes between samples of the same growth stage and found stable and predictable protein abundance patterns across time and sampling space, allowing for a set of 50 classifier proteins to be identified for use in predicting growth stages of undefined communities. Additionally, physiological changes in the dominant species, Leptospirillum Group II, were analysed as biofilms mature. During early growth stages, this population responds to abiotic stresses related to growth on the acid mine drainage solution. Enzymes involved in protein synthesis, cell division and utilization of 1- and 2-carbon compounds were more abundant in early growth stages, suggesting rapid growth and a reorganization of metabolism during biofilm initiation. As biofilms thicken and diversify, external stresses arise from competition for dwindling resources, which may inhibit cell division of Leptospirillum Group II through the SOS response. This population also represses translation and synthesizes more complex carbohydrates and amino acids in mature biofilms. These findings provide unprecedented insight into the physiological changes that may result from competitive interactions within communities in natural environments.

  11. Proteome changes in the initial bacterial colonist during ecological succession in an acid mine drainage biofilm community.

    PubMed

    Mueller, Ryan S; Dill, Brian D; Pan, Chongle; Belnap, Christopher P; Thomas, Brian C; VerBerkmoes, Nathan C; Hettich, Robert L; Banfield, Jillian F

    2011-08-01

    Proteomes of acid mine drainage biofilms at different stages of ecological succession were examined to understand microbial responses to changing community membership. We evaluated the degree of reproducibility of the community proteomes between samples of the same growth stage and found stable and predictable protein abundance patterns across time and sampling space, allowing for a set of 50 classifier proteins to be identified for use in predicting growth stages of undefined communities. Additionally, physiological changes in the dominant species, Leptospirillum Group II, were analysed as biofilms mature. During early growth stages, this population responds to abiotic stresses related to growth on the acid mine drainage solution. Enzymes involved in protein synthesis, cell division and utilization of 1- and 2-carbon compounds were more abundant in early growth stages, suggesting rapid growth and a reorganization of metabolism during biofilm initiation. As biofilms thicken and diversify, external stresses arise from competition for dwindling resources, which may inhibit cell division of Leptospirillum Group II through the SOS response. This population also represses translation and synthesizes more complex carbohydrates and amino acids in mature biofilms. These findings provide unprecedented insight into the physiological changes that may result from competitive interactions within communities in natural environments.

  12. Matrix formulation of a universal microbial transcript profiling system

    SciTech Connect

    Fitch, J P; Ng, J; Sokhansanj, B A

    2000-11-01

    DNA chips and microarrays are used to profile gene transcription. Unfortunately, the initial fabrication cost for a chip and the reagent costs to amplify thousands of open reading frames for a microarray are over $100K for a typical 4 Mbase bacterial genome. To avoid these expensive steps, a matrix formulation of a universal hybrid chip-microarray approach to transcript profiling is demonstrated for synthetic data. Initial considerations for application to the 4.3 Mbase bacterium Yersinia pestis are also presented. This approach can be applied to arbitrary bacteria by recalculating a matrix and pseudoinverse. This approach avoids the large upfront expenses associated with DNA chips and microarrays.

  13. Emx2 homeodomain transcription factor interacts with eukaryotic translation initiation factor 4E (eIF4E) in the axons of olfactory sensory neurons.

    PubMed

    Nédélec, Stéphane; Foucher, Isabelle; Brunet, Isabelle; Bouillot, Colette; Prochiantz, Alain; Trembleau, Alain

    2004-07-20

    We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.

  14. Stimulation of the lambda pR promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation.

    PubMed

    Łyzeń, Robert; Wegrzyn, Grzegorz; Wegrzyn, Alicja; Szalewska-Pałasz, Agnieszka

    2006-10-01

    Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage lambda promoters p(I), p(aQ) and p(R). While SeqA-mediated stimulation of p(I) and p(aQ) occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p(R) remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p(R) transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p(R) was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p(R) transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.

  15. Nucleotide sequence of RNA2 of Lettuce big-vein virus and evidence for a possible transcription termination/initiation strategy similar to that of rhabdoviruses.

    PubMed

    Sasaya, Takahide; Kusaba, Shinnosuke; Ishikawa, Koichi; Koganezawa, Hiroki

    2004-09-01

    Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210-1403 on the viral RNA), ORF2 (nt 1493-2494), ORF3 (nt 2617-3489), ORF4 (nt 3843-4337) and ORF5 (nt 4530-5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3' end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3'-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5'-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

  16. Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation[C][W

    PubMed Central

    Wang, Youning; Wang, Lixiang; Zou, Yanmin; Chen, Liang; Cai, Zhaoming; Zhang, Senlei; Zhao, Fang; Tian, Yinping; Jiang, Qiong; Ferguson, Brett J.; Gresshoff, Peter M.; Li, Xia

    2014-01-01

    MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean. PMID:25549672

  17. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

    PubMed

    Wilson, S A; Sieiro-Vazquez, C; Edwards, N J; Iourin, O; Byles, E D; Kotsopoulou, E; Adamson, C S; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    1999-08-15

    The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans.

  18. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...

  19. Transcription Regulation in Archaea

    PubMed Central

    Gehring, Alexandra M.; Walker, Julie E.

    2016-01-01

    The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. PMID:27137495

  20. CpsR, a GntR family regulator, transcriptionally regulates capsular polysaccharide biosynthesis and governs bacterial virulence in Streptococcus pneumoniae

    PubMed Central

    Wu, Kaifeng; Xu, Hongmei; Zheng, Yuqiang; Wang, Libin; Zhang, Xuemei; Yin, Yibing

    2016-01-01

    Transcriptional regulation of capsule expression is critical for pneumococcal transition from carriage to infection, yet the underlying mechanism remains incompletely understood. Here, we describe the regulation of capsular polysaccharide, one of the most important pneumococcal virulence factor by a GntR family regulator, CpsR. Electrophoretic mobility-shift assays have shown the direct interaction between CpsR and the cps promoter (cpsp), and their interaction could be competitively interfered by glucose. DNase I footprinting assays localized the binding site to a region −146 to −114 base pairs relative to the transcriptional start site of the cps locus in S. pneumoniae D39. We found that CpsR negatively controlled the transcription of the cps locus and hence CPS production, which was confirmed by fine-tuning expression of CpsR in a ΔcpsR complemented strain. Increased expression of CpsR in complemented strain led to a decreased resistance to the whole-blood-mediated killing, suggesting a protective role for CpsR-cpsp interaction in the establishment of invasive infection. Finally, animal experiments showed that CpsR-cpsp interaction was necessary for both pneumococcal colonization and invasive infection. Taken together, our results provide a thorough insight into the regulation of capsule production mediated by CpsR and its important roles in pneumococcal pathogenesis. PMID:27386955

  1. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  2. Spectrometric study of the folding process of i-motif-forming DNA sequences upstream of the c-kit transcription initiation site.

    PubMed

    Bucek, Pavel; Gargallo, Raimundo; Kudrev, Andrei

    2010-12-17

    The c-kit oncogene shows a cytosine-rich DNA region upstream of the transcription initiation site which forms an i-motif structure at slightly acidic pH values (Bucek et al. [5]). In the present study, the pH-induced formation of i-motif - forming sequences 5'-CCC CTC CCT CGC GCC CGC CCG-3' (ckitC1, native), 5'-CCC TTC CCT TGT GCC CGC CCG-3' (ckitC2) and 5'-CCCTT CCC TTTTT CCC T CCC T-3' (ckitC3) was studied by spectroscopic techniques, such as UV molecular absorption and circular dichroism (CD), in tandem with two multivariate data analysis methods, the hard modelling-based matrix method and the soft modelling-based MCR-ALS approach. Use of the hard chemical modelling enabled us to propose the equilibrium model, which describes spectral changes as functions of solution acidity. Additionally, the intrinsic protonation constant, K(in), and the cooperativity parameters, ω(c), and ω(a), were calculated from the fitting procedure of the coupled CD and molecular absorption spectra. In the case of ckitC2 and ckitC3, the hard model correctly reproduced the spectral variations observed experimentally. The results indicated that folding was accompanied by a cooperative process, i.e. the enhancement of protonated structure stability upon protonation. In contrast, unfolding was accompanied by an anticooperative process. Finally, folding of the native sequence, ckitC1, seemed to follow a more complex mechanism.

  3. Novel roles of SoxR, a transcriptional regulator from Xanthomonas campestris, in sensing redox-cycling drugs and regulating a protective gene that have overall implications for bacterial stress physiology and virulence on a host plant.

    PubMed

    Mahavihakanont, Aekkapol; Charoenlap, Nisanart; Namchaiw, Poommaree; Eiamphungporn, Warawan; Chattrakarn, Sorayut; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

    2012-01-01

    In Xanthomonas campestris pv. campestris, SoxR likely functions as a sensor of redox-cycling drugs and as a transcriptional regulator. Oxidized SoxR binds directly to its target site and activates the expression of xcc0300, a gene that has protective roles against the toxicity of redox-cycling compounds. In addition, SoxR acts as a noninducible repressor of its own expression. X. campestris pv. campestris requires SoxR both for protection against redox-cycling drugs and for full virulence on a host plant. The X. campestris model of the gene regulation and physiological roles of SoxR represents a novel variant of existing bacterial SoxR models.

  4. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed Central

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor. Images PMID:8264628

  5. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

  6. Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

    PubMed Central

    van Nues, Rob W.; Castro-Roa, Daniel; Yuzenkova, Yulia; Zenkin, Nikolay

    2016-01-01

    Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins. PMID:26609136

  7. Molecular characterization of collagen IV evidences early transcription expression related to the immune response against bacterial infection in the red abalone (Haliotis rufescens).

    PubMed

    Chovar-Vera, Ornella; Valenzuela-Muñoz, Valentina; Gallardo-Escárate, Cristian

    2015-02-01

    Collagen IV has been described as a structural protein of the basement membrane, which as a whole forms a specialized extracellular matrix. Recent studies have indicated a possible relationship between collagen IV and the innate immune response of invertebrate organisms. The present study characterized the alpha-1 chain of collagen IV in the red abalone Haliotis rufescens (Hr-ColIV) and evaluated its association with the innate immune response against Vibrio anguillarum. To further evidence the immune response, the matrix metalloproteinase-1 (Hr-MMP-1) and C-type lectin (Hr-CLEC) genes were also assessed. The complete sequence of Hr-ColIV was composed of 6658 bp, with a 5'UTR of 154 bp, a 3'UTR of 1177 bp, and an ORF of 5327 bp that coded for 1776 amino acids. The innate immune response generated against V. anguillarum resulted in a significant increase in the transcript levels of Hr-ColIV between 3 and 6 hpi, whereas Hr-MMP-1 and Hr-CLEC had the highest transcript activity 6 and 12 hpi, respectively. The results obtained in this study propose a putative biological function for collagen IV involved in the early innate immune response of the red abalone H. rufescens.

  8. A model for genesis of transcription systems.

    PubMed

    Burton, Zachary F; Opron, Kristopher; Wei, Guowei; Geiger, James H

    2016-01-01

    Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ-β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., (-12)TATAATG(-6)) so closely resemble TATA boxes (i.e., (-31)TATAAAAG(-24)) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., (-35)TTGACA(-30)) and archaeal (BRE(up); BRE for TFB recognition element) promoters is potentially explained. The evolution of BRE(down) elements of archaeal promoters is potentially explained.

  9. A model for genesis of transcription systems

    PubMed Central

    Burton, Zachary F.; Opron, Kristopher; Wei, Guowei; Geiger, James H.

    2016-01-01

    ABSTRACT Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ−β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., −12TATAATG−6) so closely resemble TATA boxes (i.e., −31TATAAAAG−24) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., −35TTGACA−30) and archaeal (BREup; BRE for TFB recognition element) promoters is potentially explained. The evolution of BREdown elements of archaeal promoters is potentially explained. PMID:26735411

  10. The mutant phenotype associated with P-element alleles of the vestigial locus in Drosophila melanogaster may be caused by a readthrough transcript initiated at the P-element promoter.

    PubMed

    Hodgetts, R B; O'Keefe, S L

    2001-04-01

    We report here the isolation of a new P-element-induced allele of the vestigial locus vg(2a33), the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was vg(21), which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing vg(2a33) in a dysgenic cross, we isolated the vg(2a33) allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3'-proximal copy of the 11-bp internal repeat from the P element of vg(21). To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5'-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from vg(21). Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.

  11. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

    SciTech Connect

    Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit

    2015-09-15

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR.

  12. Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection.

    PubMed

    Kumar, Swati; Wang, Lihua; Fan, Jiang; Kraft, Andrea; Bose, Michael E; Tiwari, Sagarika; Van Dyke, Meredith; Haigis, Robert; Luo, Tingquo; Ghosh, Madhushree; Tang, Huong; Haghnia, Marjan; Mather, Elizabeth L; Weisburg, William G; Henrickson, Kelly J

    2008-09-01

    Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked "surrogate" clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

  13. Promoters of the Broad Host Range Plasmid Rk2: Analysis of Transcription (Initiation) in Five Species of Gram-Negative Bacteria

    PubMed Central

    Greener, A.; Lehman, S. M.; Helinski, D. R.

    1992-01-01

    A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined. PMID:1732166

  14. Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

    PubMed

    Bermejo, Daniela A; Jackson, Shaun W; Gorosito-Serran, Melisa; Acosta-Rodriguez, Eva V; Amezcua-Vesely, Maria C; Sather, Blythe D; Singh, Akhilesh K; Khim, Socheath; Mucci, Juan; Liggitt, Denny; Campetella, Oscar; Oukka, Mohamed; Gruppi, Adriana; Rawlings, David J

    2013-05-01

    Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

  15. Nucleosome-mediated disruption of transcription factor-chromatin initiation complexes at the mouse mammary tumor virus long terminal repeat in vivo.

    PubMed Central

    Lee, H L; Archer, T K

    1994-01-01

    Glucocorticoid induction of mouse mammary tumor virus (MMTV) is short lived, returning to base levels within 24 h despite the continued presence of hormone. MMTV DNA sequences assembled as chromatin require hormone for binding by nuclear factor 1 (NF1) and octamer proteins (OCT). However, in the same cells, NF1 and OCT factors are bound to transiently introduced DNA in the absence of hormone. In contrast, recruitment of the TATA-binding protein and a novel DNA-binding protein, which we have designated FDT, for factor downstream of the TATA-binding protein, is hormone dependent for both stable and transient templates. Furthermore, transient DNA templates, but not nucleosomal templates, retain these transcription factors over the course of 24 h. This finding suggests that MMTV chromatin structure contributes to activation and cessation of transcription in vivo. Images PMID:8264599

  16. An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Park, Hae-Chul; Lee, Jehee

    2016-03-01

    Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 clade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5'-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in

  17. Single-molecule analysis of the full transcription cycle

    NASA Astrophysics Data System (ADS)

    Strick, Terence

    2005-03-01

    By monitoring the extension of a mechanically stretched, supercoiled DNA molecule containing a single bacterial promoter, we have been able to directly observe in real time the change in DNA extension associated with topological unwinding of ˜1 helical turn of promoter DNA by RNAP during transcription initiation. We find that this stage of transcription initiation is extremely sensitive to the torque acting on the supercoiled DNA. Upon addition of limited sets of nucleotides, changes in the polymerase/promoter interaction which are related to the process of abortive initiation can be studied in detail. Upon addition of the full set of nucleotides, the subsequent stages of transcription -- promoter escape, productive elongation and transcription termination -- can also be observed in real-time. The changes in DNA topology which occur at each of these stages have been determined, and these results provide for the first global view of the entire transcription cycle at the resolution of single molecules. Co-authors: Richard H. Ebright, Chen-Yu Liu and Andrey Revyakin, HHMI & Waksman Institute, Rutgers University.

  18. The fundamental ribosomal RNA transcription initiation factor-IB (TIF-IB, SL1, factor D) binds to the rRNA core promoter primarily by minor groove contacts.

    PubMed

    Geiss, G K; Radebaugh, C A; Paule, M R

    1997-11-14

    Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.

  19. Bacterial ice nucleation: significance and molecular basis.

    PubMed

    Gurian-Sherman, D; Lindow, S E

    1993-11-01

    Several bacterial species are able to catalyze ice formation at temperatures as warm as -2 degrees C. These microorganisms efficiently catalyze ice formation at temperatures much higher than most organic or inorganic substances. Because of their ubiquity on the surfaces of frost-sensitive plants, they are responsible for initiating ice formation, which results in frost injury. The high temperature of ice catalysis conferred by bacterial ice nuclei makes them useful in ice nucleation-limited processes such as artificial snow production, the freezing of some food products, and possibly in future whether modification schemes. The rarity of other ice nuclei active at high subfreezing temperature, and the ease and sensitivity with which ice nuclei can be quantified, have made the use of a promoterless bacterial ice nucleation gene valuable as a reporter of transcription. Target genes to which this promoter is fused can be used in cells in natural habitats. Warm-temperature ice nucleation sites have also been extensively studied at a molecular level. Nucleation sites active at high temperatures (above -5 degrees C) are probably composed of bacterial ice nucleation protein molecules that form functionally aligned aggregates. Models of ice nucleation proteins predict that they form a planar array of hydrogen binding groups that closely complement that of an ice crystal face. Moreover, interdigitation of these molecules may produce a large contiguous template for ice formation.

  20. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

    PubMed Central

    de Vera, M E; Shapiro, R A; Nussler, A K; Mudgett, J S; Simmons, R L; Morris, S M; Billiar, T R; Geller, D A

    1996-01-01

    The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene. Images Fig. 1 Fig. 2 PMID:8577713

  1. Escherichia coli removal in biochar-augmented biofilter: effect of infiltration rate, initial bacterial concentration, biochar particle size, and presence of compost.

    PubMed

    Mohanty, Sanjay K; Boehm, Alexandria B

    2014-10-07

    Bioretention systems and biofilters are used in low impact development to passively treat urban stormwater. However, these engineered natural systems are not efficient at removing fecal indicator bacteria, the contaminants responsible for a majority of surface water impairments. The present study investigates the efficacy of biochar-augmented model sand biofilters for Escherichia coli removal under a variety of stormwater bacterial concentrations and infiltration rates. Additionally, we test the role of biochar particle size and "presence of compost on model" biofilter performance. Our results show that E. coli removal in a biochar-augmented sand biofilter is ∼ 96% and is not greatly affected by increases in stormwater infiltration rates and influent bacterial concentrations, particularly within the ranges expected in field. Removal of fine (<125 μm) biochar particles from the biochar-sand biofilter decreased the removal capacity from 95% to 62%, indicating biochar size is important. Addition of compost to biochar-sand biofilters not only lowered E. coli removal capacity but also increased the mobilization of deposited bacteria during intermittent infiltration. This result is attributed to exhaustion of attachment sites on biochar by the dissolved organic carbon leached from compost. Overall, our study indicates that biochar has potential to remove bacteria from stormwater under a wide range of field conditions, but for biochar to be effective, the size should be small and biochar should be applied without compost. Although the results aid in the optimization of biofilter design, further studies are needed to examine biochar potential in the field over an entire rainy season.

  2. Bacterial Sialidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

  3. Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity.

    PubMed

    Fine, Frédéric; Gervais, Patrick

    2005-01-01

    Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.

  4. RNA polymerase II transcription: structure and mechanism.

    PubMed

    Liu, Xin; Bushnell, David A; Kornberg, Roger D

    2013-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  5. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B

    PubMed Central

    Hart-Smith, Gene; Tay, Ying Jin; Tng, Wei-Quan; Wilkins, Marc; Ryan, Daniel

    2017-01-01

    The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron—exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z. PMID:28234895

  6. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.

    PubMed

    Soboleva, Tatiana A; Parker, Brian J; Nekrasov, Maxim; Hart-Smith, Gene; Tay, Ying Jin; Tng, Wei-Quan; Wilkins, Marc; Ryan, Daniel; Tremethick, David J

    2017-02-01

    The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

  7. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  8. Sequence and expression analysis of two T helper master transcription factors, T-bet and GATA3, in rainbow trout Oncorhynchus mykiss and analysis of their expression during bacterial and parasitic infection.

    PubMed

    Wang, Tiehui; Holland, Jason W; Martin, Sam A M; Secombes, Christopher J

    2010-11-01

    The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71-72%) but low identities to mammalian T-bet genes (41-42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73-88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial (Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-gamma and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression

  9. A global analysis of transcription reveals two modes of Spt4/5 recruitment to archaeal RNA polymerase.

    PubMed

    Smollett, Katherine; Blombach, Fabian; Reichelt, Robert; Thomm, Michael; Werner, Finn

    2017-03-01

    The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase (RNAP) II system, while archaeal genomes are more similar to bacteria with densely packed genes organized in operons. This makes understanding transcription in archaea vital, both in terms of molecular mechanisms and evolution. Very little is known about how archaeal cells orchestrate transcription on a systems level. We have characterized the genome-wide occupancy of the Methanocaldococcus jannaschii transcription machinery and its transcriptome. Our data reveal how the TATA and BRE promoter elements facilitate recruitment of the essential initiation factors TATA-binding protein and transcription factor B, respectively, which in turn are responsible for the loading of RNAP into the transcription units. The occupancies of RNAP and Spt4/5 strongly correlate with each other and with RNA levels. Our results show that Spt4/5 is a general elongation factor in archaea as its presence on all genes matches RNAP. Spt4/5 is recruited proximal to the transcription start site on the majority of transcription units, while on a subset of genes, including rRNA and CRISPR loci, Spt4/5 is recruited to the transcription elongation complex during early elongation within 500 base pairs of the transcription start site and akin to its bacterial homologue NusG.

  10. Tracing bacterial metabolism using multi-nuclear (1H, 2H, and 13C) Solid State NMR: Realizing an Idea Initiated by James Scott

    NASA Astrophysics Data System (ADS)

    Cody, G.; Fogel, M. L.; Jin, K.; Griffen, P.; Steele, A.; Wang, Y.

    2011-12-01

    Approximately 6 years ago, while at the Geophysical Laboratory, James Scott became interested in the application of Solid State Nuclear Magnetic Resonance Spectroscopy to study bacterial metabolism. As often happens, other experiments intervened and the NMR experiments were not pursued. We have revisited Jame's question and find that using a multi-nuclear approach (1H, 2H, and 13C Solid State NMR) on laboratory cell culture has some distinct advantages. Our experiments involved batch cultures of E. coli (MG1655) harvested at stationary phase. In all experiments the growth medium consisted of MOPS medium for enterobacteria, where the substrate is glucose. In one set of experiments, 10 % of the water was D2O; in another 10 % of the glucose was per-deuterated. The control experiment used both water and glucose at natural isotopic abundance. A kill control of dead E. coli immersed in pure D2O for an extended period exhibited no deuterium incorporation. In both deuterium enriched experiments, considerable incorporation of deuterium into E. coli's biomolecular constituents was detected via 2H Solid State NMR. In the case of the D2O enriched experiment, 58 % of the incorporated deuterium is observed in a sharp peak at a frequency of 0.31 ppm, consistent with D incorporation in the cell membrane lipids, the remainder is observed in a broad peak at a higher frequency (centered at 5.4 ppm, but spanning out to beyond 10 ppm) that is consistent with D incorporation into predominantly DNA and RNA. In the case of the D-glucose experiments, 61 % of the deuterium is observed in a sharp resonance peak at 0.34 ppm, also consistent with D incorporation into membrane lipids, the remainder of the D is observed at a broad resonance peak centered at 4.3 ppm, consistent with D enrichment in glycogen. Deuterium abundance in the E. coli cells grown in 10 % D2O is nearly 2X greater than that grown with 10 % D-glucose. Very subtle differences are observed in both the 1H and 13C solid

  11. The role of the lid element in transcription by E. coli RNA polymerase.

    PubMed

    Toulokhonov, Innokenti; Landick, Robert

    2006-08-25

    The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAP's RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing sigma), the lid interacts with sigma region 3 and encloses an extended linker between sigma region 3 and sigma region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Deltalid RNAP exhibited dramatically reduced activity during initiation from -35-dependent and -35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Deltalid RNAP. Importantly, Deltalid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.

  12. Messenger RNA degradation in bacterial cells.

    PubMed

    Hui, Monica P; Foley, Patricia L; Belasco, Joel G

    2014-01-01

    mRNA degradation is an important mechanism for controlling gene expression in bacterial cells. This process involves the orderly action of a battery of cellular endonucleases and exonucleases, some universal and others present only in certain species. These ribonucleases function with the assistance of ancillary enzymes that covalently modify the 5' or 3' end of RNA or unwind base-paired regions. Triggered by initiating events at either the 5' terminus or an internal site, mRNA decay occurs at diverse rates that are transcript specific and governed by RNA sequence and structure, translating ribosomes, and bound sRNAs or proteins. In response to environmental cues, bacteria are able to orchestrate widespread changes in mRNA lifetimes by modulating the concentration or specific activity of cellular ribonucleases or by unmasking the mRNA-degrading activity of cellular toxins.

  13. Pervasive transcription: detecting functional RNAs in bacteria.

    PubMed

    Lybecker, Meghan; Bilusic, Ivana; Raghavan, Rahul

    2014-01-01

    Pervasive, or genome-wide, transcription has been reported in all domains of life. In bacteria, most pervasive transcription occurs antisense to protein-coding transcripts, although recently a new class of pervasive RNAs was identified that originates from within annotated genes. Initially considered to be non-functional transcriptional noise, pervasive transcription is increasingly being recognized as important in regulating gene expression. The function of pervasive transcription is an extensively debated question in the field of transcriptomics and regulatory RNA biology. Here, we highlight the most recent contributions addressing the purpose of pervasive transcription in bacteria and discuss their implications.

  14. A unified model for yeast transcript definition.

    PubMed

    de Boer, Carl G; van Bakel, Harm; Tsui, Kyle; Li, Joyce; Morris, Quaid D; Nislow, Corey; Greenblatt, Jack F; Hughes, Timothy R

    2014-01-01

    Identifying genes in the genomic context is central to a cell's ability to interpret the genome. Yet, in general, the signals used to define eukaryotic genes are poorly described. Here, we derived simple classifiers that identify where transcription will initiate and terminate using nucleic acid sequence features detectable by the yeast cell, which we integrate into a Unified Model (UM) that models transcription as a whole. The cis-elements that denote where transcription initiates function primarily through nucleosome depletion, and, using a synthetic promoter system, we show that most of these elements are sufficient to initiate transcription in vivo. Hrp1 binding sites are the major characteristic of terminators; these binding sites are often clustered in terminator regions and can terminate transcription bidirectionally. The UM predicts global transcript structure by modeling transcription of the genome using a hidden Markov model whose emissions are the outputs of the initiation and termination classifiers. We validated the novel predictions of the UM with available RNA-seq data and tested it further by directly comparing the transcript structure predicted by the model to the transcription generated by the cell for synthetic DNA segments of random design. We show that the UM identifies transcription start sites more accurately than the initiation classifier alone, indicating that the relative arrangement of promoter and terminator elements influences their function. Our model presents a concrete description of how the cell defines transcript units, explains the existence of nongenic transcripts, and provides insight into genome evolution.

  15. A unified model for yeast transcript definition

    PubMed Central

    de Boer, Carl G.; van Bakel, Harm; Tsui, Kyle; Li, Joyce; Morris, Quaid D.; Nislow, Corey; Greenblatt, Jack F.; Hughes, Timothy R.

    2014-01-01

    Identifying genes in the genomic context is central to a cell's ability to interpret the genome. Yet, in general, the signals used to define eukaryotic genes are poorly described. Here, we derived simple classifiers that identify where transcription will initiate and terminate using nucleic acid sequence features detectable by the yeast cell, which we integrate into a Unified Model (UM) that models transcription as a whole. The cis-elements that denote where transcription initiates function primarily through nucleosome depletion, and, using a synthetic promoter system, we show that most of these elements are sufficient to initiate transcription in vivo. Hrp1 binding sites are the major characteristic of terminators; these binding sites are often clustered in terminator regions and can terminate transcription bidirectionally. The UM predicts global transcript structure by modeling transcription of the genome using a hidden Markov model whose emissions are the outputs of the initiation and termination classifiers. We validated the novel predictions of the UM with available RNA-seq data and tested it further by directly comparing the transcript structure predicted by the model to the transcription generated by the cell for synthetic DNA segments of random design. We show that the UM identifies transcription start sites more accurately than the initiation classifier alone, indicating that the relative arrangement of promoter and terminator elements influences their function. Our model presents a concrete description of how the cell defines transcript units, explains the existence of nongenic transcripts, and provides insight into genome evolution. PMID:24170600

  16. Bacterial Tracheitis

    MedlinePlus

    ... as a complication of croup (see Croup ) or endotracheal intubation (insertion of a plastic breathing tube through the ... irregularities that distinguish bacterial tracheitis from croup. Treatment Endotracheal intubation Antibiotics With treatment, most children recover completely. Very ...

  17. On schemes of combinatorial transcription logic.

    PubMed

    Buchler, Nicolas E; Gerland, Ulrich; Hwa, Terence

    2003-04-29

    Cells receive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate specific genetic responses. Here we explore theoretically the potentials and limitations of combinatorial signal integration at the level of cis-regulatory transcription control. Our analysis suggests that many complex transcription-control functions of the type encountered in higher eukaryotes are already implementable within the much simpler bacterial transcription system. Using a quantitative model of bacterial transcription and invoking only specific protein-DNA interaction and weak glue-like interaction between regulatory proteins, we show explicit schemes to implement regulatory logic functions of increasing complexity by appropriately selecting the strengths and arranging the relative positions of the relevant protein-binding DNA sequences in the cis-regulatory region. The architectures that emerge are naturally modular and evolvable. Our results suggest that the transcription regulatory apparatus is a "programmable" computing machine, belonging formally to the class of Boltzmann machines. Crucial to our results is the ability to regulate gene expression at a distance. In bacteria, this can be achieved for isolated genes via DNA looping controlled by the dimerization of DNA-bound proteins. However, if adopted extensively in the genome, long-distance interaction can cause unintentional intergenic cross talk, a detrimental side effect difficult to overcome by the known bacterial transcription-regulation systems. This may be a key factor limiting the genome-wide adoption of complex transcription control in bacteria. Implications of our findings for combinatorial transcription control in eukaryotes are discussed.

  18. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    PubMed Central

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  19. TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection

    PubMed Central

    Cavalla, Franco; Biguetti, Claudia Cristina; Colavite, Priscila Maria; Silveira, Elcia Varise; Martins, Walter; Letra, Ariadne; Trombone, Ana Paula Favaro; Silva, Renato Menezes; Garlet, Gustavo Pompermaier

    2015-01-01

    Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis. PMID:25832120

  20. Acute intermittent porphyria: identification and expression of exonic mutations in the hydroxymethylbilane synthase gene. An initiation codon missense mutation in the housekeeping transcript causes "variant acute intermittent porphyria" with normal expression of the erythroid-specific enzyme.

    PubMed Central

    Chen, C H; Astrin, K H; Lee, G; Anderson, K E; Desnick, R J

    1994-01-01

    Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme, hydroxymethylbilane synthase (EC 4.3.1.8). Diagnosis of AIP heterozygotes is essential to prevent acute, life-threatening neurologic attacks by avoiding various precipitating factors. Since biochemical diagnosis is problematic, the identification of hydroxymethylbilane synthase mutations has facilitated the detection of AIP heterozygotes. Molecular analyses of unrelated AIP patients revealed six exonic mutations: an initiating methionine to isoleucine substitution (M1I) in a patient with variant AIP, which precluded translation of the housekeeping, but not the erythroid-specific isozyme; four missense mutations in classical AIP patients, V93F, R116W, R201W, C247F; and a nonsense mutation W283X in a classical AIP patient, which truncated the housekeeping and erythroid-specific isozymes. Each mutation was confirmed in genomic DNA from family members. The W283X lesion was found in another unrelated AIP family. Expression of each mutation in Escherichia coli revealed that R201W, C247F, and W283X had residual activity. In vitro transcription/translation studies indicated that the M1I allele produced only the erythroid-specific enzyme, while the other mutant alleles encoded both isozymes. These mutations provide insight into the molecular pathology of classic and variant AIP and facilitate molecular diagnosis in AIP families. Images PMID:7962538

  1. Genomic localization of the human genes TAF1A, TAF1B and TAF1C, encoding TAF(I)48, TAF(I)63 and TAF(I)110 subunits of class I general transcription initiation factor SL1.

    PubMed

    Di Pietro, C; Rapisarda, A; Amico, V; Bonaiuto, C; Viola, A; Scalia, M; Motta, S; Amato, A; Engel, H; Messina, A; Sichel, G; Grzeschik, K; Purrello, M

    2000-01-01

    Human SL1 is a general transcription initiation factor (GTF) essential for RNA polymerase I to start rRNA synthesis at class I promoters. It is comprised of the TATA box-binding protein (TBP) and three TBP-associated factors (TAF(I)48, TAF(I)63 and TAF(I)110). We have determined that the human genes TAF1A, TAF1B and TAF1C, encoding these three TAF(I) polypeptides, are localized at lq42, 2p25 and 16q24, respectively. All three genes are present as single copies in the human genome and map to different chromosomes, as shown by somatic cell hybrid panel and radiation hybrid panel analysis and FISH. Two of these genes, TAF1C and TAF1B, are transcribed into multiple RNAs, as determined through Northern analysis of mRNA from various human organs and cell lines. If translated into different polypeptides, this could result in production of variant isoforms of SL1 with different activation potentials.

  2. pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

    PubMed

    Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

    2009-11-23

    Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

  3. Bacterial rheotaxis.

    PubMed

    Marcos; Fu, Henry C; Powers, Thomas R; Stocker, Roman

    2012-03-27

    The motility of organisms is often directed in response to environmental stimuli. Rheotaxis is the directed movement resulting from fluid velocity gradients, long studied in fish, aquatic invertebrates, and spermatozoa. Using carefully controlled microfluidic flows, we show that rheotaxis also occurs in bacteria. Excellent quantitative agreement between experiments with Bacillus subtilis and a mathematical model reveals that bacterial rheotaxis is a purely physical phenomenon, in contrast to fish rheotaxis but in the same way as sperm rheotaxis. This previously unrecognized bacterial taxis results from a subtle interplay between velocity gradients and the helical shape of flagella, which together generate a torque that alters a bacterium's swimming direction. Because this torque is independent of the presence of a nearby surface, bacterial rheotaxis is not limited to the immediate neighborhood of liquid-solid interfaces, but also takes place in the bulk fluid. We predict that rheotaxis occurs in a wide range of bacterial habitats, from the natural environment to the human body, and can interfere with chemotaxis, suggesting that the fitness benefit conferred by bacterial motility may be sharply reduced in some hydrodynamic conditions.

  4. Disease notes - Bacterial root rot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial root rot initiated by lactic acid bacteria, particularly Leuconostoc, occurs every year in Idaho sugarbeet fields. Hot fall weather seems to make the problem worse. Although Leuconostoc initiates the rot, other bacteria and yeast frequently invade the tissue as well. The acetic acid bac...

  5. Bacterial Chromosome Organization and Segregation

    PubMed Central

    Toro, Esteban; Shapiro, Lucy

    2010-01-01

    Bacterial chromosomes are generally ∼1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. Such organization is transmitted from one generation to the next by progressive segregation of daughter chromosomes and anchoring of DNA to the cell envelope. Active segregation by a mitotic machinery appears to be common; however, the mode of chromosome segregation varies significantly from species to species. PMID:20182613

  6. The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate.

    PubMed Central

    Hryniewicz, M M; Kredich, N M

    1991-01-01

    The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine, N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind

  7. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death.

    PubMed

    Botella, Laure; Vaubourgeix, Julien; Livny, Jonathan; Schnappinger, Dirk

    2017-03-28

    Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3' ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase.

  8. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death

    PubMed Central

    Botella, Laure; Vaubourgeix, Julien; Livny, Jonathan; Schnappinger, Dirk

    2017-01-01

    Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3′ ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase. PMID:28348398

  9. Making temporal maps using bacterial luciferase: Bacteriophage

    NASA Astrophysics Data System (ADS)

    Kuhn, Jonathan; Broza, Rachel; Verkin, Ekaterina

    2004-06-01

    A method for making temporal maps in bacteria, plasmids and bacteriophages is described. A cassette containing both the genes for bacterial luciferase and kanamycin resistance can be introduced at precise sites. The technique involves clonging followed by genetic recombination. The result is formation of structures that have the luciferase genes in place of the normal DNA and this allows the very precise measurement of transcription/translation of the substituted regions. Very low levels of transcription as well as the kinetics of induction can be easily ascertained. As a specific demonstration of this general method, the technique was used with bacteriophage λ, one of the best known organisms. By measuring light emission, the expression of luciferase was followed after induction for both early and late genes. The exact timing of initial expression of genes was also determined by sampling at very short intervals. The results show that the early genes express almost without delay implying that the function of the N antitermination system is not temporal regulation.

  10. The Role of Multiple Transcription Factors In Archaeal Gene Expression

    SciTech Connect

    Charles J. Daniels

    2008-09-23

    Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcanii was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of specific tfb

  11. Monitoring Abortive Initiation

    PubMed Central

    Hsu, Lilian M.

    2009-01-01

    Abortive initiation, when first discovered, was an enigmatic phenomenon, but fully three decades hence, it has been shown to be an integral step in the transcript initiation process intimately tied to the promoter escape reaction undergone by RNA polymerase at the initiation-elongation transition. A detailed understanding of abortive initiation-promoter escape has brought within reach a full description of the transcription initiation mechanism. This enormous progress was the result of convergent biochemical, genetic, and biophysical investigations propelled by parallel advances in quantitation technology. This chapter discusses the knowledge gained through the biochemical approach and a high-resolution method that yields quantitative and qualitative information regarding abortive initiation-promoter escape at a promoter. PMID:18948204

  12. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  13. Transcription of mitochondrial DNA.

    PubMed

    Tabak, H F; Grivell, L A; Borst, P

    1983-01-01

    While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.

  14. Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

    PubMed

    Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

    2016-01-15

    Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator.

  15. Transcription of Trypanosoma brucei maxicircles

    SciTech Connect

    Michelotti, E.F.; Hajduk, S.L.

    1987-05-01

    Trypanosoma brucei is a protozoan parasite which developmentally regulates mitochondrial activity. In the mammal T. brucei produces ATP entirely by glycolysis while cytochrome mediated respiration resumes in the life-stage in the midgut of the insect vector. Using quantitative S1 nuclease protection assays two types of regulation of the steady state levels of the mitochondrial transcripts were found. Transcription of cytochrome b, cytochrome oxidase, and the rRNA genes is repressed in early bloodstream developmental stages, undergoes dramatic activation in later bloodstream stages, and finally a lesser activation in the insect developmental stage. Transcription of NADH dehydrogenase genes, however, is unregulated. Mitochondrial transcripts with a 5' triphosphate terminus, representing the site of transcription initiation, were capped using guanylyl transferase. The in vitro capped RNA hybridized to only one of eight mitochondrial restriction fragments on a Southern blot, however, hybridization of Southern blots with RNA from ..cap alpha..-/sup 32/P-UTP pulsed mitochondria labelled all restriction fragments equally. These results suggest that each DNA strand has a single promoter which directs the transcription of a full-length RNA which is subsequently processed. Different mitochondrial genes, despite being expressed on the same precursor RNA molecule, are independently regulated by both transcription initiation and RNA processing.

  16. Bacterial Sigma Factors and Anti-Sigma Factors: Structure, Function and Distribution.

    PubMed

    Paget, Mark S

    2015-06-26

    Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoters as well as the initial steps in RNA synthesis. This review focuses on the structure and function of the major sigma-70 class that includes the housekeeping sigma factor (Group 1) that directs the bulk of transcription during active growth, and structurally-related alternative sigma factors (Groups 2-4) that control a wide variety of adaptive responses such as morphological development and the management of stress. A recurring theme in sigma factor control is their sequestration by anti-sigma factors that occlude their RNAP-binding determinants. Sigma factors are then released through a wide variety of mechanisms, often involving branched signal transduction pathways that allow the integration of distinct signals. Three major strategies for sigma release are discussed: regulated proteolysis, partner-switching, and direct sensing by the anti-sigma factor.

  17. Evolutionary aspects of plastid proteins involved in transcription: the transcription of a tiny genome is mediated by a complicated machinery.

    PubMed

    Yagi, Yusuke; Shiina, Takashi

    2012-01-01

    Chloroplasts in land plants have a small genome consisting of only 100 genes encoding partial sets of proteins for photosynthesis, transcription and translation. Although it has been thought that chloroplast transcription is mediated by a basically cyanobacterium-derived system, due to the endosymbiotic origin of plastids, recent studies suggest the existence of a hybrid transcription machinery containing non-bacterial proteins that have been newly acquired during plant evolution. Here, we highlight chloroplast-specific non-bacterial transcription mechanisms by which land plant chloroplasts have gained novel functions.

  18. Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

    PubMed

    Palmer, Adam C; Egan, J Barry; Shearwin, Keith E

    2011-01-01

    Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

  19. GTPases involved in bacterial ribosome maturation.

    PubMed

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  20. Epigenetics and bacterial infections.

    PubMed

    Bierne, Hélène; Hamon, Mélanie; Cossart, Pascale

    2012-12-01

    Epigenetic mechanisms regulate expression of the genome to generate various cell types during development or orchestrate cellular responses to external stimuli. Recent studies highlight that bacteria can affect the chromatin structure and transcriptional program of host cells by influencing diverse epigenetic factors (i.e., histone modifications, DNA methylation, chromatin-associated complexes, noncoding RNAs, and RNA splicing factors). In this article, we first review the molecular bases of the epigenetic language and then describe the current state of research regarding how bacteria can alter epigenetic marks and machineries. Bacterial-induced epigenetic deregulations may affect host cell function either to promote host defense or to allow pathogen persistence. Thus, pathogenic bacteria can be considered as potential epimutagens able to reshape the epigenome. Their effects might generate specific, long-lasting imprints on host cells, leading to a memory of infection that influences immunity and might be at the origin of unexplained diseases.

  1. Bacterial population autowave patterns: spontaneous symmetry bursting

    NASA Astrophysics Data System (ADS)

    Medvinsky, A. B.; Tsyganov, M. A.; Karpov, V. A.; Kresteva, I. B.; Shakhbazian, V. Yu.; Ivanitsky, G. R.

    1994-12-01

    Bacteria are known to form autowave patterns (population waves) like those formed by propagating nerve impulses, phase transitions, concentration waves in the Belousov-Zhabotinsky reaction, etc. The formation of bacterial waves is due to the ability of bacteria to drift (through chemotaxis) into the regions with higher attractant concentration. As a result, in contrast to other types of autowaves, bacterial population waves have not only a diffusion component of a bacterial flow but a chemotaxis flow as well. We present the experimental results of the study of spontaneous symmetry loss of bacterial autowave patterns. We show that this phenomenon can be simulated with a simple cellular automata model, and symmetry bursting depends on the parameters characterizing chemotactic sensitivity and motility of the cells forming the population wave. In the experiments in vivo we show that the distortion of a bacterial wave shape can be initiated by bacterial density fluctuations in the parent population that the bacterial waves flake off from.

  2. Bacterial biofilms: prokaryotic adventures in multicellularity.

    PubMed

    Webb, Jeremy S; Givskov, Michael; Kjelleberg, Staffan

    2003-12-01

    The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity.

  3. Effects of heavy fuel oil on the bacterial community structure of a pristine microbial mat.

    PubMed

    Bordenave, Sylvain; Goñi-Urriza, María Soledad; Caumette, Pierre; Duran, Robert

    2007-10-01

    The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by "Bacilli" and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution.

  4. Bacterial Hydrodynamics

    NASA Astrophysics Data System (ADS)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  5. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    PubMed

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.

  6. Collapsing bacterial cylinders

    NASA Astrophysics Data System (ADS)

    Betterton, M. D.; Brenner, Michael P.

    2001-12-01

    Under special conditions bacteria excrete an attractant and aggregate. The high density regions initially collapse into cylindrical structures, which subsequently destabilize and break up into spherical aggregates. This paper presents a theoretical description of the process, from the structure of the collapsing cylinder to the spacing of the final aggregates. We show that cylindrical collapse involves a delicate balance in which bacterial attraction and diffusion nearly cancel, leading to corrections to the collapse laws expected from dimensional analysis. The instability of a collapsing cylinder is composed of two distinct stages: Initially, slow modulations to the cylinder develop, which correspond to a variation of the collapse time along the cylinder axis. Ultimately, one point on the cylinder pinches off. At this final stage of the instability, a front propagates from the pinch into the remainder of the cylinder. The spacing of the resulting spherical aggregates is determined by the front propagation.

  7. Exploiting Transcriptions of Identical Subject Content Lessons

    ERIC Educational Resources Information Center

    Harfitt, Gary James

    2008-01-01

    This article describes a strategy employed on a teacher training course in Hong Kong involving the use of lesson transcriptions. Transcriptions from two course participants' English lessons were used to arouse greater classroom language awareness and promote reflection in one of the teachers, who was initially very reluctant to accept comments and…

  8. Mercury Detoxification by Bacteria: Simulations of Transcription Activation and Mercury-Carbon Bond Cleavage

    SciTech Connect

    Guo, Hao-Bo; Parks, Jerry M; Johs, Alexander; Smith, Jeremy C

    2011-01-01

    In this chapter, we summarize recent work from our laboratory and provide new perspective on two important aspects of bacterial mercury resistance: the molecular mechanism of transcriptional regulation by MerR, and the enzymatic cleavage of the Hg-C bond in methylmercury by the organomercurial lyase, MerB. Molecular dynamics (MD) simulations of MerR reveal an opening-and-closing dynamics, which may be involved in initiating transcription of mercury resistance genes upon Hg(II) binding. Density functional theory (DFT) calculations on an active-site model of the enzyme reveal how MerB catalyzes the Hg-C bond cleavage using cysteine coordination and acid-base chemistry. These studies provide insight into the detailed mechanisms of microbial gene regulation and defense against mercury toxicity.

  9. Bacterial vaginosis.

    PubMed Central

    Spiegel, C A

    1991-01-01

    Bacterial vaginosis (BV) is the most common of the vaginitides affecting women of reproductive age. It appears to be due to an alteration in the vaginal ecology by which Lactobacillus spp., the predominant organisms in the healthy vagina, are replaced by a mixed flora including Prevotella bivia, Prevotella disiens, Porphyromonas spp., Mobiluncus spp., and Peptostreptococcus spp. All of these organisms except Mobiluncus spp. are also members of the endogenous vaginal flora. While evidence from treatment trials does not support the notion that BV is sexually transmitted, recent studies have shown an increased risk associated with multiple sexual partners. It has also been suggested that the pathogenesis of BV may be similar to that of urinary tract infections, with the rectum serving as a reservoir for some BV-associated flora. The organisms associated with BV have also been recognized as agents of female upper genital tract infection, including pelvic inflammatory disease, and the syndrome BV has been associated with adverse outcome of pregnancy, including premature rupture of membranes, chorioamnionitis, and fetal loss; postpartum endometritis; cuff cellulitis; and urinary tract infections. The mechanisms by which the BV-associated flora causes the signs of BV are not well understood, but a role for H2O2-producing Lactobacillus spp. in protecting against colonization by catalase-negative anaerobic bacteria has been recognized. These and other aspects of BV are reviewed. PMID:1747864

  10. Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals

    PubMed Central

    Stokes, Bethany A.; Yadav, Shruti; Shokal, Upasana; Smith, L. C.; Eleftherianos, Ioannis

    2015-01-01

    In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies. PMID:25674081

  11. Small bowel bacterial overgrowth

    MedlinePlus

    Overgrowth - intestinal bacteria; Bacterial overgrowth - intestine; Small intestinal bacterial overgrowth; SIBO ... intestine does not have a high number of bacteria. Excess bacteria in the small intestine may use ...

  12. Structural characterization of human general transcription factor TFIIF in solution

    PubMed Central

    Akashi, Satoko; Nagakura, Shinjiro; Yamamoto, Seiji; Okuda, Masahiko; Ohkuma, Yoshiaki; Nishimura, Yoshifumi

    2008-01-01

    Human general transcription factor IIF (TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of α and β subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size >200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an αβ heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy (EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human α and β subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC. PMID:18218714

  13. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

  14. Changes in rhizosphere bacterial gene expression following glyphosate treatment.

    PubMed

    Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

    2016-05-15

    In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria.

  15. Theoretical analysis of transcription process with polymerase stalling

    NASA Astrophysics Data System (ADS)

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  16. Theoretical analysis of transcription process with polymerase stalling.

    PubMed

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  17. Identification of a Transcription Factor That Regulates Host Cell Exit and Virulence of Mycobacterium tuberculosis

    PubMed Central

    Srinivasan, Lalitha; Gurses, Serdar A.; Hurley, Benjamin E.; Miller, Jessica L.; Karakousis, Petros C.; Briken, Volker

    2016-01-01

    The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb. PMID:27191591

  18. Full transcription of the chloroplast genome in photosynthetic eukaryotes

    PubMed Central

    Shi, Chao; Wang, Shuo; Xia, En-Hua; Jiang, Jian-Jun; Zeng, Fan-Chun; Gao, Li-Zhi

    2016-01-01

    Prokaryotes possess a simple genome transcription system that is different from that of eukaryotes. In chloroplasts (plastids), it is believed that the prokaryotic gene transcription features govern genome transcription. However, the polycistronic operon transcription model cannot account for all the chloroplast genome (plastome) transcription products at whole-genome level, especially regarding various RNA isoforms. By systematically analyzing transcriptomes of plastids of algae and higher plants, and cyanobacteria, we find that the entire plastome is transcribed in photosynthetic green plants, and that this pattern originated from prokaryotic cyanobacteria — ancestor of the chloroplast genomes that diverged about 1 billion years ago. We propose a multiple arrangement transcription model that multiple transcription initiations and terminations combine haphazardly to accomplish the genome transcription followed by subsequent RNA processing events, which explains the full chloroplast genome transcription phenomenon and numerous functional and/or aberrant pre-RNAs. Our findings indicate a complex prokaryotic genome regulation when processing primary transcripts. PMID:27456469

  19. Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

    PubMed Central

    Turner, Matthew L.; Cronin, James G.; Healey, Gareth D.

    2014-01-01

    Bacteria often infect the endometrium of cattle to cause endometritis, uterine disease, and infertility. Lipopeptides are commonly found among bacteria and are detected by the Toll-like receptor (TLR) cell surface receptor TLR2 on immune cells. Heterodimers of TLR2 with TLR1 or TLR6 activate MAPK and nuclear factor-κB intracellular signaling pathways to stimulate inflammatory responses. In the endometrium, epithelial and stromal cells are the first to encounter invading bacteria, so the present study explored whether endometrial cells can also mount inflammatory responses to bacterial lipopeptides via TLRs. The supernatants of pure populations of primary bovine endometrial epithelial and stromal cells accumulated the cytokine IL-6 and the chemokine IL-8 in response to triacylated or diacylated bacterial lipopeptides. The accumulation of IL-6 and IL-8 in response to triacylated lipopeptides was reduced by small interfering RNA targeting TLR2 or TLR1 but not TLR6, whereas cellular responses to diacylated lipopeptide were reduced by small interfering RNA targeting TLR2, TLR1, or TLR6. Both lipopeptides induced rapid phosphorylation of ERK1/2, p38, and nuclear factor-κB in endometrial cells, and inhibitors of ERK1/2 or p38 limited the accumulation of IL-6. The ovarian steroids estradiol and progesterone had little impact on inflammatory responses to lipopeptides. The endometrial epithelial and stromal cell responses to lipopeptides via TLR2, TLR1, and TLR6 provide a mechanism linking a wide range of bacterial infections to inflammation of the endometrium. PMID:24437488

  20. Epithelial and stromal cells of bovine endometrium have roles in innate immunity and initiate inflammatory responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2, TLR1, and TLR6.

    PubMed

    Turner, Matthew L; Cronin, James G; Healey, Gareth D; Sheldon, Iain Martin

    2014-04-01

    Bacteria often infect the endometrium of cattle to cause endometritis, uterine disease, and infertility. Lipopeptides are commonly found among bacteria and are detected by the Toll-like receptor (TLR) cell surface receptor TLR2 on immune cells. Heterodimers of TLR2 with TLR1 or TLR6 activate MAPK and nuclear factor-κB intracellular signaling pathways to stimulate inflammatory responses. In the endometrium, epithelial and stromal cells are the first to encounter invading bacteria, so the present study explored whether endometrial cells can also mount inflammatory responses to bacterial lipopeptides via TLRs. The supernatants of pure populations of primary bovine endometrial epithelial and stromal cells accumulated the cytokine IL-6 and the chemokine IL-8 in response to triacylated or diacylated bacterial lipopeptides. The accumulation of IL-6 and IL-8 in response to triacylated lipopeptides was reduced by small interfering RNA targeting TLR2 or TLR1 but not TLR6, whereas cellular responses to diacylated lipopeptide were reduced by small interfering RNA targeting TLR2, TLR1, or TLR6. Both lipopeptides induced rapid phosphorylation of ERK1/2, p38, and nuclear factor-κB in endometrial cells, and inhibitors of ERK1/2 or p38 limited the accumulation of IL-6. The ovarian steroids estradiol and progesterone had little impact on inflammatory responses to lipopeptides. The endometrial epithelial and stromal cell responses to lipopeptides via TLR2, TLR1, and TLR6 provide a mechanism linking a wide range of bacterial infections to inflammation of the endometrium.

  1. Responses of Active Bacterial and Fungal Communities in Soils under Winter Wheat to Different Fertilizer and Pesticide Regimens

    PubMed Central

    Girvan, Martina S.; Bullimore, Juliet; Ball, Andrew S.; Pretty, Jules N.; Osborn, A. Mark

    2004-01-01

    The composition of the active microbial (bacterial and fungal) soil community in an arable wheat field subjected to different management practices was examined at five times during a 1-year period. Field sections were fertilized either at good agricultural practice (GAP) levels or at reduced levels (0.5× GAP) and were inoculated with vesicular arbuscular mycorrhizae (VAM) at the same time. Field subsections were treated either with or without pesticides. Changes in the active microbial communities were investigated by denaturing gradient gel electrophoresis analysis of reverse transcription-PCR-amplified 16S and 18S rRNA. Microbial community structure was primarily determined by season, and the seasonal trends were similar for the fungal and bacterial components. Between-sample microbial heterogeneity decreased under a mature crop in the summer but increased following harvesting and plowing. Although similar overall trends were seen for the two microbial components, sample variability was greater for the fungal community than for the bacterial community. The greatest management effects were due to GAP fertilization, which caused increases in the bacterial numbers in the total and culturable communities. Microbial biomass similarly increased. GAP fertilization also caused large shifts in both the active bacterial community structure and the active fungal community structure and additionally resulted in a decrease in the heterogeneity of the active bacterial community. Pesticide addition did not significantly affect bacterial numbers or heterogeneity, but it led to major shifts in the active soil bacterial community structure. PCR primers specific for Glomales 25S rRNA genes were used to monitor the VAM population following inoculation. Glomales were detected initially only in VAM-inoculated field sections but were subsequently detected in noninoculated field sections as the season progressed. After plowing, the level of Glomales was reduced in noninoculated field

  2. Mechanism of Bacterial Oligosaccharyltransferase

    PubMed Central

    Gerber, Sabina; Lizak, Christian; Michaud, Gaëlle; Bucher, Monika; Darbre, Tamis; Aebi, Markus; Reymond, Jean-Louis; Locher, Kaspar P.

    2013-01-01

    N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation. PMID:23382388

  3. Genetic interactions with C-terminal domain (CTD) kinases and the CTD of RNA Pol II suggest a role for ESS1 in transcription initiation and elongation in Saccharomyces cerevisiae.

    PubMed Central

    Wilcox, Cathy B; Rossettini, Anne; Hanes, Steven D

    2004-01-01

    Ess1 is an essential prolyl isomerase that binds the C-terminal domain (CTD) of Rpb1, the large subunit of RNA polymerase II. Ess1 is proposed to control transcription by isomerizing phospho-Ser-Pro peptide bonds within the CTD repeat. To determine which step(s) in the transcription cycle might require Ess1, we examined genetic interactions between ESS1 and genes encoding the known CTD kinases (KIN28, CTK1, BUR1, and SRB10). Although genetic interactions were identified between ESS1 and all four kinases, the clearest interactions were with CTK1 and SRB10. Reduced dosage of CTK1 rescued the growth defect of ess1(ts) mutants, while overexpression of CTK1 enhanced the growth defects of ess1(ts) mutants. Deletion of SRB10 suppressed ess1(ts) and ess1Delta mutants. The interactions suggest that Ess1 opposes the functions of these kinases, which are thought to function in preinitiation and elongation. Using a series of CTD substitution alleles, we also identified Ser5-Pro6 as a potential target for Ess1 isomerization within the first "half" of the CTD repeats. On the basis of the results, we suggest a model in which Ess1-directed conformational changes promote dephosphorylation of Ser5 to stimulate preinitiation complex formation and, later, to inhibit elongation. PMID:15166139

  4. The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation

    PubMed Central

    Shang, Yingli; Coppo, Maddalena; He, Teng; Ning, Fei; Yu, Li; Kang, Lan; Zhang, Bin; Ju, Chanyang; Qiao, Yu; Zhao, Baohong; Gessler, Manfred; Rogatsky, Inez; Hu, Xiaoyu

    2016-01-01

    Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription initiation steps and evidence for regulation of inflammatory gene expression post initiation remains scarce. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses production of CXCL1, a chemokine crucial for recruiting neutrophils. Hes1 negatively regulates neutrophil recruitment in vivo in a manner that is dependent on macrophage-produced CXCL1 and attenuates severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 does not involve modification of transcription initiation. Instead, Hes1 inhibits signal-induced recruitment of positive transcription elongation complex P-TEFb, thereby preventing phosphorylation of RNA polymerase II on serine-2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses which exerts its suppressive function by regulating transcription elongation. PMID:27322654

  5. Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference.

    PubMed

    Ard, Ryan; Allshire, Robin C

    2016-12-15

    Long non-coding RNA (lncRNA) transcription into a downstream promoter frequently results in transcriptional interference. However, the mechanism of this repression is not fully understood. We recently showed that drug tolerance in fission yeast Schizosaccharomyces pombe is controlled by lncRNA transcription upstream of the tgp1(+) permease gene. Here we demonstrate that transcriptional interference of tgp1(+) involves several transcription-coupled chromatin changes mediated by conserved elongation factors Set2, Clr6CII, Spt6 and FACT. These factors are known to travel with RNAPII and establish repressive chromatin in order to limit aberrant transcription initiation from cryptic promoters present in gene bodies. We therefore conclude that conserved RNAPII-associated mechanisms exist to both suppress intragenic cryptic promoters during genic transcription and to repress gene promoters by transcriptional interference. Our analyses also demonstrate that key mechanistic features of transcriptional interference are shared between S. pombe and the highly divergent budding yeast Saccharomyces cerevisiae Thus, transcriptional interference is an ancient, conserved mechanism for tightly controlling gene expression. Our mechanistic insights allowed us to predict and validate a second example of transcriptional interference involving the S. pombe pho1(+) gene. Given that eukaryotic genomes are pervasively transcribed, transcriptional interference likely represents a more general feature of gene regulation than is currently appreciated.

  6. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  7. Quantitative characterization of gene regulation by Rho dependent transcription termination.

    PubMed

    Hussein, Razika; Lee, Tiffany Y; Lim, Han N

    2015-08-01

    Rho factor dependent transcription termination (RTT) is common within the coding sequences of bacterial genes and it acts to couple transcription and translation levels. Despite the importance of RTT for gene regulation, its effects on mRNA and protein concentrations have not been quantitatively characterized. Here we demonstrate that the exogenous cfp gene encoding the cyan fluorescent protein can serve as a model for gene regulation by RTT. This was confirmed by showing that Psu and bicyclomycin decrease RTT and increase full length cfp mRNAs (but remarkably they have little effect on protein production). We then use cfp to characterize the relationship between its protein and full length mRNA concentrations when the translation initiation rate is varied by sequence modifications of the translation initiation region (TIR). These experiments reveal that the fold change in protein concentration (RP) and the fold change in full length mRNA concentration (Rm) have the relationship RP≈Rm(b), where b is a constant. The average value of b was determined from three separate data sets to be ~3.6. We demonstrate that the above power law function can predict how altering the translation initiation rate of a gene in an operon will affect the mRNA concentrations of downstream genes and specify a lower bound for the associated changes in protein concentrations. In summary, this study defines a simple phenomenological model to help program expression from single genes and operons that are regulated by RTT, and to guide molecular models of RTT.

  8. miR-15a/16 regulates macrophage phagocytosis after bacterial infection.

    PubMed

    Moon, Hyung-Geun; Yang, Jincheng; Zheng, Yijie; Jin, Yang

    2014-11-01

    Bacterial infection and its associated sepsis are devastating clinical entities that lead to high mortality and morbidity in critically ill patients. Phagocytosis, along with other innate immune responses, exerts crucial impacts on the outcomes of these patients. MicroRNAs (miRNAs) are a novel class of regulatory noncoding RNAs that target specific mRNAs for modulation of translation and expression of a targeted protein. The roles of miRNAs in host defense against bacterial sepsis remain unclear. We found that bacterial infections and/or bacterial-derived LPS enhanced the level of miR-15a/16 in bone marrow-derived macrophages (BMDMs). Deletion of miR-15a/16 (miR-15a/16(-/-)) in myeloid cells significantly decreased the bacterial infection-associated mortality in sepsis mouse models. Moreover, miR-15a/16 deficiency (miR-15a/16(-/-)) resulted in augmented phagocytosis and generation of mitochondrial reactive oxygen species in BMDMs. Supportively, overexpression of miR-15a/16 using miRNA mimics led to decreased phagocytosis and decreased generation of mitochondrial reactive oxygen species. Mechanistically, deletion of miR-15a/16 upregulated the expression of TLR4 via targeting the principle transcriptional regulator PU.1 locating on the promoter region of TLR4, and further modulated the downstream signaling molecules of TLR4, including Rho GTPase Cdc 42 and TRAF6. In addition, deficiency of miR-15a/16 also facilitated TLR4-mediated proinflammatory cytokine/chemokine release from BMDMs at the initial phase of infections. Taken together, miR-15a/16 altered phagocytosis and bacterial clearance by targeting, at least partially, on the TLR4-associated pathways, subsequently affecting the survival of septic mice.

  9. Genes for human general transcription initiation factors TFIIIB, TFIIIB-associated proteins, TFIIIC2 and PTF/SNAPC: functional and positional candidates for tumour predisposition or inherited genetic diseases?

    PubMed

    Purrello, M; Di Pietro, C; Rapisarda, A; Amico, V; Giunta, V; Engel, H; Stevens, S; Hsieh, Y; Teichman, M; Wang, Z; Sichel, G; Roeder, R; Grzeschik, K H

    2001-08-09

    TFIIIB, TFIIIC2, and PTF/SNAPC are heteromultimeric general transcription factors (GTFs) needed for expression of genes encoding small cytoplasmic (scRNAs) and small nuclear RNAs (snRNAs). Their activity is stimulated by viral oncogenes, such as SV40 large T antigen and Adenovirus E1A, and is repressed by specific transcription factors (STFs) acting as anti-oncogenes, such as p53 and pRb. GTFs role as final targets of critical signal transduction pathways, that control cell proliferation and differentiation, and their involvement in gene expression regulation suggest that the genes encoding them are potential proto-oncogenes or anti-oncogenes or may be otherwise involved in the pathogenesis of inherited genetic diseases. To test our hypothesis through the positional candidate gene approach, we have determined the physical localization in the human genome of the 11 genes, encoding the subunits of these GTFs, and of three genes for proteins associated with TFIIIB (GTF3BAPs). Our data, obtained by chromosomal in situ hybridization, radiation hybrids and somatic cell hybrids analysis, demonstrate that these genes are present in the human genome as single copy sequences and that some cluster to the same cytogenetic band, alone or in combination with class II GTFs. Intriguingly, some of them are localized within chromosomal regions where recurrent, cytogenetically detectable mutations are seen in specific neoplasias, such as neuroblastoma, uterine leyomioma, mucoepidermoid carcinoma of the salivary glands and hemangiopericytoma, or where mutations causing inherited genetic diseases map, such as Peutz-Jeghers syndrome. Their molecular function and genomic position make these GTF genes interesting candidates for causal involvement in oncogenesis or in the pathogenesis of inherited genetic diseases.

  10. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    PubMed Central

    Chandra, Govind; Bibb, Maureen J.; Findlay, Kim C.; Buttner, Mark J.

    2016-01-01

    ABSTRACT WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo. PMID:27094333

  11. DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus

    PubMed Central

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-01-01

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (−145 and −85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis. PMID:25158097

  12. DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

    SciTech Connect

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-08-23

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.

  13. DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

    DOE PAGES

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; ...

    2014-08-23

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsible for themore » first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.« less

  14. Transcriptional enhancers: Transcription, function and flexibility.

    PubMed

    Melamed, Philippa; Yosefzon, Yahav; Rudnizky, Sergei; Pnueli, Lilach

    2016-01-01

    Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

  15. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

  16. Molecular structure and dynamics in bacterial mercury resistance

    SciTech Connect

    Johs, Alexander; Shi, Liang; Miller, Susan M; Summers, Anne O; Liang, Liyuan

    2008-01-01

    Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a metal-responsive regulator protein on the level of transcription. In vitro studies have shown that MerR forms a non-transcribing pre-initiation complex with RNA polymerase and the promoter DNA. Binding of Hg(II) induces conformational changes in MerR and other components of the complex resulting in the transcription of mer operon genes. As part of ongoing investigations on allosteric conformational changes induced by Hg(II) in dimeric MerR, and the implications on the binding of RNA polymerase to the promoter of the mer operon, we applied small angle scattering to study the regulatory mechanism of MerR in the presence and absence of Hg(II). Our results show that in the presence of Hg(II) the MerR dimer undergoes a significant reorientation from a compact state to a conformation revealing two distinct domains. Bacterial reduction of Hg(II) can also occur at concentrations too low to induce mer operon functions. Dissimilatory metal reducing bacteria, such as Shewanella and Geobacter are able to reduce Hg(II) in the presence of mineral oxides. This process has been linked to the activity of outer membrane multiheme cytochromes. We isolated and purified a decaheme outer membrane cytochrome OmcA from Shewanella oneidensis MR-1 and characterized its envelope shape in solution by small angle x-ray scattering. Structural features were identified and compared to homology models. These results show that OmcA is an elongated macromolecule consisting of separate modules, which may be connected by flexible linkers.

  17. Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

    PubMed Central

    Hadizadeh, Nastaran; Johnson, Reid C.

    2016-01-01

    ABSTRACT Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant (kexch) of ≈104 M−1 s−1, comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo. IMPORTANCE Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function. PMID:27044624

  18. Transcriptional response of nitrifying communities to wetting of dry soil.

    PubMed

    Placella, Sarah A; Firestone, Mary K

    2013-05-01

    The first rainfall following a severe dry period provides an abrupt water potential change that is both an acute physiological stress and a defined stimulus for the reawakening of soil microbial communities. We followed the responses of indigenous communities of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and nitrite-oxidizing bacteria to the addition of water to laboratory incubations of soils taken from two California annual grasslands following a typically dry Mediterranean summer. By quantifying transcripts for a subunit of bacterial and archaeal ammonia monooxygenases (amoA) and a bacterial nitrite oxidoreductase (nxrA) in soil from 15 min to 72 h after water addition, we identified transcriptional response patterns for each of these three groups of nitrifiers. An increase in quantity of bacterial amoA transcripts was detectable within 1 h of wet-up and continued until the size of the ammonium pool began to decrease, reflecting a possible role of transcription in upregulation of nitrification after drought-induced stasis. In one soil, the pulse of amoA transcription lasted for less than 24 h, demonstrating the transience of transcriptional pools and the tight coupling of transcription to the local soil environment. Analysis of 16S rRNA using a high-density microarray suggested that nitrite-oxidizing Nitrobacter spp. respond in tandem with ammonia-oxidizing bacteria while nitrite-oxidizing Nitrospina spp. and Nitrospira bacteria may not. Archaeal ammonia oxidizers may respond slightly later than bacterial ammonia oxidizers but may maintain elevated transcription longer. Despite months of desiccation-induced inactivation, we found rapid transcriptional response by all three groups of soil nitrifiers.

  19. Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris.

    PubMed

    Hsiao, Yi-Min; Song, Wan-Ling; Liao, Chao-Tsai; Lin, I-Hsuan; Pan, Mei-Ying; Lin, Ching-Fen

    2012-04-01

    The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

  20. Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis

    PubMed Central

    Nikolakakis, K.; Lehnert, E.

    2015-01-01

    The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. PMID:25956763

  1. Transcription of the ftsZ gene and cell division in Escherichia coli.

    PubMed Central

    Robin, A; Joseleau-Petit, D; D'Ari, R

    1990-01-01

    The ftsZ gene of Escherichia coli, which lies in a cluster of cell division genes at 2 min on the genetic map, codes for a protein which is thought to play a key role in triggering cell division. Using an ftsZ::lacZ operon fusion, we have studied the transcription of the ftsZ gene under conditions in which cell division was either inhibited or synchronized in the bacterial population. In ftsZ, ftsA, ftsQ, and ftsI (or pbpB) mutants, there was no change in the differential rate of expression of the ftsZ gene in nonpermissive conditions, when cell division was completely blocked. Although the FtsZ protein is thought to be limiting for cell division, in synchronized cultures the ftsZ gene was expressed not only at the moment of septation initiation but throughout the cell cycle. Its expression, however, was not exponential but linear, with a rapid doubling in rate at a specific cell age; this age, about 20 min after division in a 60-min cycle, was different from the age at which the ftsZ::lacZ operon was duplicated. However, it was close to the age at which replication initiated and at which the rate of phospholipid synthesis doubled. During the transient division inhibition after a nutritional shift-up, ftsZ transcription again became linear, with two doublings in rate at intervals equal to the mass doubling time in the rich medium; it adopted the exponential rate typical of rich medium about 60 min after the shift-up, just before the bacterial population resumed cell division. The doubling in the rate of ftsZ transcription once per cycle in synchronized cultures and once per mass doubling time during the transition period after a nutritional shift-up reflects a new cell cycle event. PMID:2106510

  2. Transcription inactivation through local refolding of the RNA polymerase structure

    SciTech Connect

    Belogurov, Georgiy A.; Vassylyeva, Marina N.; Sevostyanova, Anastasiya; Appleman, James R.; Xiang, Alan X.; Lira, Ricardo; Webber, Stephen E.; Klyuyev, Sergiy; Nudler, Evgeny; Artsimovitch, Irina; Vassylyev, Dmitry G.

    2009-02-12

    Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx - a desmethyl derivative of myxopyronin B - complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the {beta}'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex - the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

  3. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  4. The transcriptional control of the perforin locus

    PubMed Central

    Pipkin, Matthew E.; Rao, Anjana; Lichtenheld, Mathias G.

    2013-01-01

    Summary Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use cytotoxic granules containing perforin and granzymes to lyse infected or malignant host cells and provide immunity to intracellular microbes and tumors. Perforin is essential for cytotoxic granule-mediated killing. Perforin expression is regulated transcriptionally and correlates tightly with the development of cells that can exhibit cytotoxic activity. Although a number of genes transcribed by T cells and NK cells have been studied, the cell-specificity of perforin gene expression makes it an ideal model system in which to clarify the transcriptional mechanisms that guide the development and activation of cytotoxic lymphocytes. In this review, we discuss what is known about perforin expression and its regulation, then elaborate on recent studies that utilized chromosome transfer and bacterial artificial chromosome (BAC) transgenics to define a comprehensive set of cis-regulatory regions that control transcription of the human PRF1 gene in a near-physiological context. In addition, we compare the human and murine Prf1 loci and discuss how transcription factors known to be important for driving CTL differentiation might also directly regulate the cis-acting domains that control Prf1. Our review emphasizes how studies of PRF1/Prf1 gene transcription can illuminate not only the mechanisms of cytotoxic lymphocyte differentiation but also some basic principles of transcriptional regulation. PMID:20536555

  5. Structural basis for transcription elongation by bacterial RNA polymerase.

    PubMed

    Vassylyev, Dmitry G; Vassylyeva, Marina N; Perederina, Anna; Tahirov, Tahir H; Artsimovitch, Irina

    2007-07-12

    The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

  6. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  7. CHD chromatin remodelers and the transcription cycle.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by "opening" or "closing" chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

  8. Our evolving knowledge of the transcriptional landscape.

    PubMed

    Hume, David A

    2008-01-01

    The development of a genome-scale approach to identification of the 5' ends of capped mRNAs (CAGE) has given new insights into many aspects of mammalian RNApolII transcription control. They include the identification of the minimal initiator motif, the different types of proximal promoter architecture, the promoters of noncoding RNAs, the transcription of retrotransposons, and the extensive impact of alternative promoters on the proteome. CAGE also offers applications as a form of expression profiling that measures promoter use, allowing more precise development of transcriptional network models.

  9. Composing a Tumor Specific Bacterial Promoter

    PubMed Central

    Deyneko, Igor V.; Kasnitz, Nadine; Leschner, Sara; Weiss, Siegfried

    2016-01-01

    Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy. PMID:27171245

  10. Engineered cerium oxide nanoparticles: Effects on bacterial growth and viability

    SciTech Connect

    Pelletier, Dale A; Suresh, Anil K; Holton, Gregory A; McKeown, Catherine K; Wang, Wei; Gu, Baohua; Mortensen, Ninell P; Allison, David P; Joy, David Charles; Allison, Martin R; Brown, Steven D; Phelps, Tommy Joe; Doktycz, Mitchel John

    2010-01-01

    Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear. Further, standardized methods for assessing such interactions are lacking. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. In this study the effects of cerium oxide nanoparticles on the growth and viability of Gram-negative Escherichia coli and Shewanella oneidensis, a metal-reducing bacteria, and Gram-positive Bacillus subtilis were examined relative to particle size, growth media, pH, and dosage. A hydrothermal based synthesis procedure was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined by minimum inhibitory concentration, colony forming units, disc diffusion tests and Live/Dead assays. In growth inhibition experiments involving E. coli and B. subtilis, a clear strain and size-dependent inhibition was observed. S. oneidensis appeared to be unaffected by the cerium oxide nanoparticles. Transmission electron microscopy along with microarray-based transcriptional profiling have been used to understand the response mechanism of the bacteria. The use of multiple analytical approaches adds confidence to toxicity assessments while the use of different bacterial systems highlights the potential wide-ranging effects of

  11. Near-atomic resolution visualization of human transcription promoter opening.

    PubMed

    He, Yuan; Yan, Chunli; Fang, Jie; Inouye, Carla; Tjian, Robert; Ivanov, Ivaylo; Nogales, Eva

    2016-05-19

    In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA-RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

  12. The Transcription Unit Architecture of the Escherichia Coli Genome

    SciTech Connect

    Cho, Byung-Kwan; Zengler, Karsten; Qiu, Yu; Park, Young S.; Knight, Eric M.; Barrett, Christian; Gao, Yuan; Palsson, Bernhard O.

    2009-11-01

    Under EMSL User Proposal 25660, the authors reported that bacterial genomes are organized by structural and functional elements, including promoters, transcription start and termination sites, open reading frames, regulatory noncoding regions, untranslated regions and transcription units. Here, we iteratively integrate high-throughput, genome-wide measurements of RNA polymerase binding locations and mRNA transcript abundance, 5' sequences and translation into proteins to determine the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational elements provides an experimentally annotated transcription unit architecture, including alternative transcription start sites, 5' untranslated region, boundaries and open reading frames of each transcription unit. A total of 4,661 transcription units were identified, representing an increase of >530% over current knowledge. This comprehensive transcription unit architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome scale. Furthermore, the transcription unit architecture provides a foundation on which to construct genome-scale transcriptional and translational regulatory networks.

  13. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

  14. Transcription of foreign DNA in Escherichia coli.

    PubMed

    Warren, René L; Freeman, John D; Levesque, Roger C; Smailus, Duane E; Flibotte, Stephane; Holt, Robert A

    2008-11-01

    Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.

  15. Bacterial phospholipases C.

    PubMed Central

    Titball, R W

    1993-01-01

    A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C. PMID:8336671

  16. Evolution of Bacterial Suicide

    NASA Astrophysics Data System (ADS)

    Tchernookov, Martin; Nemenman, Ilya

    2013-03-01

    While active, controlled cellular suicide (autolysis) in bacteria is commonly observed, it has been hard to argue that autolysis can be beneficial to an individual who commits it. We propose a theoretical model that predicts that bacterial autolysis is evolutionarily advantageous to an individualand would fixate in physically structured environments for stationary phase colonies. We perform spatially resolved agent-based simulations of the model, which predict that lower mixing in the environment results in fixation of a higher autolysis rate from a single mutated cell, regardless of the colony's genetic diversity. We argue that quorum sensing will fixate as well, even if initially rare, if it is coupled to controlling the autolysis rate. The model does not predict a strong additional competitive advantage for cells where autolysis is controlled by quorum sensing systems that distinguish self from nonself. These predictions are broadly supported by recent experimental results in B. subtilisand S. pneumoniae. Research partially supported by the James S McDonnell Foundation grant No. 220020321 and by HFSP grant No. RGY0084/2011.

  17. Bacterial endocarditis prophylaxis.

    PubMed

    Blanco-Carrión, Andrés

    2004-01-01

    Bacterial endocarditis (BE) is a disease resulting from the association of morphological alterations of the heart and bacteraemia originating from different sources that at times can be indiscernible (infectious endocarditis). It is classified on the basis of the morphological alteration involved, depending on the clinical manifestations and course of illness, which varies according to the causative microorganism and host conditions (for example, it is characteristic in I.V. drug users). The most common microorganisms involved are: Streptococcus viridans (55%), Staphylococcus aureus (30%), Enterococcus (6%) and HACEK bacteria (corresponding to the initials: Haemophilus, Actinobacillus, Cardiobacterium, Eikenella and Kingella), although on occasions it can also be caused by fungi. The oral microbiological flora plays a very important role in the aetiopathogenesis of BE, given that the condition may be of oral or dental origin. This paper will deal with the prevention of said bacteraemia. Prophylaxis will be undertaken using amoxicillin or clindamycin according to action protocols, with special emphasis placed on oral hygiene in patients with structural defects of the heart.

  18. Formation and dissolution of bacterial colonies.

    PubMed

    Weber, Christoph A; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  19. Formation and dissolution of bacterial colonies

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  20. Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism.

    PubMed

    Koul, Anil; Vranckx, Luc; Dhar, Neeraj; Göhlmann, Hinrich W H; Özdemir, Emre; Neefs, Jean-Marc; Schulz, Melanie; Lu, Ping; Mørtz, Ejvind; McKinney, John D; Andries, Koen; Bald, Dirk

    2014-02-26

    Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.

  1. The role of mRNA structure in bacterial translational regulation.

    PubMed

    Meyer, Michelle M

    2017-01-01

    The characteristics of bacterial messenger RNAs (mRNAs) that influence translation efficiency provide many convenient handles for regulation of gene expression, especially when coupled with the processes of transcription termination and mRNA degradation. An mRNA's structure, especially near the site of initiation, has profound consequences for how readily it is translated. This property allows bacterial gene expression to be altered by changes to mRNA structure induced by temperature, or interactions with a wide variety of cellular components including small molecules, other RNAs (such as sRNAs and tRNAs), and RNA-binding proteins. This review discusses the links between mRNA structure and translation efficiency, and how mRNA structure is manipulated by conditions and signals within the cell to regulate gene expression. The range of RNA regulators discussed follows a continuum from very complex tertiary structures such as riboswitch aptamers and ribosomal protein-binding sites to thermosensors and mRNA:sRNA interactions that involve only base-pairing interactions. Furthermore, the high degrees of diversity observed for both mRNA structures and the mechanisms by which inhibition of translation occur have significant consequences for understanding the evolution of bacterial translational regulation. WIREs RNA 2017, 8:e1370. doi: 10.1002/wrna.1370 For further resources related to this article, please visit the WIREs website.

  2. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    PubMed

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  3. The human cut homeodomain protein represses transcription from the c-myc promoter.

    PubMed Central

    Dufort, D; Nepveu, A

    1994-01-01

    Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene. Images PMID:8196661

  4. Demonstrating Bacterial Flagella.

    ERIC Educational Resources Information Center

    Porter, John R.; And Others

    1992-01-01

    Describes an effective laboratory method for demonstrating bacterial flagella that utilizes the Proteus mirabilis organism and a special harvesting technique. Includes safety considerations for the laboratory exercise. (MDH)

  5. Innate immunity and inflammation: a transcriptional paradigm.

    PubMed

    Hawiger, J

    2001-01-01

    The innate immune response and the process of inflammation are interwoven. Excessive and continuing cytokine production in response to bacterial lipopolysacharides (LPS) or superantigens is a hallmark of the systemic inflammatory response (IR), which can be life-threatening. Dissemination of these bacterial products induces waves of proinflammatory cytokines that cause vascular injury and multiple organ dysfunction. Both LPS and superantigens induce signaling to the nucleus in mononuclear phagocytes and T cells, respectively. These signaling pathways are mediated by NF-kappaB and other stress-responsive transcription factors (SRTFs), which play a critical role in reprogramming gene expression. The nuclear import of NF-kappaB allows transcriptional activation of over 100 genes that encode mediators of inflammatory and immune responses. We have developed a novel method to block nuclear import of NF-kappaB through cell-permeable peptide transduction in monocytes, macrophages, T lymphocytes, and endothelial cells. Strikingly, a cell-permeable peptide that antagonizes nuclear import of NF-kappaB and other SRTFs, suppressed the systemic production of proinflammatory cytokines (TNFalpha and interferon gamma) in mice challenged with a lethal dose of LPS, and increased their survival by at least 90%. Thus, systemic inflammatory responses are critically dependent on the transcriptional activation ofcytokine genes that are controlled by NF-kappaB and other SRTFs.

  6. Transcription upregulation via force-induced direct stretching of chromatin

    NASA Astrophysics Data System (ADS)

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-12-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

  7. Transcription activation by the siderophore sensor Btr is mediated by ligand-dependent stimulation of promoter clearance

    PubMed Central

    Gaballa, Ahmed; MacLellan, Shawn; Helmann, John D.

    2012-01-01

    Bacterial transcription factors often function as DNA-binding proteins that selectively activate or repress promoters, although the biochemical mechanisms vary. In most well-understood examples, activators function by either increasing the affinity of RNA polymerase (RNAP) for the target promoter, or by increasing the isomerization of the initial closed complex to the open complex. We report that Bacillus subtilis Btr, a member of the AraC family of activators, functions principally as a ligand-dependent activator of promoter clearance. In the presence of its co-activator, the siderophore bacillibactin (BB), the Btr:BB complex enhances productive transcription, while having only modest effects on either RNAP promoter association or the production of abortive transcripts. Btr binds to two direct repeat sequences adjacent to the −35 region; recognition of the downstream motif is most important for establishing a productive interaction between the Btr:BB complex and RNAP. The resulting Btr:BB dependent increase in transcription enables the production of the ferric-BB importer to be activated by the presence of its cognate substrate. PMID:22210890

  8. Ports Initiative

    EPA Pesticide Factsheets

    The purpose of the ports initiative is to assist EPA and other participants to formulate strategies for moving forward on actions addressing the transportation, air quality and climate issues raised in the National Conversations.

  9. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  10. Sry is a transcriptional activator.

    PubMed

    Dubin, R A; Ostrer, H

    1994-09-01

    The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  11. Acute bacterial parotitis following acute stroke.

    PubMed

    Lee, V K; Kimbrough, D J; Jarquin-Valdivia, A A

    2009-06-01

    Acute bacterial parotitis (ABP) is a relatively uncommon condition that tends to occur in debilitated older patients. We report a case of an older woman that presented with an acute intracerebral hemorrhage who developed ABP. This morbidity led to endotracheal intubation, mechanical ventilation, tracheostomy and gastrostomy, all of which were not initially needed. We discuss the proposed physiopathology and etiopathogenesis of ABP in adults.

  12. The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

    PubMed Central

    Goldman, Seth R; Nair, Nikhil U; Wells, Christopher D; Nickels, Bryce E; Hochschild, Ann

    2015-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001 PMID:26371553

  13. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.

    PubMed

    Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C

    2015-09-01

    The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

  14. Biophysical models of transcription in cells

    NASA Astrophysics Data System (ADS)

    Choubey, Sandeep

    Cells constantly face environmental challenges and deal with them by changing their gene expression patterns. They make decisions regarding which genes to express and which genes not to express based on intra-cellular and environmental cues. These decisions are often made by regulating the process of transcription. While the identities of the different molecules that take part in regulating transcription have been determined for a number of different genes, their dynamics inside the cell are still poorly understood. One key feature of these regulatory dynamics is that the numbers of the bio-molecules involved is typically small, resulting in large temporal fluctuations in transcriptional outputs (mRNA and protein). In this thesis I show that measurements of the cell-to-cell variability of the distribution of transcribing RNA polymerases along a gene provide a previously unexplored method for deciphering the mechanism of its transcription in vivo. First, I propose a simple kinetic model of transcription initiation and elongation from which I calculate transcribing RNA polymerase copy-number fluctuations. I test my theory against published data obtained for yeast genes and propose a novel mechanism of transcription. Rather than transcription being initiated through a single rate-limiting step, as was previously proposed, my single-cell analysis reveals the presence of at least two rate limiting steps. Second, I compute the distribution of inter-polymerase distance distribution along a gene and propose a method for analyzing inter-polymerase distance distributions acquired in experiments. By applying this method to images of polymerases transcribing ribosomal genes in E.coli I show that one model of regulation of these genes is consistent with inter-polymerase distance data while a number of other models are not. The analytical framework described in this thesis can be used to extract quantitative information about the dynamics of transcription from single

  15. A continuum model of transcriptional bursting

    PubMed Central

    Corrigan, Adam M; Tunnacliffe, Edward; Cannon, Danielle; Chubb, Jonathan R

    2016-01-01

    Transcription occurs in stochastic bursts. Early models based upon RNA hybridisation studies suggest bursting dynamics arise from alternating inactive and permissive states. Here we investigate bursting mechanism in live cells by quantitative imaging of actin gene transcription, combined with molecular genetics, stochastic simulation and probabilistic modelling. In contrast to early models, our data indicate a continuum of transcriptional states, with a slowly fluctuating initiation rate converting the gene between different levels of activity, interspersed with extended periods of inactivity. We place an upper limit of 40 s on the lifetime of fluctuations in elongation rate, with initiation rate variations persisting an order of magnitude longer. TATA mutations reduce the accessibility of high activity states, leaving the lifetime of on- and off-states unchanged. A continuum or spectrum of gene states potentially enables a wide dynamic range for cell responses to stimuli. DOI: http://dx.doi.org/10.7554/eLife.13051.001 PMID:26896676

  16. ppGpp couples transcription to DNA repair in E. coli.

    PubMed

    Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley; Proshkin, Sergey; Mironov, Alexander; Cashel, Michael; Nudler, Evgeny

    2016-05-20

    The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

  17. Vimentin in Bacterial Infections

    PubMed Central

    Mak, Tim N.; Brüggemann, Holger

    2016-01-01

    Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection. PMID:27096872

  18. Collective decision making in bacterial viruses.

    PubMed

    Weitz, Joshua S; Mileyko, Yuriy; Joh, Richard I; Voit, Eberhard O

    2008-09-15

    For many bacterial viruses, the choice of whether to kill host cells or enter a latent state depends on the multiplicity of coinfection. Here, we present a mathematical theory of how bacterial viruses can make collective decisions concerning the fate of infected cells. We base our theory on mechanistic models of gene regulatory dynamics. Unlike most previous work, we treat the copy number of viral genes as variable. Increasing the viral copy number increases the rate of transcription of viral mRNAs. When viral regulation of cell fate includes nonlinear feedback loops, very small changes in transcriptional rates can lead to dramatic changes in steady-state gene expression. Hence, we prove that deterministic decisions can be reached, e.g., lysis or latency, depending on the cellular multiplicity of infection within a broad class of gene regulatory models of viral decision-making. Comparisons of a parameterized version of the model with molecular studies of the decision structure in the temperate bacteriophage lambda are consistent with our conclusions. Because the model is general, it suggests that bacterial viruses can respond adaptively to changes in population dynamics, and that features of collective decision-making in viruses are evolvable life history traits.

  19. TAL effector-mediated susceptibility to bacterial blight of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial blight of cotton (BBC) caused by Xanthomonas campestris pv. malvacearum (Xcm) is a destructive disease that has recently re-emerged in the U.S. Xcm injects transcription activator-like (TAL) effectors that directly induce the expression of host susceptibility (S) or resistance (R) genes. ...

  20. Specificity mechanisms in the control of transcription.

    PubMed

    von Hippel, P H; Rees, W A; Rippe, K; Wilson, K S

    1996-04-16

    In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription. Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase. In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex. Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions. However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes. Cis effectors, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision. Examples, drawn from studies of phage lambda N-dependent antitermination and E. coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants. The generality of such regulatory principles are stressed.

  1. Septins and Bacterial Infection

    PubMed Central

    Torraca, Vincenzo; Mostowy, Serge

    2016-01-01

    Septins, a unique cytoskeletal component associated with cellular membranes, are increasingly recognized as having important roles in host defense against bacterial infection. A role for septins during invasion of Listeria monocytogenes into host cells was first proposed in 2002. Since then, work has shown that septins assemble in response to a wide variety of invasive bacterial pathogens, and septin assemblies can have different roles during the bacterial infection process. Here we review the interplay between septins and bacterial pathogens, highlighting septins as a structural determinant of host defense. We also discuss how investigation of septin assembly in response to bacterial infection can yield insight into basic cellular processes including phagocytosis, autophagy, and mitochondrial dynamics. PMID:27891501

  2. Nuclear gadgets in mitochondrial DNA replication and transcription.

    PubMed

    Clayton, D A

    1991-03-01

    In mammalian mitochondrial DNA, activation of the light-strand promoter mediates both priming of leading-strand replication and initiation of light-strand transcription. Accurate and efficient transcription requires at least two proteins: mitochondrial RNA polymerase and a separable transcription factor that can function across species boundaries. Subsequently, primer RNAs are cleaved by a site-specific ribonucleoprotein endoribonuclease that recognizes short, highly conserved sequence elements in the RNA substrate.

  3. Gene transcription and electromagnetic fields

    SciTech Connect

    Henderson, A.S.

    1992-01-01

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  4. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  5. Proteomics in the Study of Bacterial Keratitis

    PubMed Central

    Bouhenni, Rachida; Dunmire, Jeffrey; Rowe, Theresa; Bates, James

    2015-01-01

    Bacterial keratitis is a serious ocular infection that can cause severe visual loss if treatment is not initiated at an early stage. It is most commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, or Serratia species. Depending on the invading organism, bacterial keratitis can progress rapidly, leading to corneal destruction and potential blindness. Common risk factors for bacterial keratitis include contact lens wear, ocular trauma, ocular surface disease, ocular surgery, lid deformity, chronic use of topical steroids, contaminated ocular medications or solutions, and systemic immunosuppression. The pathogenesis of bacterial keratitis, which depends on the bacterium-host interaction and the virulence of the invading bacterium, is complicated and not completely understood. This review highlights some of the proteomic technologies that have been used to identify virulence factors and the host response to infections of bacterial keratitis in order to understand the disease process and develop improved methods of diagnosis and treatment. Although work in this field is not abundant, proteomic technologies have provided valuable information toward our current knowledge of bacterial keratitis. More studies using global proteomic approaches are warranted because it is an important tool to identify novel targets for intervention and prevention of corneal damage caused by these virulent microorganisms. PMID:28248282

  6. Proteomics in the Study of Bacterial Keratitis.

    PubMed

    Bouhenni, Rachida; Dunmire, Jeffrey; Rowe, Theresa; Bates, James

    2015-12-14

    Bacterial keratitis is a serious ocular infection that can cause severe visual loss if treatment is not initiated at an early stage. It is most commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, or Serratia species. Depending on the invading organism, bacterial keratitis can progress rapidly, leading to corneal destruction and potential blindness. Common risk factors for bacterial keratitis include contact lens wear, ocular trauma, ocular surface disease, ocular surgery, lid deformity, chronic use of topical steroids, contaminated ocular medications or solutions, and systemic immunosuppression. The pathogenesis of bacterial keratitis, which depends on the bacterium-host interaction and the virulence of the invading bacterium, is complicated and not completely understood. This review highlights some of the proteomic technologies that have been used to identify virulence factors and the host response to infections of bacterial keratitis in order to understand the disease process and develop improved methods of diagnosis and treatment. Although work in this field is not abundant, proteomic technologies have provided valuable information toward our current knowledge of bacterial keratitis. More studies using global proteomic approaches are warranted because it is an important tool to identify novel targets for intervention and prevention of corneal damage caused by these virulent microorganisms.

  7. Bacterial Sigma Factors and Anti-Sigma Factors: Structure, Function and Distribution

    PubMed Central

    Paget, Mark S.

    2015-01-01

    Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoters as well as the initial steps in RNA synthesis. This review focuses on the structure and function of the major sigma-70 class that includes the housekeeping sigma factor (Group 1) that directs the bulk of transcription during active growth, and structurally-related alternative sigma factors (Groups 2–4) that control a wide variety of adaptive responses such as morphological development and the management of stress. A recurring theme in sigma factor control is their sequestration by anti-sigma factors that occlude their RNAP-binding determinants. Sigma factors are then released through a wide variety of mechanisms, often involving branched signal transduction pathways that allow the integration of distinct signals. Three major strategies for sigma release are discussed: regulated proteolysis, partner-switching, and direct sensing by the anti-sigma factor. PMID:26131973

  8. [Dual promoters enhance heterologous enzyme production from bacterial phage based recombinant Bacillus subtilis].

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

    The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production.

  9. Divergent RNA transcription: a role in promoter unwinding?

    PubMed

    Naughton, Catherine; Corless, Samuel; Gilbert, Nick

    2013-01-01

    New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance.

  10. ASTP Onboard Voice Transcription

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The transcription is presented of the Apollo-Soyuz Test Project voice communications as recorded on the command module data storage equipment. Data from this recorder are telemetered (dumped) to Space Tracking and Data Network sites for retransmission to the Johnson Space Center. The transcript is divided into three columns -- time, speaker, and text. The Greenwich mean time column consists of three two-digit numbers representing hours, minutes, and seconds (e.g., 22 34 14) for the Julian dates shown at the top of the page on which a new day begins. The speaker column indicates the source of a transmission; the text column contains the verbatim transcript of the communications.

  11. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  12. Youth Initiatives.

    ERIC Educational Resources Information Center

    Employment and Training Administration (DOL), Washington, DC. Office of Youth Programs.

    Summarizing the first eight months of the planning, design, and implementation of the new federal youth programs created by the Youth Employment and Demonstration Projects Act of 1977 (YEDPA) and the efforts for expansion and enrichment of the Job Corps, this report explains the charters of the two projects and their initial successes in and…

  13. Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

    PubMed

    Krzeslak, Joanna; Papaioannou, Evelina; van Merkerk, Ronald; Paal, Krisztina A; Bischoff, Rainer; Cool, Robbert H; Quax, Wim J

    2012-04-01

    Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.

  14. Electrokinetic control of bacterial deposition and transport.

    PubMed

    Qin, Jinyi; Sun, Xiaohui; Liu, Yang; Berthold, Tom; Harms, Hauke; Wick, Lukas Y

    2015-05-05

    Microbial biofilms can cause severe problems in technical installations where they may give rise to microbially influenced corrosion and clogging of filters and membranes or even threaten human health, e.g. when they infest water treatment processes. There is, hence, high interest in methods to prevent microbial adhesion as the initial step of biofilm formation. In environmental technology it might be desired to enhance bacterial transport through porous matrices. This motivated us to test the hypothesis that the attractive interaction energy allowing cells to adhere can be counteracted and overcome by the shear force induced by electroosmotic flow (EOF, i.e. the water flow over surfaces exposed to a weak direct current (DC) electric field). Applying EOF of varying strengths we quantified the deposition of Pseudomonas fluorescens Lp6a in columns containing glass collectors and on a quartz crystal microbalance. We found that the presence of DC reduced the efficiency of initial adhesion and bacterial surface coverage by >85%. A model is presented which quantitatively explains the reduction of bacterial adhesion based on the extended Derjaguin, Landau, Verwey, and Overbeek (XDLVO) theory of colloid stability and the EOF-induced shear forces acting on a bacterium. We propose that DC fields may be used to electrokinetically regulate the interaction of bacteria with surfaces in order to delay initial adhesion and biofilm formation in technical installations or to enhance bacterial transport in environmental matrices.

  15. Formaldehyde Stress Responses in Bacterial Pathogens

    PubMed Central

    Chen, Nathan H.; Djoko, Karrera Y.; Veyrier, Frédéric J.; McEwan, Alastair G.

    2016-01-01

    Formaldehyde is the simplest of all aldehydes and is highly cytotoxic. Its use and associated dangers from environmental exposure have been well documented. Detoxification systems for formaldehyde are found throughout the biological world and they are especially important in methylotrophic bacteria, which generate this compound as part of their metabolism of methanol. Formaldehyde metabolizing systems can be divided into those dependent upon pterin cofactors, sugar phosphates and those dependent upon glutathione. The more prevalent thiol-dependent formaldehyde detoxification system is found in many bacterial pathogens, almost all of which do not metabolize methane or methanol. This review describes the endogenous and exogenous sources of formaldehyde, its toxic effects and mechanisms of detoxification. The methods of formaldehyde sensing are also described with a focus on the formaldehyde responsive transcription factors HxlR, FrmR, and NmlR. Finally, the physiological relevance of detoxification systems for formaldehyde in bacterial pathogens is discussed. PMID:26973631

  16. DNA supercoiling during transcription

    PubMed Central

    Ma, Jie; Wang, Michelle D.

    2017-01-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  17. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  18. Linking Core Promoter Classes to Circadian Transcription

    PubMed Central

    Westermark, Pål O.

    2016-01-01

    Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs), is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription. PMID:27504829

  19. Transcription and cancer.

    PubMed Central

    Cox, P. M.; Goding, C. R.

    1991-01-01

    The normal growth, development and function of an organism requires precise and co-ordinated control of gene expression. A major part of this control is exerted by regulating messenger RNA (mRNA) production and involves complex interactions between an array of transcriptionally active proteins and specific regulatory DNA sequences. The combination of such proteins and DNA sequences is specific for given gene or group of genes in a particular cell type and the proteins regulating the same gene may vary between cell types. In addition the expression or activity of these regulatory proteins may be modified depending on the state of differentiation of a cell or in response to an external stimulus. Thus, the differentiation of embryonic cells into diverse tissues is achieved and the mature structure and function of the organism is maintained. This review focusses on the role of perturbations of these transcriptional controls in neoplasia. Deregulation of transcription may result in the failure to express genes responsible for cellular differentiation, or alternatively, in the transcription of genes involved in cell division, through the inappropriate expression or activation of positively acting transcription factors and nuclear oncogenes. Whether the biochemical abnormalities that lead to the disordered growth and differentiation of a malignant tumour affect cell surface receptors, membrane or cytoplasmic signalling proteins or nuclear transcription factors, the end result is the inappropriate expression of some genes and failure to express others. Current research is starting to elucidate which of the elements of this complicated system are important in neoplasia. PMID:1645561

  20. Nascent RNA sequencing reveals distinct features in plant transcription

    PubMed Central

    Hetzel, Jonathan; Duttke, Sascha H.; Benner, Christopher; Chory, Joanne

    2016-01-01

    Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole. PMID:27729530

  1. SCB initiator

    DOEpatents

    Bickes Jr., Robert W.; Renlund, Anita M.; Stanton, Philip L.

    1994-11-01

    A detonator for high explosives initiated by mechanical impact includes a cylindrical barrel, a layer of flyer material mechanically covering the barrel at one end, and a semiconductor bridge ignitor including a pair of electrically conductive pads connected by a semiconductor bridge. The bridge is in operational contact with the layer, whereby ignition of said bridge forces a portion of the layer through the barrel to detonate the explosive. Input means are provided for igniting the semiconductor bridge ignitor.

  2. SCB initiator

    SciTech Connect

    Bickes, Jr., Robert W.; Renlund, Anita M.; Stanton, Philip L.

    1994-01-01

    A detonator for high explosives initiated by mechanical impact includes a cylindrical barrel, a layer of flyer material mechanically covering the barrel at one end, and a semiconductor bridge ignitor including a pair of electrically conductive pads connected by a semiconductor bridge. The bridge is in operational contact with the layer, whereby ignition of said bridge forces a portion of the layer through the barrel to detonate the explosive. Input means are provided for igniting the semiconductor bridge ignitor.

  3. Bacterial surface adaptation

    NASA Astrophysics Data System (ADS)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  4. Bacterial Wound Culture

    MedlinePlus

    ... and services. Advertising & Sponsorship: Policy | Opportunities Bacterial Wound Culture Share this page: Was this page helpful? Also known as: Aerobic Wound Culture; Anaerobic Wound Culture Formal name: Culture, wound Related ...

  5. Experimental Bacterial Endocarditis

    PubMed Central

    Durack, David T.; Beeson, Paul B.

    1972-01-01

    A method has been developed for assessing metabolic activity of bacteria in the vegetations of bacterial endocarditis using a labelled metabolite and autoradiography. Evidence provided by this technique suggests that there are different degrees of activity between superficial and more deeply placed bacterial colonies, and that variations in activity also exist within a single group of organisms. The possible relevance of these findings to the antibiotic therapy of endocarditis is discussed. ImagesFigs. 1-3Figs. 4-5 PMID:4111329

  6. [Diagnosis of bacterial vaginosis].

    PubMed

    Djukić, Slobodanka; Ćirković, Ivana; Arsić, Biljana; Garalejić, Eliana

    2013-01-01

    Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2-producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent's scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up-to-date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short-term and long-term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  7. Transcription-dependent and transcription-independent nucleosome disruption induced by dioxin

    SciTech Connect

    Morgan, J.E.; Whitlock, J.P. Jr. )

    1992-12-01

    In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosomes is independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation. 38 refs., 5 figs.

  8. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  9. Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

    PubMed

    Arndt, Karen M; Reines, Daniel

    2015-01-01

    The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.

  10. Bacterial cell division proteins as antibiotic targets.

    PubMed

    den Blaauwen, Tanneke; Andreu, José M; Monasterio, Octavio

    2014-08-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of proteins and the availability of crystal structures has increased our knowledge on bacterial cell division considerably in this century. Consequently, bacterial cell division proteins are more and more recognized as potential new antibiotic targets. An international effort to find small molecules that inhibit the cell division initiating protein FtsZ has yielded many compounds of which some are promising as leads for preclinical use. The essential transglycosylase activity of peptidoglycan synthases has recently become accessible to inhibitor screening. Enzymatic assays for and structural information on essential integral membrane proteins such as MraY and FtsW involved in lipid II (the peptidoglycan building block precursor) biosynthesis have put these proteins on the list of potential new targets. This review summarises and discusses the results and approaches to the development of lead compounds that inhibit bacterial cell division.

  11. Regulation of Transcript Elongation

    PubMed Central

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  12. Optimising Antibiotic Usage to Treat Bacterial Infections

    NASA Astrophysics Data System (ADS)

    Paterson, Iona K.; Hoyle, Andy; Ochoa, Gabriela; Baker-Austin, Craig; Taylor, Nick G. H.

    2016-11-01

    The increase in antibiotic resistant bacteria poses a threat to the continued use of antibiotics to treat bacterial infections. The overuse and misuse of antibiotics has been identified as a significant driver in the emergence of resistance. Finding optimal treatment regimens is therefore critical in ensuring the prolonged effectiveness of these antibiotics. This study uses mathematical modelling to analyse the effect traditional treatment regimens have on the dynamics of a bacterial infection. Using a novel approach, a genetic algorithm, the study then identifies improved treatment regimens. Using a single antibiotic the genetic algorithm identifies regimens which minimise the amount of antibiotic used while maximising bacterial eradication. Although exact treatments are highly dependent on parameter values and initial bacterial load, a significant common trend is identified throughout the results. A treatment regimen consisting of a high initial dose followed by an extended tapering of doses is found to optimise the use of antibiotics. This consistently improves the success of eradicating infections, uses less antibiotic than traditional regimens and reduces the time to eradication. The use of genetic algorithms to optimise treatment regimens enables an extensive search of possible regimens, with previous regimens directing the search into regions of better performance.

  13. Optimising Antibiotic Usage to Treat Bacterial Infections

    PubMed Central

    Paterson, Iona K.; Hoyle, Andy; Ochoa, Gabriela; Baker-Austin, Craig; Taylor, Nick G. H.

    2016-01-01

    The increase in antibiotic resistant bacteria poses a threat to the continued use of antibiotics to treat bacterial infections. The overuse and misuse of antibiotics has been identified as a significant driver in the emergence of resistance. Finding optimal treatment regimens is therefore critical in ensuring the prolonged effectiveness of these antibiotics. This study uses mathematical modelling to analyse the effect traditional treatment regimens have on the dynamics of a bacterial infection. Using a novel approach, a genetic algorithm, the study then identifies improved treatment regimens. Using a single antibiotic the genetic algorithm identifies regimens which minimise the amount of antibiotic used while maximising bacterial eradication. Although exact treatments are highly dependent on parameter values and initial bacterial load, a significant common trend is identified throughout the results. A treatment regimen consisting of a high initial dose followed by an extended tapering of doses is found to optimise the use of antibiotics. This consistently improves the success of eradicating infections, uses less antibiotic than traditional regimens and reduces the time to eradication. The use of genetic algorithms to optimise treatment regimens enables an extensive search of possible regimens, with previous regimens directing the search into regions of better performance. PMID:27892497

  14. The transcription factor encyclopedia.

    PubMed

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

  15. The Transcription Factor Encyclopedia

    PubMed Central

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe. PMID:22458515

  16. Collective decisions among bacterial viruses

    NASA Astrophysics Data System (ADS)

    Joh, Richard; Mileyko, Yuriy; Voit, Eberhard; Weitz, Joshua

    2010-03-01

    For many temperate bacteriophages, the decision of whether to kill hosts or enter a latent state depends on the multiplicity of infection. In this talk, I present a quantitative model of gene regulatory dynamics to describe how phages make collective decisions within host cells. Unlike most previous studies, the copy number of viral genomes is treated as a variable. In the absence of feedback loops, viral mRNA transcription is expected to be proportional to the viral copy number. However, when there are nonlinear feedback loops in viral gene regulation, our model shows that gene expression patterns are sensitive to changes in viral copy number and there can be a domain of copy number where the system becomes bistable. Hence, the viral copy number is a key control parameter determining host cell fates. This suggests that bacterial viruses can respond adaptively to changes in population dynamics, and can make alternative decisions as a bet-hedging strategy. Finally, I present a stochastic version of viral gene regulation and discuss speed-accuracy trade-offs in the context of cell fate determination by viruses.

  17. The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease

    PubMed Central

    O’Connor, Leigh; Gilmour, Jane; Bonifer, Constanze

    2016-01-01

    Sp1 belongs to the 26 member strong Sp/KLF family of transcription factors. It is a paradigm for a ubiquitously expressed transcription factor and is involved in regulating the expression of genes associated with a wide range of cellular processes in mammalian cells. Sp1 can interact with a range of proteins, including other transcription factors, members of the transcription initiation complex and epigenetic regulators, enabling tight regulation of its target genes. In this review, we discuss the mechanisms involved in Sp1-mediated transcriptional regulation, as well as how a ubiquitous transcription factor can be involved in establishing a tissue-specific pattern of gene expression and mechanisms by which its activity may be regulated. We also consider the role of Sp1 in human diseases, such as cancer. PMID:28018142

  18. Bacterial oncogenesis in the colon

    PubMed Central

    Dejea, Christine; Wick, Elizabeth; Sears, Cynthia L

    2013-01-01

    The human colon plays host to a diverse and metabolically complex community of microorganisms. While the colonic microbiome has been suggested to contribute to the development of colorectal cancer (CRC), a definitive link has not been made. The role in which the colon microflora could contribute to the initiation and/or progression of CRC is explored in this review. Potential mechanisms of bacterial oncogenesis are presented, along with lines of evidence derived from animal models of microbially induced CRC. Particular focus is given to the oncogenic capabilities of enterotoxigenic Bacteroides fragilis. Recent progress in defining the microbiome of CRC in the human population is evaluated, and the future challenges of linking specific etiologic agents to CRC are emphasized. PMID:23534358

  19. The essential activities of the bacterial sigma factor.

    PubMed

    Davis, Maria C; Kesthely, Christopher A; Franklin, Emily A; MacLellan, Shawn R

    2017-02-01

    Transcription is the first and most heavily regulated step in gene expression. Sigma (σ) factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. σ Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when σ factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the σ(70) family of σ proteins and the essential roles they play in the transcription process.

  20. The transcriptional regulation of the glyoxylate cycle in SAR11 in response to iron fertilization in the Southern Ocean.

    PubMed

    Beier, Sara; Gálvez, María J; Molina, Veronica; Sarthou, Géraldine; Quéroué, Fabien; Blain, Stephane; Obernosterer, Ingrid

    2015-06-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway that is present in all aerobic organisms and initiates the respiration of organic material. The glyoxylate cycle is a variation of the TCA cycle, where organic material is recycled for subsequent assimilation into cell material instead of being released as carbon dioxide. Despite the importance for the fate of organic matter, the environmental factors that induce the glyoxylate cycle in microbial communities remain poorly understood. In this study, we assessed the expression of isocitrate lyase, the enzyme that induces the switch to the glyoxylate cycle, of the ubiquitous SAR11 clade in response to natural iron fertilization in the Southern Ocean. The cell-specific transcriptional regulation of the glyoxylate cycle, as determined by the ratio between copy numbers of isocitrate lyase gene transcripts and isocitrate genes, was consistently lower in iron fertilized than in high-nutrient, low chlorophyll waters (by 2.4- to 16.5-fold). SAR11 cell-specific isocitrate lyase gene transcription was negatively correlated to chlorophyll a, and bulk bacterial heterotrophic metabolism. We conclude that the glyoxylate cycle is a metabolic strategy for SAR11 that is highly sensitive to the degree of iron and carbon limitation in the marine environment.

  1. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  2. An overview on transcriptional regulators in Streptomyces.

    PubMed

    Romero-Rodríguez, Alba; Robledo-Casados, Ivonne; Sánchez, Sergio

    2015-08-01

    Streptomyces are Gram-positive microorganisms able to adapt and respond to different environmental conditions. It is the largest genus of Actinobacteria comprising over 900 species. During their lifetime, these microorganisms are able to differentiate, produce aerial mycelia and secondary metabolites. All of these processes are controlled by subtle and precise regulatory systems. Regulation at the transcriptional initiation level is probably the most common for metabolic adaptation in bacteria. In this mechanism, the major players are proteins named transcription factors (TFs), capable of binding DNA in order to repress or activate the transcription of specific genes. Some of the TFs exert their action just like activators or repressors, whereas others can function in both manners, depending on the target promoter. Generally, TFs achieve their effects by using one- or two-component systems, linking a specific type of environmental stimulus to a transcriptional response. After DNA sequencing, many streptomycetes have been found to have chromosomes ranging between 6 and 12Mb in size, with high GC content (around 70%). They encode for approximately 7000 to 10,000 genes, 50 to 100 pseudogenes and a large set (around 12% of the total chromosome) of regulatory genes, organized in networks, controlling gene expression in these bacteria. Among the sequenced streptomycetes reported up to now, the number of transcription factors ranges from 471 to 1101. Among these, 315 to 691 correspond to transcriptional regulators and 31 to 76 are sigma factors. The aim of this work is to give a state of the art overview on transcription factors in the genus Streptomyces.

  3. Pioneer transcription factors in cell reprogramming.

    PubMed

    Iwafuchi-Doi, Makiko; Zaret, Kenneth S

    2014-12-15

    A subset of eukaryotic transcription factors possesses the remarkable ability to reprogram one type of cell into another. The transcription factors that reprogram cell fate are invariably those that are crucial for the initial cell programming in embryonic development. To elicit cell programming or reprogramming, transcription factors must be able to engage genes that are developmentally silenced and inappropriate for expression in the original cell. Developmentally silenced genes are typically embedded in "closed" chromatin that is covered by nucleosomes and not hypersensitive to nuclease probes such as DNase I. Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as "pioneer factors" to initiate events in closed chromatin. Other reprogramming factors appear dependent on pioneer factors for engaging nucleosomes and closed chromatin. However, certain genomic domains in which nucleosomes are occluded by higher-order chromatin structures, such as in heterochromatin, are resistant to pioneer factor binding. Understanding the means by which pioneer factors can engage closed chromatin and how heterochromatin can prevent such binding promises to advance our ability to reprogram cell fates at will and is the topic of this review.

  4. Coupled effects of chemotaxis and growth on traveling bacterial waves.

    PubMed

    Yan, Zhifeng; Bouwer, Edward J; Hilpert, Markus

    2014-08-01

    Traveling bacterial waves are capable of improving contaminant remediation in the subsurface. It is fairly well understood how bacterial chemotaxis and growth separately affect the formation and propagation of such waves. However, their interaction is not well understood. We therefore perform a modeling study to investigate the coupled effects of chemotaxis and growth on bacterial migration, and examine their effects on contaminant remediation. We study the waves by using different initial electron acceptor concentrations for different bacteria and substrate systems. Three types of traveling waves can occur: a chemotactic wave due to the biased movement of chemotactic bacteria resulting from metabolism-generated substrate concentration gradients; a growth/decay/motility wave due to a dynamic equilibrium between bacterial growth, decay and random motility; and an integrated wave due to the interaction between bacterial chemotaxis and growth. Chemotaxis hardly enhances the bacterial propagation if it is too weak to form a chemotactic wave or its wave speed is less than half of the growth/decay/motility wave speed. However, chemotaxis significantly accelerates bacterial propagation once its wave speed exceeds the growth/decay/motility wave speed. When convection occurs, it speeds up the growth/decay/motility wave but slows down or even eliminates the chemotactic wave due to the dispersion. Bacterial survival proves particularly important for bacterial propagation. Therefore we develop a conceptual model to estimate the speed of growth/decay/motility waves.

  5. Crystal structure of enterococcus faecalis sly A-like transcriptional factor.

    SciTech Connect

    Wu, R.; Zhang, R.; Zagnitko, O.; Dementieva, I.; Maltsev, N.; Watson, J. D.; Laskowski, R.; Gornicki, P.; Joachimiak, A.; Univ. of Chicago; European Bioinformatics Inst.

    2003-05-30

    The crystal structure of a SlyA transcriptional regulator at 1.6 {angstrom} resolution is presented, and structural relationships between members of the MarR/SlyA family are discussed. The SlyA family, which includes SlyA, Rap, Hor, and RovA proteins, is widely distributed in bacterial and archaeal genomes. Current evidence suggests that SlyA-like factors act as repressors, activators, and modulators of gene transcription. These proteins have been shown to up-regulate the expression of molecular chaperones, acid-resistance proteins, and cytolysin, and down-regulate several biosynthetic enzymes. The structure of SlyA from Enterococcus faecalis, determined as a part of an ongoing structural genomics initiative (www.mcsg.anl.gov), revealed the same winged helix DNA-binding motif that was recently found in the MarR repressor from Escherichia coli and the MexR repressor from Pseudomonas aeruginosa, a sequence homologue of MarR. Phylogenetic analysis of the MarR/SlyA family suggests that Sly is placed between the SlyA and MarR subfamilies and shows significant sequence similarity to members of both subfamilies.

  6. Sticky Situations: Key Components That Control Bacterial Surface Attachment

    PubMed Central

    Petrova, Olga E.

    2012-01-01

    The formation of bacterial biofilms is initiated by cells transitioning from the free-swimming mode of growth to a surface. This review is aimed at highlighting the common themes that have emerged in recent research regarding the key components, signals, and cues that aid in the transition and those involved in establishing a more permanent surface association during initial attachment. PMID:22389478

  7. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  8. Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in Escherichia coli via Regulation of the csgD Gene

    PubMed Central

    Prigent-Combaret, Claire; Brombacher, Eva; Vidal, Olivier; Ambert, Arnaud; Lejeune, Philippe; Landini, Paolo; Dorel, Corinne

    2001-01-01

    The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop. PMID:11717281

  9. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene.

    PubMed

    Prigent-Combaret, C; Brombacher, E; Vidal, O; Ambert, A; Lejeune, P; Landini, P; Dorel, C

    2001-12-01

    The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

  10. Bacterial transfer RNAs

    PubMed Central

    Shepherd, Jennifer; Ibba, Michael

    2015-01-01

    Transfer RNA is an essential adapter molecule that is found across all three domains of life. The primary role of transfer RNA resides in its critical involvement in the accurate translation of messenger RNA codons during protein synthesis and, therefore, ultimately in the determination of cellular gene expression. This review aims to bring together the results of intensive investigations into the synthesis, maturation, modification, aminoacylation, editing and recycling of bacterial transfer RNAs. Codon recognition at the ribosome as well as the ever-increasing number of alternative roles for transfer RNA outside of translation will be discussed in the specific context of bacterial cells. PMID:25796611

  11. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation.

    PubMed

    Ross, C; Samuel, M; Broitman, S L

    1992-12-30

    We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7. Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter. These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide. It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

  12. Mapping yeast transcriptional networks.

    PubMed

    Hughes, Timothy R; de Boer, Carl G

    2013-09-01

    The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

  13. Fungal CSL transcription factors

    PubMed Central

    Převorovský, Martin; Půta, František; Folk, Petr

    2007-01-01

    Background The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. Results We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. Conclusion Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans. PMID:17629904

  14. Focus on Refugees. Transcript.

    ERIC Educational Resources Information Center

    Brandel, Sarah; And Others

    This is the transcript of the "Focus on Refugees," proqram conducted by the Overseas Development Council. Remarks from the following participants are included: (1) Sarah Brandel, Associate Fellow at the Overseas Development Council; (2) Gary Perkins, Chief of Mission of the Washington Office of the United Nations High Commissioner for Refugees…

  15. Openness initiative

    SciTech Connect

    Duncan, S.S.

    1995-12-31

    Although antinuclear campaigns seem to be effective, public communication and education efforts on low-level radioactive waste have mixed results. Attempts at public information programs on low-level radioactive waste still focus on influencing public opinion. A question then is: {open_quotes}Is it preferable to have a program focus on public education that will empower individuals to make informed decisions rather than trying to influence them in their decisions?{close_quotes} To address this question, a case study with both quantitative and qualitative data will be used. The Ohio Low-Level Radioactive Waste Education Program has a goal to provide people with information they want/need to make their own decisions. The program initiated its efforts by conducting a statewide survey to determine information needed by people and where they turned for that information. This presentation reports data from the survey and then explores the program development process in which programs were designed and presented using the information. Pre and post data from the programs reveal attitude and knowledge shifts.

  16. Bacterial β-glucosidase function and metabolic activity depend on soil management in semiarid rainfed agriculture

    PubMed Central

    Cañizares, Rosa; Moreno, Beatriz; Benitez, Emilio

    2012-01-01

    Genomic and transcriptomic approaches were used to gain insights into the relationship between soil management and bacterial-mediated functions in an olive orchard agroecosystem. Four management practices were assessed in a 30-year trial in a semiarid Mediterranean region. Transcriptional activity of bacterial 16S rRNA genes increased in noncovered soils, indicating higher microbial maintenance requirements to thrive in less favorable environmental conditions. The 16S rRNA transcript:gene copy ratio confirmed this assumption and pointed toward a much higher constitutive expression from rRNA operons in noncovered soils and to even higher expression levels when spontaneous vegetation was removed chemically. As described for 16S rRNA, potential transcription did not reveal the real transcription of bacterial β-glucosidase genes, and higher gene expression in noncovered soils plus herbicides was evidenced. Since no relationship between total or soluble organic carbon and bacterial β-glucosidase transcription was found, the above hypothesis could indicate either that soluble organic carbon is not the main pool of enzyme-inducing substrates or that constitutive production of bacterial β-glucosidase enzymes increases as soil conditions worsen. PMID:22837821

  17. Problem-Solving Test: Attenuation--A Mechanism to Regulate Bacterial Tryptophan Biosynthesis

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: tryptophan, transcription unit, operon, "trp" repressor, corepressor, operator, promoter, palindrome, initiation, elongation, and termination of transcription, open reading frame, coupled transcription/translation, chromosome-polysome complex. (Contains 2 figures and 1 footnote.)

  18. Nano Particles: Emerging Warheads Against Bacterial Superbugs.

    PubMed

    Dwivedi, Gaurav Raj; Sanchita; Singh, D P; Sharma, Ashok; Darokar, Mahendra P; Srivastava, Santosh K

    2016-01-01

    Infectious diseases are one of the major causes of morbidity and mortality in children in developing and underdeveloped countries. Limited knowledge of targets (cell wall synthesis, replication, transcription, protein synthesis) for antibiotics and lack of novel antibiotics have lead to an emergence of different level of resistance in bacterial pathogens. Multidrug resistance is the phenomenon by which the bacteria exerts resistance against the two or more structurally unrelated drugs/antibiotics. A common goal in the post-genomic era is to identify novel targets/drugs for various life threatening bacterial pathogens. Nanoparticles are broadly defined as submicron colloidal particles of size less than 1μm. Nanoparticles of size less than 100nm are the most promising warheads to overcome microbial drug resistance because they can act as antibacterial/antibiotic modulating agents at the site of infection and may have more than one mode of action. These nanoparticles will be of immense help in transporting drugs directly at the infected sites. Thus prevent drug resistance development to a great extent. In this review, the key mechanisms of resistance in bacterial superbugs have been discussed as well as how nanoparticles can overcome them. It is hypothesized that the nanoparticles can overcome the drug resistance via a novel mechanism of action. Additionaly, nanopaticles may also work synergistically with antibiotics via increased uptake, decreased efflux and inhibition of biofilm formation. The degradation by metallo beta lactamases and synthesis of porins may also be facilitated through these nanoparticles.

  19. Methylation of an intragenic alternative promoter regulates transcription of GARP.

    PubMed

    Haupt, Sonja; Söntgerath, Viktoria Sophie Apollonia; Leipe, Jan; Schulze-Koops, Hendrik; Skapenko, Alla

    2016-02-01

    Alternative promoter usage has been proposed as a mechanism regulating transcriptional and translational diversity in highly elaborated systems like the immune system in humans. Here, we report that transcription of human glycoprotein A repetitions predominant (GARP) in regulatory CD4 T cells (Tregs) is tightly regulated by two alternative promoters. An intragenic promoter contains several CpGs and acts as a weak promoter that is demethylated and initiates transcription Treg-specifically. The strong up-stream promoter containing a CpG-island is, in contrast, fully demethylated throughout tissues. Transcriptional activity of the strong promoter was surprisingly down-regulated upon demethylation of the weak promoter. This demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression and correlated with disease activity in rheumatoid arthritis. Treg-specific GARP transcription was initiated by synergistic interaction of forkhead box protein 3 (Foxp3) with nuclear factor of activated T cells (NFAT) and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. Our findings describe a novel function of alternative promoters in regulating the extent of transcription. Moreover, since GARP functions as a transporter of transforming growth factor β (TGFβ), a cytokine with broad pleiotropic traits, GARP transcriptional attenuation by alternative promoters might provide a mechanism regulating peripheral TGFβ to avoid unwanted harmful effects.

  20. RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways1[OPEN

    PubMed Central

    Bequette, Carlton J.; Fu, Zheng Qing; Loraine, Ann E.

    2016-01-01

    AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae. These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense. PMID:27208279

  1. Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors.

    PubMed

    Leedjärv, Anu; Ivask, Angela; Virta, Marko; Kahru, Anne

    2006-09-01

    A whole-cell recombinant bacterial sensor for the detection of phenolic compounds was constructed and used for the analysis of bioavailable phenols in natural samples. The sensor Pseudomonas fluorescens OS8(pDNdmpRlux) contains luxCDABE operon as a reporter under the control of phenol-inducible Po promoter from Pseudomonas sp. CF600. Expression of lux genes from the Po promoter, and thus the production of bioluminescence is controlled by the transcriptional activator DmpR, which initiates transcription in the presence of phenolic compounds. To take into account possible quenching (turbidity, toxicity) and/or stimulating effects of the environmental samples on the bacterial luminescence, control bacteria comparable to the sensors but lacking the phenol recognising elements were constructed and used in parallel in assays. The sensor bacteria were inducible with phenol, methylphenols, 2,3-, 2,4-, 2,6- and 3,4-dimethylphenol, resorcinol and 5-methylresorcinol but not with 2,5-dimethylresorcinol. The detection limits for different phenols varied from 0.03 mg/l (2-methylphenol) to 42.7 mg/l (5-methylresorcinol), being 0.08 mg/l for phenol, the most abundant phenolic contaminant in the environment. Different phenolic compounds had an additive effect on the inducibility of the sensor. The constructed sensor bacteria were applied on groundwaters and semi-coke leachates to estimate the bioavailable fraction of phenols. The sensor-determined amount of phenols in different samples varied from 6% to 95% of total phenol content depending on the nature of the sample. As the phenol-recognising unit in the sensor originates from a natural phenol biodegradation pathway, the sensor-determined amount of phenols corresponds to the biodegradable amount of phenolic pollutants in the samples and therefore this sensor could be used to estimate the natural biodegradation potential of phenolic compounds in the complex environmental mixtures and matrixes.

  2. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  3. Bacterial microflora of nectarines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microflora of fruit surfaces has been the best source of antagonists against fungi causing postharvest decays of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grapes, apples, and citrus fruit. We characterized bacterial microflora on nectarine f...

  4. Bacterial leaf spot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial leaf spot has been reported in Australia (Queensland), Egypt, El Salvador, India, Japan, Nicaragua, Sudan, and the United States (Florida, Iowa, Kansas, Maryland, and Wisconsin). It occasionally causes locally severe defoliation and post-emergence damping-off and stunting. The disease is...

  5. Bacterial inclusion body purification.

    PubMed

    Seras-Franzoso, Joaquin; Peternel, Spela; Cano-Garrido, Olivia; Villaverde, Antonio; García-Fruitós, Elena

    2015-01-01

    Purification of bacterial inclusion bodies (IBs) is gaining importance due to the raising of novel applications for this type of submicron particulate protein clusters, with potential uses in the biomedical field among others. Here, we present two optimized methods to purify IBs adapting classical procedures to the material nature as well as the requirements of its final application.

  6. Proteases in bacterial pathogenesis.

    PubMed

    Ingmer, Hanne; Brøndsted, Lone

    2009-11-01

    Bacterial pathogens rely on proteolysis for protein quality control under adverse conditions experienced in the host, as well as for the timely degradation of central virulence regulators. We have focused on the contribution of the conserved Lon, Clp, HtrA and FtsH proteases to pathogenesis and have highlighted common biological processes for which their activities are important for virulence.

  7. BACTERIAL WATERBORNE PATHOGENS

    EPA Science Inventory

    Bacterial pathogens are examples of classical etiological agents of waterborne disease. While these agents no longer serve as major threats to U.S. water supplies, they are still important pathogens in areas with substandard sanitation and poor water treatment facilities. In th...

  8. The Bacterial Growth Curve.

    ERIC Educational Resources Information Center

    Paulton, Richard J. L.

    1991-01-01

    A procedure that allows students to view an entire bacterial growth curve during a two- to three-hour student laboratory period is described. Observations of the lag phase, logarithmic phase, maximum stationary phase, and phase of decline are possible. A nonpathogenic, marine bacterium is used in the investigation. (KR)

  9. Transcriptional Regulation in Mammalian Cells by Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Mitchell, Pamela J.; Tjian, Robert

    1989-07-01

    The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

  10. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  11. Microdrill Initiative - Initial Market Evaluation

    SciTech Connect

    Spears & Associates, Inc

    2003-07-01

    The U.S. Department of Energy (DOE) is launching a major research and development initiative to create a small, fast, inexpensive and environmentally friendly rig for drilling 5000 feet boreholes to investigate potential oil and gas reservoirs. DOE wishes to get input from petroleum industry operators, service companies and equipment suppliers on the operation and application of this coiled-tubing-based drilling unit. To that end, DOE has asked Spears & Associates, Inc. (SAI) to prepare a special state-of-the-market report and assist during a DOE-sponsored project-scoping workshop in Albuquerque near the end of April 2003. The scope of the project is four-fold: (1) Evaluate the history, status and future of demand for very small bore-hole drilling; (2) Measure the market for coiled tubing drilling and describe the state-of-the-art; (3) Identify companies and individuals who should have an interest in micro drilling and invite them to the DOE workshop; and (4) Participate in 3 concurrent workshop sessions, record and evaluate participant comments and report workshop conclusions.

  12. Land use type significantly affects microbial gene transcription in soil.

    PubMed

    Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

    2014-05-01

    Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

  13. Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

    PubMed Central

    Castro, Joana; Alves, Patrícia; Sousa, Cármen; Cereija, Tatiana; França, Ângela; Jefferson, Kimberly K.; Cerca, Nuno

    2015-01-01

    Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development. PMID:26113465

  14. The nature of mutations induced by replication–transcription collisions.

    PubMed

    Sankar, T Sabari; Wastuwidyaningtyas, Brigitta D; Dong, Yuexin; Lewis, Sarah A; Wang, Jue D

    2016-07-07

    The DNA replication and transcription machineries share a common DNA template and thus can collide with each other co-directionally or head-on. Replication–transcription collisions can cause replication fork arrest, premature transcription termination, DNA breaks, and recombination intermediates threatening genome integrity. Collisions may also trigger mutations, which are major contributors to genetic disease and evolution. However, the nature and mechanisms of collision-induced mutagenesis remain poorly understood. Here we reveal the genetic consequences of replication–transcription collisions in actively dividing bacteria to be two classes of mutations: duplications/deletions and base substitutions in promoters. Both signatures are highly deleterious but are distinct from the previously well-characterized base substitutions in the coding sequence. Duplications/deletions are probably caused by replication stalling events that are triggered by collisions; their distribution patterns are consistent with where the fork first encounters a transcription complex upon entering a transcription unit. Promoter substitutions result mostly from head-on collisions and frequently occur at a nucleotide that is conserved in promoters recognized by the major σ factor in bacteria. This substitution is generated via adenine deamination on the template strand in the promoter open complex, as a consequence of head-on replication perturbing transcription initiation. We conclude that replication–transcription collisions induce distinct mutation signatures by antagonizing replication and transcription, not only in coding sequences but also in gene regulatory elements.

  15. The Nature of Mutations Induced by Replication-Transcription Collisions

    PubMed Central

    Sankar, T. Sabari; Wastuwidyaningtyas, Brigitta D.; Dong, Yuexin; Lewis, Sarah A.; Wang, Jue D.

    2016-01-01

    Summary The DNA replication and transcription machineries share a common DNA template and thus can collide with each other co-directionally or head-on1,2. Replication-transcription collisions can cause replication fork arrest, premature transcription termination, DNA breaks, and recombination intermediates threatening genome integrity1–10. Collisions may also trigger mutations, which are major contributors of genetic disease and evolution5,7,11. However, the nature and mechanisms of collision-induced mutagenesis remain poorly understood. Here we reveal the genetic consequence of replication-transcription collisions in actively dividing bacteria to be two classes of mutations: duplications/deletions and base substitutions in promoters. Both signatures are highly deleterious but are distinct from the well-characterized base substitutions in coding sequence. Duplications/deletions are likely caused by replication stalling events that are triggered by collisions; their distribution patterns are consistent with where the fork first encounters a transcription complex upon entering a transcription unit. Promoter substitutions result mostly from head-on collisions and frequently occur at a nucleotide conserved in promoters recognized by the major sigma factor in bacteria. This substitution is generated via adenine deamination on the template strand in the promoter open complex, as a consequence of head-on replication perturbing transcription initiation. We conclude that replication-transcription collisions induce distinct mutation signatures by antagonizing replication and transcription, not only in coding sequences but also in gene regulatory elements. PMID:27362223

  16. Machine Transcription--Practically Speaking.

    ERIC Educational Resources Information Center

    Clippinger, Dorinda A.

    1984-01-01

    Draws transcription teaching principles from Gagne's theories about learning. Recommends 12-16 weeks of instruction, pre-transcription development of related skills, frequent feedback, and use of teaching materials that are arranged to take advantage of learning cycles. (SK)

  17. Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

    PubMed Central

    Brandt, Stephanie L.; Ke, Wujian; Reid, Tara B.; Molini, Barbara J.; Iverson-Cabral, Stefanie; Ciccarese, Giulia; Drago, Francesco; Lukehart, Sheila A.; Centurion-Lara, Arturo

    2015-01-01

    An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis. PMID:25802057

  18. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

    PubMed Central

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E.; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-01-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  19. Nuclear stability and transcriptional directionality separate functionally distinct RNA species.

    PubMed

    Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind; Core, Leighton J; Bornholdt, Jette; Boyd, Mette; Heick Jensen, Torben; Sandelin, Albin

    2014-11-12

    Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

  20. Dynamic usage of transcription start sites within core promoters

    PubMed Central

    Kawaji, Hideya; Frith, Martin C; Katayama, Shintaro; Sandelin, Albin; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide

    2006-01-01

    Background Mammalian promoters do not initiate transcription at single, well defined base pairs, but rather at multiple, alternative start sites spread across a region. We previously characterized the static structures of transcription start site usage within promoters at the base pair level, based on large-scale sequencing of transcript 5' ends. Results In the present study we begin to explore the internal dynamics of mammalian promoters, and demonstrate that start site selection within many mouse core promoters varies among tissues. We also show that this dynamic usage of start sites is associated with CpG islands, broad and multimodal promoter structures, and imprinting. Conclusion Our results reveal a new level of biologic complexity within promoters - fine-scale regulation of transcription starting events at the base pair level. These events are likely to be related to epigenetic transcriptional regulation. PMID:17156492

  1. Bacterial community reconstruction using compressed sensing.

    PubMed

    Amir, Amnon; Zuk, Or

    2011-11-01

    Bacteria are the unseen majority on our planet, with millions of species and comprising most of the living protoplasm. We propose a novel approach for reconstruction of the composition of an unknown mixture of bacteria using a single Sanger-sequencing reaction of the mixture. Our method is based on compressive sensing theory, which deals with reconstruction of a sparse signal using a small number of measurements. Utilizing the fact that in many cases each bacterial community is comprised of a small subset of all known bacterial species, we show the feasibility of this approach for determining the composition of a bacterial mixture. Using simulations, we show that sequencing a few hundred base-pairs of the 16S rRNA gene sequence may provide enough information for reconstruction of mixtures containing tens of species, out of tens of thousands, even in the presence of realistic measurement noise. Finally, we show initial promising results when applying our method for the reconstruction of a toy experimental mixture with five species. Our approach may have a potential for a simple and efficient way for identifying bacterial species compositions in biological samples. All supplementary data and the MATLAB code are available at www.broadinstitute.org/?orzuk/publications/BCS/.

  2. Effect of aerosolization on subsequent bacterial survival.

    PubMed Central

    Walter, M V; Marthi, B; Fieland, V P; Ganio, L M

    1990-01-01

    To determine whether aerosolization could impair bacterial survival, Pseudomonas syringae and Erwinia herbicola were aerosolized in a greenhouse, the aerosol was sampled at various distances from the site of release by using all-glass impingers, and bacterial survival was followed in the impingers for 6 h. Bacterial survival subsequent to aerosolization of P. syringae and E. herbicola was not impaired 1 m from the site of release. P. syringae aerosolized at 3 to 15 m from the site of release at a temperature of 12 degrees C and a relative humidity of 80% survived 35- to 65-fold better than P. syringae released at 27 degrees C and a relative humidity of 40%. No difference was observed in the survival of P. syringae and E. herbicola following aerosolization at the same temperature and relative humidity. Bacteria sprayed directly onto bean and oat plants established stable populations at comparable numbers on both plants over an 8-day period following inoculation. Bacteria that inoculated adjacent plants by drifting downwind up to 5 m were detectable at an initial population of 10(2) CFU/g on oats and 10(5) CFU/g on beans 2 h after the spray. However, bacterial populations on both plants were undetectable within 48 h. PMID:2268157

  3. The Lrp family of transcription regulators in archaea.

    PubMed

    Peeters, Eveline; Charlier, Daniel

    2010-11-30

    Archaea possess a eukaryotic-type basal transcription apparatus that is regulated by bacteria-like transcription regulators. A universal and abundant family of transcription regulators are the bacterial/archaeal Lrp-like regulators. The Lrp family is one of the best studied regulator families in archaea, illustrated by investigations of proteins from the archaeal model organisms: Sulfolobus, Pyrococcus, Methanocaldococcus, and Halobacterium. These regulators are extremely versatile in their DNA-binding properties, response to effector molecules, and molecular regulatory mechanisms. Besides being involved in the regulation of the amino acid metabolism, they also regulate central metabolic processes. It appears that these regulatory proteins are also involved in large regulatory networks, because of hierarchical regulations and the possible combinatorial use of different Lrp-like proteins. Here, we discuss the recent developments in our understanding of this important class of regulators.

  4. Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

    PubMed

    Metcalf, Kevin J; Tullman-Ercek, Danielle

    2017-01-01

    Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

  5. Formation of bacterial nanocells

    NASA Astrophysics Data System (ADS)

    Vainshtein, Mikhail; Kudryashova, Ekaterina; Suzina, Natalia; Ariskina, Elena; Voronkov, Vadim

    1998-07-01

    Existence of nanobacteria received increasing attention both in environmental microbiology/geomicro-biology and in medical microbiology. In order to study a production of nanoforms by typical bacterial cells. Effects of different physical factors were investigated. Treatment of bacterial cultures with microwave radiation, or culturing in field of electric current resulted in formation a few types of nanocells. The number and type of nanoforms were determined with type and dose of the treatment. The produced nanoforms were: i) globules, ii) clusters of the globules--probably produced by liaison, iii) nanocells coated with membrane. The viability of the globules is an object opened for doubts. The nanocells discovered multiplication and growth on solidified nutrient media. The authors suggest that formation of nanocells is a common response of bacteria to stress-actions produced by different agents.

  6. Bacterial Genome Instability

    PubMed Central

    Darmon, Elise

    2014-01-01

    SUMMARY Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease. PMID:24600039

  7. Flagella and bacterial pathogenicity.

    PubMed

    Duan, Qiangde; Zhou, Mingxu; Zhu, Liqian; Zhu, Guoqiang

    2013-01-01

    As locomotive organelles, flagella allow bacteria to move toward favorable environments. A flagellum consists of three parts: the basal structure (rotary motor), the hook (universal joint), and the filament (helical propeller). For ages, flagella have been generally regarded as important virulence factors, mainly because of their motility property. However, flagella are getting recognized to play multiple roles with more functions besides motility and chemotaxis. Recent evidence has pinpointed that the bacterial flagella participate in many additional processes including adhesion, biofilm formation, virulence factor secretion, and modulation of the immune system of eukaryotic cells. This mini-review summarizes data from recent studies that elucidated how flagella, as a virulence factor, contribute to bacterial pathogenicity.

  8. Bacterial ratchet motors

    PubMed Central

    Di Leonardo, R.; Angelani, L.; Dell’Arciprete, D.; Ruocco, G.; Iebba, V.; Schippa, S.; Conte, M. P.; Mecarini, F.; De Angelis, F.; Di Fabrizio, E.

    2010-01-01

    Self-propelling bacteria are a nanotechnology dream. These unicellular organisms are not just capable of living and reproducing, but they can swim very efficiently, sense the environment, and look for food, all packaged in a body measuring a few microns. Before such perfect machines can be artificially assembled, researchers are beginning to explore new ways to harness bacteria as propelling units for microdevices. Proposed strategies require the careful task of aligning and binding bacterial cells on synthetic surfaces in order to have them work cooperatively. Here we show that asymmetric environments can produce a spontaneous and unidirectional rotation of nanofabricated objects immersed in an active bacterial bath. The propulsion mechanism is provided by the self-assembly of motile Escherichia coli cells along the rotor boundaries. Our results highlight the technological implications of active matter’s ability to overcome the restrictions imposed by the second law of thermodynamics on equilibrium passive fluids. PMID:20457936

  9. Bacterial transformation of terpenoids

    NASA Astrophysics Data System (ADS)

    Grishko, V. V.; Nogovitsina, Y. M.; Ivshina, I. B.

    2014-04-01

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references.

  10. Transcription regulation by distal enhancers: who's in the loop?

    PubMed

    Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

    2012-01-01

    Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.

  11. Positively regulated bacterial expression systems

    PubMed Central

    Brautaset, Trygve; Lale, Rahmi; Valla, Svein

    2009-01-01

    Summary Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high‐level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC‐XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (l‐arabinose, l‐rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone‐related compounds, ε‐caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC‐XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/PBAD, RhaR‐RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications. PMID:21261879

  12. Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association.

    PubMed

    Chun, Carlene K; Troll, Joshua V; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E; Bonaldo, Maria de Fatima; Casavant, Thomas L; Soares, M Bento; Ruby, Edward G; McFall-Ngai, Margaret J

    2008-08-12

    The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

  13. Bacterial nitric oxide synthases.

    PubMed

    Crane, Brian R; Sudhamsu, Jawahar; Patel, Bhumit A

    2010-01-01

    Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.

  14. Bacterial infections in cirrhosis.

    PubMed

    Garcia-Tsao, Guadalupe

    2004-06-01

    Hospitalized patients with cirrhosis are at increased risk of developing bacterial infections, the most common being spontaneous bacterial peritonitis (SBP) and urinary tract infections. Independent predictors of the development of bacterial infections in hospitalized cirrhotic patients are poor liver synthetic function and admission for gastrointestinal hemorrhage. Short term (seven-day) prophylaxis with norfloxacin reduces the rate of infections and improves survival and should therefore be administered to all patients with cirrhosis and variceal hemorrhage. Cirrhotic patients who develop abdominal pain, tenderness, fever, renal failure or hepatic encephalopathy should undergo diagnostic paracentesis, and those who meet the criterion for SBP (eg, an ascites neutrophil count greater than 250/mm3) should receive antibiotics, preferably a third-generation cephalosporin. In addition to antibiotic therapy, albumin infusions have been shown to reduce the risk of renal failure and mortality in patients with SBP, particularly in those with renal dysfunction and hyperbilirubinemia at the time of diagnosis. Patients who recover from an episode of SBP should be given long term prophylaxis with norfloxacin and should be assessed for liver transplantation.