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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. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  3. The sabotage of the bacterial transcription machinery by a small bacteriophage protein.

    PubMed

    Liu, Bing; Shadrin, Andrey; Sheppard, Carol; Mekler, Vladimir; Xu, Yingqi; Severinov, Konstantin; Matthews, Steve; Wigneshweraraj, Sivaramesh

    2014-01-01

    Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research. PMID:24701369

  4. A dual switch controls bacterial enhancer-dependent transcription

    PubMed Central

    Wiesler, Simone C.; Burrows, Patricia C.; Buck, Martin

    2012-01-01

    Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation. PMID:22965125

  5. 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

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

    PubMed

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

    2016-03-01

    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

  7. 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

  8. Transcription initiation complex structures elucidate DNA opening.

    PubMed

    Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

    2016-05-19

    Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts. PMID:27193681

  9. Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex

    PubMed Central

    Mekler, Vladimir; Minakhin, Leonid; Borukhov, Sergei; Mustaev, Arkady; Severinov, Konstantin

    2014-01-01

    Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT-rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis. PMID:25311862

  10. 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. PMID:21326901

  11. Spatial organization of bacterial transcription and translation.

    PubMed

    Castellana, Michele; Hsin-Jung Li, Sophia; Wingreen, Ned S

    2016-08-16

    In bacteria such as Escherichia coli, DNA is compacted into a nucleoid near the cell center, whereas ribosomes-molecular complexes that translate mRNAs into proteins-are mainly localized to the poles. We study the impact of this spatial organization using a minimal reaction-diffusion model for the cellular transcriptional-translational machinery. Although genome-wide mRNA-nucleoid segregation still lacks experimental validation, our model predicts that [Formula: see text] of mRNAs are segregated to the poles. In addition, our analysis reveals a "circulation" of ribosomes driven by the flux of mRNAs, from synthesis in the nucleoid to degradation at the poles. We show that our results are robust with respect to multiple, biologically relevant factors, such as mRNA degradation by RNase enzymes, different phases of the cell division cycle and growth rates, and the existence of nonspecific, transient interactions between ribosomes and mRNAs. Finally, we confirm that the observed nucleoid size stems from a balance between the forces that the chromosome and mRNAs exert on each other. This suggests a potential global feedback circuit in which gene expression feeds back on itself via nucleoid compaction. PMID:27486246

  12. 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. PMID:26733675

  13. Listening Instruction and Initial Machine Transcription Performance.

    ERIC Educational Resources Information Center

    Remp, Ann M.

    1984-01-01

    The problem of this study was to determine whether introductory machine transcription materials that incorporate listening principles and techniques would facilitate the fusion of keyboarding, language application, and proofreading skills. (JOW)

  14. Fidaxomicin Is an Inhibitor of the Initiation of Bacterial RNA Synthesis

    PubMed Central

    Artsimovitch, Irina; Seddon, Jaime; Sears, Pamela

    2012-01-01

    Fidaxomicin was recently approved for the treatment of Clostridium difficile infection. It inhibits transcription by bacterial RNA polymerase. Because transcription is a multistep process, experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step. DNA footprinting experiments were also conducted to further elucidate the stage inhibited. Fidaxomicin blocks initiation only if added before the formation of the “open promoter complex,” in which the template DNA strands have separated but RNA synthesis has not yet begun. Binding of fidaxomicin precludes the initial separation of DNA strands that is prerequisite to RNA synthesis. These studies show that it has a mechanism distinct from that of elongation inhibitors, such as streptolydigin, and from the transcription initiation inhibitors myxopyronin and the rifamycins. PMID:22752861

  15. An Ising model of transcription polarity in bacterial chromosomes

    NASA Astrophysics Data System (ADS)

    Baran, Robert H.; Ko, Hanseok

    2006-04-01

    Bacterial genes form clusters of the same transcription polarity and typically exhibit a preference to be coded on the leading strand of replication. An Ising model is proposed to quantify these two phenomena by analogy to the behavior of magnetic dipoles (spins) in a one-dimensional lattice. Corresponding to magnetic forces that co-orient adjacent spins and align them with an externally applied field, we imagine pseudo-forces that influence transcription polarity. Bonds of uniform strength {1}/{2} J between adjacent sites will model the adhesive (or repulsive) interactions while a polarity entraining force of strength H has the direction of replication. Ten bacterial chromosomes are reduced to spin configurations from which the model parameters are estimated by the method of maximum likelihood under the assumption of thermal equilibrium, following the application of established methods to locate replication origins and termini. χ 2-tests show that the model fits the data well in about half the cases but cluster size exhibits excess variance in general. These findings lead to a speculative interpretation of the pseudo-forces as the net effects of numerous insertions and deletions that succeed or fail according to their impact on the motions of enzymatic complexes involved in replication and transcription.

  16. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    PubMed Central

    Kort, Remco; Keijser, Bart J; Caspers, Martien PM; Schuren, Frank H; Montijn, Roy

    2008-01-01

    Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability. PMID:19061518

  17. 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

  18. A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing

    PubMed Central

    Berdygulova, Zhanna; Esyunina, Daria; Miropolskaya, Nataliya; Mukhamedyarov, Damir; Kuznedelov, Konstantin; Nickels, Bryce E.; Severinov, Konstantin; Kulbachinskiy, Andrey; Minakhin, Leonid

    2012-01-01

    Gp39, a small protein encoded by Thermus thermophilus phage P23–45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity. PMID:22238378

  19. The 26S Proteasome and Initiation of Gene Transcription

    PubMed Central

    Durairaj, Geetha; Kaiser, Peter

    2014-01-01

    Transcription activation is the foremost step of gene expression and is modulated by various factors that act in synergy. Misregulation of this process and its associated factors has severe effects and hence requires strong regulatory control. In recent years, growing evidence has highlighted the 26S proteasome as an important contributor to the regulation of transcription initiation. Well known for its role in protein destruction, its contribution to protein synthesis was initially viewed with skepticism. However, studies over the past several years have established the proteasome as an important component of transcription initiation through proteolytic and non-proteolytic activities. In this review, we discuss findings made so far in understanding the connections between transcription initiation and the 26S proteasome complex. PMID:25211636

  20. Dynamic competition between transcription initiation and repression: Role of nonequilibrium steps in cell-to-cell heterogeneity.

    PubMed

    Mitarai, Namiko; Semsey, Szabolcs; Sneppen, Kim

    2015-08-01

    Transcriptional repression may cause transcriptional noise by a competition between repressor and RNA polymerase binding. Although promoter activity is often governed by a single limiting step, we argue here that the size of the noise strongly depends on whether this step is the initial equilibrium binding or one of the subsequent unidirectional steps. Overall, we show that nonequilibrium steps of transcription initiation systematically increase the cell-to-cell heterogeneity in bacterial populations. In particular, this allows also weak promoters to give substantial transcriptional noise. PMID:26382435

  1. 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

  2. Organization of the human mitochondrial transcription initiation complex

    PubMed Central

    Yakubovskaya, Elena; Guja, Kip E.; Eng, Edward T.; Choi, Woo Suk; Mejia, Edison; Beglov, Dmitri; Lukin, Mark; Kozakov, Dima; Garcia-Diaz, Miguel

    2014-01-01

    Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein–protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex. PMID:24413562

  3. Structural insights into transcription initiation by RNA polymerase II

    PubMed Central

    Grünberg, Sebastian; Hahn, Steven

    2013-01-01

    Transcriptional regulation is one of the most important steps in control of cell identity, growth, differentiation and development. Many signaling pathways controlling these processes ultimately target the core transcription machinery that, for protein coding genes, consists of RNA polymerase II (Pol II) and the general transcription factors (GTFs). New studies on the structure and mechanism of the core assembly and how it interfaces with promoter DNA and coactivator complexes have given tremendous insight into early steps in the initiation process, genome-wide binding, and mechanisms conserved for all nuclear and archaeal Pols. Here we review recent developments in dissecting the architecture of the Pol II core machinery with a focus on early and regulated steps in transcription initiation. PMID:24120742

  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. Altered Nucleosome Positioning at the Transcription Start Site and Deficient Transcriptional Initiation in Friedreich Ataxia*

    PubMed Central

    Chutake, Yogesh K.; Costello, Whitney N.; Lam, Christina; Bidichandani, Sanjay I.

    2014-01-01

    Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation. PMID:24737321

  6. Bacterial effectors target the plant cell nucleus to subvert host transcription

    PubMed Central

    Canonne, Joanne; Rivas, Susana

    2012-01-01

    In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells. PMID:22353865

  7. The impact of structure dimensions on initial bacterial adhesion.

    PubMed

    Helbig, Ralf; Günther, Denise; Friedrichs, Jens; Rößler, Florian; Lasagni, Andrés; Werner, Carsten

    2016-07-21

    Substrate topography can have profound effects on initial bacterial adhesion during biofilm formation. We applied Staphylococcus epidermidis and Escherichia coli cells onto periodically structured substrates with different structure dimensions, structure types and wetting properties. We found a strong dependence of cell retention on the structure dimensions of the applied substrates. Periodicities in the range of the cell size increased, whereas smaller periodicities decreased cell retention, independent of contact time (minutes to hours) and hydrophobicity. These novel insights on the role of surface topography on bacterial retention might facilitate the development of non-fouling surfaces in the future. PMID:27232637

  8. 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

  9. 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. PMID:27026687

  10. CRISPR-mediated control of the bacterial initiation of replication.

    PubMed

    Wiktor, Jakub; Lesterlin, Christian; Sherratt, David J; Dekker, Cees

    2016-05-01

    Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering. PMID:27036863

  11. CRISPR-mediated control of the bacterial initiation of replication

    PubMed Central

    Wiktor, Jakub; Lesterlin, Christian; Sherratt, David J.; Dekker, Cees

    2016-01-01

    Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC. We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering. PMID:27036863

  12. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  13. 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. PMID:23756343

  14. Transcriptional unit of the murine Thy-1 gene: different distribution of transcription initiation sites in brain.

    PubMed Central

    Spanopoulou, E; Giguere, V; Grosveld, F

    1988-01-01

    Structural analysis of the mouse Thy-1.2 gene has shown that the major promoter of the gene is characterized by a tissue-specific DNase I-hypersensitive site and is located within a methylation-free island. The gene is regulated at the transcriptional level, and steady-state mRNA analysis reveals that the previously reported exon Ib contributes at most 5% of the total mRNA. The major promoter uses several transcription initiation sites within a region of 100 base pairs. The frequency of usage of these sites in brain is markedly different from that in other tissues. Images PMID:2906111

  15. Domain structure of a human general transcription initiation factor, TFIIF.

    PubMed Central

    Yonaha, M; Aso, T; Kobayashi, Y; Vasavada, H; Yasukochi, Y; Weissman, S M; Kitajima, S

    1993-01-01

    The structural and functional domains of a general transcription initiation factor, TFIIF (RAP30/74, FC), have been investigated using various deletion mutants of each subunit, both in vivo and in vitro. An in vivo assay showed that the N-terminal sequence containing residues of 1-110 of RAP30 that is located close to a sigma homology region interacts with a minimum sequence of residues 62-171 of RAP74 to form a heteromeric interaction. Reconstitution of in vitro transcription activity by deletion mutants of RAP74 clearly indicated that both N-terminal residues 73-205 and C-terminal residues 356-517 are essential for full activity, the former interacting with RAP30, thus complexing with RNA polymerase II. From these data, the functional significance of domain structure of TFIIF is discussed in terms of its sigma homology sequences and complex formation with RNA polymerase II in the initiation and elongation of transcription. Images PMID:8441635

  16. 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. PMID:26934187

  17. 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

  18. Mechanisms of Bacterial Transcription Termination: All Good Things Must End.

    PubMed

    Ray-Soni, Ananya; Bellecourt, Michael J; Landick, Robert

    2016-06-01

    Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination. PMID:27023849

  19. 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

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

    SciTech Connect

    Volkman, B.F.

    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 {open_quote}receiver domain{close_quote} in the family of {open_quote}two-component{close_quote} 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.

  1. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    PubMed Central

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. Conclusions Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution. PMID:23914835

  2. Transcriptional response of Musca domestica larvae to bacterial infection.

    PubMed

    Tang, Ting; Li, Xiang; Yang, Xue; Yu, Xue; Wang, Jianhui; Liu, Fengsong; Huang, Dawei

    2014-01-01

    The house fly Musca domestica, a cosmopolitan dipteran insect, is a significant vector for human and animal bacterial pathogens, but little is known about its immune response to these pathogens. To address this issue, we inoculated the larvae with a mixture of Escherichia coli and Staphylococcus aureus and profiled the transcriptome 6, 24, and 48 h thereafter. Many genes known to controlling innate immunity in insects were induced following infection, including genes encoding pattern recognition proteins (PGRPs), various components of the Toll and IMD signaling pathways and of the proPO-activating and redox systems, and multiple antimicrobial peptides. Interestingly, we also uncovered a large set of novel immune response genes including two broad-spectrum antimicrobial peptides (muscin and domesticin), which might have evolved to adapt to house-fly's unique ecological environments. Finally, genes mediating oxidative phosphorylation were repressed at 48 h post-infection, suggesting disruption of energy homeostasis and mitochondrial function at the late stages of infection. Collectively, our data reveal dynamic changes in gene expression following bacterial infection in the house fly, paving the way for future in-depth analysis of M. domestica's immune system. PMID:25137050

  3. Transcriptional Response of Musca domestica Larvae to Bacterial Infection

    PubMed Central

    Tang, Ting; Li, Xiang; Yang, Xue; Yu, Xue; Wang, Jianhui; Liu, Fengsong; Huang, Dawei

    2014-01-01

    The house fly Musca domestica, a cosmopolitan dipteran insect, is a significant vector for human and animal bacterial pathogens, but little is known about its immune response to these pathogens. To address this issue, we inoculated the larvae with a mixture of Escherichia coli and Staphylococcus aureus and profiled the transcriptome 6, 24, and 48 h thereafter. Many genes known to controlling innate immunity in insects were induced following infection, including genes encoding pattern recognition proteins (PGRPs), various components of the Toll and IMD signaling pathways and of the proPO-activating and redox systems, and multiple antimicrobial peptides. Interestingly, we also uncovered a large set of novel immune response genes including two broad-spectrum antimicrobial peptides (muscin and domesticin), which might have evolved to adapt to house-fly's unique ecological environments. Finally, genes mediating oxidative phosphorylation were repressed at 48 h post-infection, suggesting disruption of energy homeostasis and mitochondrial function at the late stages of infection. Collectively, our data reveal dynamic changes in gene expression following bacterial infection in the house fly, paving the way for future in-depth analysis of M. domestica's immune system. PMID:25137050

  4. Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles.

    PubMed

    Traverse, Charles C; Ochman, Howard

    2016-03-22

    Errors that occur during transcription have received much less attention than the mutations that occur in DNA because transcription errors are not heritable and usually result in a very limited number of altered proteins. However, transcription error rates are typically several orders of magnitude higher than the mutation rate. Also, individual transcripts can be translated multiple times, so a single error can have substantial effects on the pool of proteins. Transcription errors can also contribute to cellular noise, thereby influencing cell survival under stressful conditions, such as starvation or antibiotic stress. Implementing a method that captures transcription errors genome-wide, we measured the rates and spectra of transcription errors in Escherichia coli and in endosymbionts for which mutation and/or substitution rates are greatly elevated over those of E. coli Under all tested conditions, across all species, and even for different categories of RNA sequences (mRNA and rRNAs), there were no significant differences in rates of transcription errors, which ranged from 2.3 × 10(-5) per nucleotide in mRNA of the endosymbiont Buchnera aphidicola to 5.2 × 10(-5) per nucleotide in rRNA of the endosymbiont Carsonella ruddii The similarity of transcription error rates in these bacterial endosymbionts to that in E. coli (4.63 × 10(-5) per nucleotide) is all the more surprising given that genomic erosion has resulted in the loss of transcription fidelity factors in both Buchnera and Carsonella. PMID:26884158

  5. 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

  6. Molecular Mechanisms of Transcription Initiation-Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation.

    PubMed

    Blombach, Fabian; Smollett, Katherine L; Grohmann, Dina; Werner, Finn

    2016-06-19

    Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the β-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery. PMID:27107643

  7. Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation

    PubMed Central

    Junier, Ivan; Rivoire, Olivier

    2016-01-01

    Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. PMID:27195891

  8. Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

    PubMed Central

    Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

    2013-01-01

    The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

  9. Structural basis of initial RNA polymerase II transcription

    PubMed Central

    Cheung, Alan C M; Sainsbury, Sarah; Cramer, Patrick

    2011-01-01

    During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II–DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site. The ITC-mimicking structures show that two invariant lysine residues anchor the 3′-proximal phosphate of short RNAs. Short DNA–RNA hybrids adopt a tilted conformation that excludes the +1 template nt from the active site. NTP binding induces complete DNA translocation and the standard hybrid conformation. Conserved NTP contacts indicate a universal mechanism of NTP selection. The essential residue Q1078 in the closed trigger loop binds the NTP 2′-OH group, explaining how the trigger loop couples catalysis to NTP selection, suppressing dNTP binding and DNA synthesis. PMID:22056778

  10. 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. PMID:16190201

  11. 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.

  12. Bacterial Sigma Factors as Targets for Engineered or Synthetic Transcriptional Control

    PubMed Central

    Tripathi, Lakshmi; Zhang, Yan; Lin, Zhanglin

    2014-01-01

    Sigma (σ) factors are the predominant constituents of transcription regulation in bacteria. σ Factors recruit the core RNA polymerase to recognize promoters with specific DNA sequences. Recently, engineering of transcriptional regulators has become a significant tool for strain engineering. The present review summarizes the recent advances in σ factor based engineering or synthetic design. The manipulation of σ factors presents insights into the bacterial stress tolerance and metabolite productivity. We envision more synthetic design based on σ factors that can be used to tune the regulatory network of bacteria. PMID:25232540

  13. Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

    PubMed Central

    Petushkov, Ivan; Pupov, Danil; Bass, Irina; Kulbachinskiy, Andrey

    2015-01-01

    During transcription, the catalytic core of RNA polymerase (RNAP) must interact with the DNA template with low-sequence specificity to ensure efficient enzyme translocation and RNA extension. Unexpectedly, recent structural studies of bacterial promoter complexes revealed specific interactions between the nontemplate DNA strand at the downstream edge of the transcription bubble (CRE, core recognition element) and a protein pocket formed by core RNAP (CRE pocket). We investigated the roles of these interactions in transcription by analyzing point amino acid substitutions and deletions in Escherichia coli RNAP. The mutations affected multiple steps of transcription, including promoter recognition, RNA elongation and termination. In particular, we showed that interactions of the CRE pocket with a nontemplate guanine immediately downstream of the active center stimulate RNA-hairpin-dependent transcription pausing but not other types of pausing. Thus, conformational changes of the elongation complex induced by nascent RNA can modulate CRE effects on transcription. The results highlight the roles of specific core RNAP–DNA interactions at different steps of RNA synthesis and suggest their importance for transcription regulation in various organisms. PMID:25990734

  14. 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. PMID:27178279

  15. Global analysis of bacterial transcription factors to predict cellular target processes.

    PubMed

    Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

    2004-03-01

    Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs. PMID:15049306

  16. 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. PMID:25431049

  17. Mechanics of Bacterial Cells and Initial Surface Colonisation.

    PubMed

    Aguayo, Sebastian; Bozec, Laurent

    2016-01-01

    The mechanical properties of bacterial cells play an important role in crucial bacterial processes such as cell growth, colonisation and biofilm formation. Recent developments in the field of nanotechnology and atomic force microscopy (AFM) have made it possible to observe, characterise and understand the nanomechanic behaviour of live bacterial cells as never before. Unlike traditional techniques, AFM makes it possible to employ living bacteria in their physiological environment with minimal or no sample preparation. The technique of AFM nanoindentation opens new possibilities to study bacterial cell wall stiffness under different mechanical and buffer conditions. Also, by attaching bacterial cells to functionalised AFM cantilevers, single-cell force spectroscopy (SCFS) can be used to measure the adhesion of bacteria to biological and non-biological substrates at the nano-newton and pico-newton scale, and provide specific information on receptor-ligand interactions. By studying the biophysics of the bacterial-surface interaction with the abovementioned techniques, it has been possible to gain new insight on the early stages of bacterial colonisation and biofilm formation. PMID:27193547

  18. A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.

    PubMed

    Shelburne, Samuel A; Olsen, Randall J; Suber, Bryce; Sahasrabhojane, Pranoti; Sumby, Paul; Brennan, Richard G; Musser, James M

    2010-03-01

    Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both DeltaccpA and DeltacovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection. PMID:20333240

  19. 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. PMID:27226527

  20. Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response

    PubMed Central

    Lamacchia, Marina; Dyrka, Witold; Breton, Annick; Saupe, Sven J.; Paoletti, Mathieu

    2016-01-01

    Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most

  1. Cysteine sulfur chemistry in transcriptional regulators at the host-bacterial pathogen interface.

    PubMed

    Luebke, Justin L; Giedroc, David P

    2015-06-01

    Hosts employ myriad weapons to combat invading microorganisms as an integral feature of the host-bacterial pathogen interface. This interface is dominated by highly reactive small molecules that collectively induce oxidative stress. Successful pathogens employ transcriptional regulatory proteins that sense these small molecules directly or indirectly via a change in the ratio of reduced to oxidized low-molecular weight (LMW) thiols that collectively comprise the redox buffer in the cytoplasm. These transcriptional regulators employ either a prosthetic group or reactive cysteine residue(s) to effect changes in the transcription of genes that encode detoxification and repair systems that is driven by regulator conformational switching between high-affinity and low-affinity DNA-binding states. Cysteine harbors a highly polarizable sulfur atom that readily undergoes changes in oxidation state in response to oxidative stress to produce a range of regulatory post-translational modifications (PTMs), including sulfenylation (S-hydroxylation), mixed disulfide bond formation with LMW thiols (S-thiolation), di- and trisulfide bond formation, S-nitrosation, and S-alkylation. Here we discuss several examples of structurally characterized cysteine thiol-specific transcriptional regulators that sense changes in cellular redox balance, focusing on the nature of the cysteine PTM itself and the interplay of small molecule oxidative stressors in mediating a specific transcriptional response. PMID:25946648

  2. The Bacterial Transcription Termination Factor Rho Coordinates Mg(2+) Homeostasis with Translational Signals.

    PubMed

    Kriner, Michelle A; Groisman, Eduardo A

    2015-12-01

    The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5' leader region or continues into the downstream coding region. Here, we report that the Mg(2+) channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5' leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive conformations that determine whether or not Rho interacts with a Rho utilization site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella's other two Mg(2+) transport genes, suggesting that Rho links Mg(2+) uptake to translational signals. PMID:26523680

  3. The Interaction Surface of a Bacterial Transcription Elongation Factor Required for Complex Formation with an Antiterminator during Transcription Antitermination*

    PubMed Central

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

    2013-01-01

    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. PMID:23913688

  4. BACTERIAL SOURCE TRACKING (BST) INITIATIVE FOR THE GULF OF MEXICO

    EPA Science Inventory

    This project will sponsor several meetings to enable scientists to discuss mutual problems and solutions regarding bacterial source tracking methods. Issues common to all the Gulf states include but are not limited to beach closures, TMDL analysis, longevity of indicator organism...

  5. Mapping in vivo initiation sites of RNA transcription and determining their relative use.

    PubMed Central

    Kessler, M; Aloni, Y

    1984-01-01

    Runoff transcripts were generated on viral transcriptional complexes cleaved with restriction enzymes and incubated in vitro with [alpha-32P]UTP under pulse-chase conditions. As viral transcriptional complexes in vitro elongated the nascent RNA preinitiated in vivo, size analysis by gel electrophoresis of the runoff transcripts allowed identification of the in vivo initiation sites. Moreover, scanning the intensities of the runoff bands as they appeared in the autoradiogram of the gel allowed determination of the relative use of these sites. A model system in which the initiation sites of simian virus 40 late RNA were identified and their relative use determined is presented. Images PMID:6090704

  6. 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. PMID:27129284

  7. Transcription initiation at the tet promoter and effect of mutations.

    PubMed Central

    Harley, C B; Lawrie, J; Betlach, M; Crea, R; Boyer, H W; Hedgpeth, J

    1988-01-01

    We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence. Images PMID:3045754

  8. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators.

    PubMed

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan

    2015-09-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration. PMID:26220449

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

    PubMed

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

    2011-06-01

    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

  10. Bacterial Transcription Inhibitor of RNA Polymerase Holoenzyme Formation by Structure-Based Drug Design: From in Silico Screening to Validation.

    PubMed

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

    2016-01-01

    Bacterial transcription is a proven target for antibacterial research. However, most of the known inhibitors targeting transcription are from natural extracts or are hits from screens where the binding site remains unidentified. Using an RNA polymerase holoenzyme homology structure from the model Gram-positive organism Bacillus subtilis, we created a pharmacophore model and used it for in silico screening of a publicly available library for compounds able to inhibit holoenzyme formation. The hits demonstrated specific affinity to bacterial RNA polymerase and excellent activity using in vitro assays and showed no binding to the equivalent structure from human RNA polymerase II. The target specificity in live cells and antibacterial activity was demonstrated in microscopy and growth inhibition experiments. This is the first example of targeted inhibitor development for a bacterial RNA polymerase, outlining a complete discovery process from virtual screening to biochemical validation. This approach could serve as an appropriate platform for the future identification of inhibitors of bacterial transcription. PMID:27622946

  11. Genomic context drives transcription of insertion sequences in the bacterial endosymbiont Wolbachia wVulC.

    PubMed

    Cerveau, Nicolas; Gilbert, Clément; Liu, Chao; Garrett, Roger A; Grève, Pierre; Bouchon, Didier; Cordaux, Richard

    2015-06-10

    Transposable elements (TEs) are DNA pieces that are present in almost all the living world at variable genomic density. Due to their mobility and density, TEs are involved in a large array of genomic modifications. In eukaryotes, TE expression has been studied in detail in several species. In prokaryotes, studies of IS expression are generally linked to particular copies that induce a modification of neighboring gene expression. Here we investigated global patterns of IS transcription in the Alphaproteobacterial endosymbiont Wolbachia wVulC, using both RT-PCR and bioinformatic analyses. We detected several transcriptional promoters in all IS groups. Nevertheless, only one of the potentially functional IS groups possesses a promoter located upstream of the transposase gene, that could lead up to the production of a functional protein. We found that the majority of IS groups are expressed whatever their functional status. RT-PCR analyses indicate that the transcription of two IS groups lacking internal promoters upstream of the transposase start codon may be driven by the genomic environment. We confirmed this observation with the transcription analysis of individual copies of one IS group. These results suggest that the genomic environment is important for IS expression and it could explain, at least partly, copy number variability of the various IS groups present in the wVulC genome and, more generally, in bacterial genomes. PMID:25813874

  12. Functional significance of the TATA element major groove in transcription initiation by RNA polymerase II.

    PubMed Central

    Lee, D K; Wang, K C; Roeder, R G

    1997-01-01

    The binding of TFIID to the TATA element initiates assembly of a preinitiation complex and thus represents one of the most important steps for transcriptional regulation. The fact that the TATA binding protein (TBP), a subunit of TFIID, exclusively contacts the minor groove of the TATA element led us to ask whether the major groove of the TATA element plays any role in transcription initiation or its regulation. Our results show that modifications of the major groove of the TATA element in the adenovirus major late promoter have no effect on TFIID binding affinity or on transcription in a cell-free system reconstituted with purified factors. However, major groove modifications do decrease the levels of both basal and activator-mediated transcription in unfractionated nuclear extracts, indicating that the intact structure of the major groove of the TATA element is functionally important for transcription initiation in a more physiological context. PMID:9336466

  13. Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

    PubMed Central

    Thao, Sandy; Chen, Chien-Sheng; Zhu, Heng; Escalante-Semerena, Jorge C.

    2010-01-01

    Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells. PMID:21217812

  14. 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

  15. 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

  16. Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

    PubMed Central

    Shashni, Rajesh; Qayyum, M. Zuhaib; Vishalini, V.; Dey, Debashish; Sen, Ranjan

    2014-01-01

    The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions. PMID:25081210

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

    PubMed

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

    2016-09-01

    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 found 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 owing 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, provide a framework with which evolution shapes transcriptomes. PMID:27455346

  18. Increasing the efficiency of bacterial transcription simulations: When to exclude the genome without loss of accuracy

    PubMed Central

    Iafolla, Marco AJ; Dong, Guang Qiang; McMillen, David R

    2008-01-01

    Background Simulating the major molecular events inside an Escherichia coli cell can lead to a very large number of reactions that compose its overall behaviour. Not only should the model be accurate, but it is imperative for the experimenter to create an efficient model to obtain the results in a timely fashion. Here, we show that for many parameter regimes, the effect of the host cell genome on the transcription of a gene from a plasmid-borne promoter is negligible, allowing one to simulate the system more efficiently by removing the computational load associated with representing the presence of the rest of the genome. The key parameter is the on-rate of RNAP binding to the promoter (k_on), and we compare the total number of transcripts produced from a plasmid vector generated as a function of this rate constant, for two versions of our gene expression model, one incorporating the host cell genome and one excluding it. By sweeping parameters, we identify the k_on range for which the difference between the genome and no-genome models drops below 5%, over a wide range of doubling times, mRNA degradation rates, plasmid copy numbers, and gene lengths. Results We assess the effect of the simulating the presence of the genome over a four-dimensional parameter space, considering: 24 min <= bacterial doubling time <= 100 min; 10 <= plasmid copy number <= 1000; 2 min <= mRNA half-life <= 14 min; and 10 bp <= gene length <= 10000 bp. A simple MATLAB user interface generates an interpolated k_on threshold for any point in this range; this rate can be compared to the ones used in other transcription studies to assess the need for including the genome. Conclusion Exclusion of the genome is shown to yield less than 5% difference in transcript numbers over wide ranges of values, and computational speed is improved by two to 24 times by excluding explicit representation of the genome. PMID:18789148

  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. Effects of Rate-Limiting Steps in Transcription Initiation on Genetic Filter Motifs

    PubMed Central

    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. PMID:23940576

  1. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

    PubMed Central

    Wiesner, Thomas; Lee, William; Obenauf, Anna C.; Ran, Leili; Murali, Rajmohan; Zhang, Qi Fan; Wong, Elissa W. P.; Hu, Wenhuo; Scott, Sasinya N.; Shah, Ronak H.; Landa, Iñigo; Button, Julia; Lailler, Nathalie; Sboner, Andrea; Gao, Dong; Murphy, Devan A.; Cao, Zhen; Shukla, Shipra; Hollmann, Travis J.; Wang, Lu; Borsu, Laetitia; Merghoub, Taha; Schwartz, Gary K.; Postow, Michael A.; Ariyan, Charlotte E.; Fagin, James A.; Zheng, Deyou; Ladanyi, Marc; Busam, Klaus J.; Berger, Michael F.; Chen, Yu; Chi, Ping

    2016-01-01

    Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation. PMID:26444240

  2. A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.

    PubMed Central

    Lofquist, A K; Garcia, A D; Sharp, S J

    1988-01-01

    We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters. Images PMID:3141790

  3. 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

  4. mioC transcription, initiation of replication, and the eclipse in Escherichia coli.

    PubMed Central

    Bogan, J A; Helmstetter, C E

    1996-01-01

    The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated. When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling. Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation. Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription. This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse. Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation. It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely. It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature. Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of Dna

  5. 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

  6. Transcriptional Responses of Resistant and Susceptible Fish Clones to the Bacterial Pathogen Flavobacterium psychrophilum

    PubMed Central

    Martin, Samuel A. M.; Jouneau, Luc; Bernardet, Jean-Francois; Houel, Armel; Lunazzi, Aurélie; Duchaud, Eric; Michel, Christian; Quillet, Edwige; Boudinot, Pierre

    2012-01-01

    Flavobacterium psychrophilum is a bacterial species that represents one of the most important pathogens for aquaculture worldwide, especially for salmonids. To gain insights into the genetic basis of the natural resistance to F. psychrophilum, we selected homozygous clones of rainbow trout with contrasted susceptibility to the infection. We compared the transcriptional response to the bacteria in the pronephros of a susceptible and a resistant line by micro-array analysis five days after infection. While the basal transcriptome of healthy fish was significantly different in the resistant and susceptible lines, the transcriptome modifications induced by the bacteria involved essentially the same genes and pathways. The response to F. psychrophilum involved antimicrobial peptides, complement, and a number of enzymes and chemokines. The matrix metalloproteases mmp9 and mmp13 were among the most highly induced genes in both genetic backgrounds. Key genes of both pro- and anti-inflammatory response such as IL1 and IL10, were up-regulated with a greater magnitude in susceptible animals where the bacterial load was also much higher. While higher resistance to F. psychrophilum does not seem to be based on extensive differences in the orientation of the immune response, several genes including complement C3 showed stronger induction in the resistant fish. They may be important for the variation of susceptibility to the infection. PMID:22720048

  7. 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.

  8. 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

  9. 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. PMID:26331540

  10. Mediator and TREX-2: Emerging links between transcription initiation and mRNA export

    PubMed Central

    Schubert, Tobias; Köhler, Alwin

    2016-01-01

    ABSTRACT Nuclear pore proteins interact dynamically with chromatin to regulate gene activities. A key question is how nucleoporin interactions mechanistically alter a gene's intranuclear position and transcriptional output. We reported recently on a direct interaction between the nuclear pore-associated TREX-2 complex and promoter-bound Mediator. This highlights how nuclear-pore associated adaptors gain regulatory access to the core transcription machinery. In this Extra View, we discuss an additional implication that arises from our work and the recent literature: how promoter elements may regulate mRNA metabolism beyond transcription initiation. PMID:27028218

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

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. PMID:24987095

  12. 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.

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

    PubMed

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E; Ha, Un-Hwan; Wu, Donghai; Wu, Weihui; Terada, Naohiro; Jin, Shouguang

    2015-08-01

    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. PMID:26062981

  14. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  15. Real-Time Observation of the Initiation of RNA Polymerase II Transcription

    PubMed Central

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

    2015-01-01

    Biochemical and structural studies have shown that the initiation of RNA polymerase II (pol II) transcription proceeds in the following stages: assembly of pol II with general transcription factors (GTFs) and promoter DNA in a “closed” preinitiation complex (PIC)1,2; unwinding about 15 bp of the promoter DNA to form an “open” complex3,4; scanning downstream to a transcription start site; synthesis of a short transcript, believed to be about 10 nucleotides; and promoter escape. We have assembled a 32-protein, 1.5 megadalton PIC5 derived from Saccharomyces cerevisiae and observed subsequent initiation processes in real time with optical tweezers6. Contrary to expectation, scanning driven by transcription factor IIH (TFIIH)7-12 entailed the rapid opening of an extended bubble, averaging 85 bp, 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. PMID:26331540

  16. 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

  17. A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

    PubMed Central

    Morozov, Yaroslav I.; Agaronyan, Karen; Cheung, Alan C. M.; Anikin, Michael; Cramer, Patrick; Temiakov, Dmitry

    2014-01-01

    The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation—TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate—a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein–protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription. PMID:24393772

  18. High-Density Transcriptional Initiation Signals Underline Genomic Islands in Bacteria

    PubMed Central

    Huang, Qianli; Cheng, Xuanjin; Cheung, Man Kit; Kiselev, Sergey S.; Ozoline, Olga N.; Kwan, Hoi Shan

    2012-01-01

    Genomic islands (GIs), frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of “alien” elements which probably undergo special temporal–spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these “exotic” regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs) in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased “non-optimal” codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for “alien” regions, but also provide hints to the special

  19. Transcriptional initiation under conditions of anoxia-induced quiescence in mitochondria from Artemia franciscana embryos.

    PubMed

    Eads, Brian D; Hand, Steven C

    2003-02-01

    In response to anoxia, embryos of the brine shrimp Artemia franciscana are able coordinately to downregulate metabolism to levels low enough to permit survival for several years at room temperature. In addition to dramatic decreases in free ATP levels and heat production, intracellular pH drops from 7.8 to 6.3 overnight. Use of isolated mitochondria to study transcriptional responses to anoxia offers several advantages: (1). the localized nature of transcript initiation, processing and degradation, all of which may be followed in organello; (2). the relatively simple cis- and trans-machinery involved and (3). the ability to provide relevant physiological treatments in vitro. In response to anoxic incubation of embryos in vivo for 4 h followed by anoxic mitochondrial isolation and anoxic transcription assay at pH 6.4, a significant decrease in overall UTP incorporation (77%) was seen after 30 min relative to normoxic, pH 7.9 controls. A less severe inhibition of transcription under anoxia (52%) was observed compared with controls when pH was raised to 7.9. Similarly, under normoxia, the incubation at low pH (6.4) reduced transcription by 59%. Ribonuclease protection assays showed that the contribution of in vitro initiation during the assay fell from 78% at pH 7.9 to approximately 32% at pH 6.4 under either normoxic or anoxic conditions. DNA footprinting of putative transcriptional promoters revealed proteins at regular intervals upstream of the 12S rRNA in the control region, which previously had been indirectly inferred to contain promoters for H-strand transcription. The area between 1230 and 12065 contains a sequence in the tRNA(leu) gene believed to bind the transcription termination factor mTERF or TERM, and we provide the first evidence that this sequence is protein-bound in A. franciscana. However, our hypothesis that initiation is reduced at low pH because of a change in DNA binding by mitochondrial transcription factors was not confirmed. We propose that

  20. RNA Transcriptional Biosignature Analysis for Identifying Febrile Infants With Serious Bacterial Infections in the Emergency Department

    PubMed Central

    Mahajan, Prashant; Kuppermann, Nathan; Suarez, Nicolas; Mejias, Asuncion; Casper, Charlie; Dean, J. Michael; Ramilo, Octavio

    2015-01-01

    Objectives To develop the infrastructure and demonstrate the feasibility of conducting microarray-based RNA transcriptional profile analyses for the diagnosis of serious bacterial infections in febrile infants 60 days and younger in a multicenter pediatric emergency research network. Methods We designed a prospective multicenter cohort study with the aim of enrolling more than 4000 febrile infants 60 days and younger. To ensure success of conducting complex genomic studies in emergency department (ED) settings, we established an infrastructure within the Pediatric Emergency Care Applied Research Network, including 21 sites, to evaluate RNA transcriptional profiles in young febrile infants. We developed a comprehensive manual of operations and trained site investigators to obtain and process blood samples for RNA extraction and genomic analyses. We created standard operating procedures for blood sample collection, processing, storage, shipping, and analyses. We planned to prospectively identify, enroll, and collect 1 mL blood samples for genomic analyses from eligible patients to identify logistical issues with study procedures. Finally, we planned to batch blood samples and determined RNA quantity and quality at the central microarray laboratory and organized data analysis with the Pediatric Emergency Care Applied Research Network data coordinating center. Below we report on establishment of the infrastructure and the feasibility success in the first year based on the enrollment of a limited number of patients. Results We successfully established the infrastructure at 21 EDs. Over the first 5 months we enrolled 79% (74 of 94) of eligible febrile infants. We were able to obtain and ship 1 mL of blood from 74% (55 of 74) of enrolled participants, with at least 1 sample per participating ED. The 55 samples were shipped and evaluated at the microarray laboratory, and 95% (52 of 55) of blood samples were of adequate quality and contained sufficient RNA for expression

  1. Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells.

    PubMed

    Jain, Aklank; Magistri, Marco; Napoli, Sara; Carbone, Giuseppina M; Catapano, Carlo V

    2010-03-01

    Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells. PMID:20045441

  2. Biochemical Analysis of Distinct Activation Functions in p300 That Enhance Transcription Initiation with Chromatin Templates

    PubMed Central

    Kraus, W. Lee; Manning, E. Tory; Kadonaga, James T.

    1999-01-01

    To investigate the mechanisms of transcriptional enhancement by the p300 coactivator, we analyzed wild-type and mutant versions of p300 with a chromatin transcription system in vitro. Estrogen receptor, NF-κB p65 plus Sp1, and Gal4-VP16 were used as different sequence-specific activators. The CH3 domain (or E1A-binding region) was found to be essential for the function of each of the activators tested. The bromodomain was also observed to be generally important for p300 coactivator activity, though to a lesser extent than the CH3 domain/E1A-binding region. The acetyltransferase activity and the C-terminal region (containing the steroid receptor coactivator/p160-binding region and the glutamine-rich region) were each found to be important for activation by estrogen receptor but not for that by Gal4-VP16. The N-terminal region of p300, which had been previously found to interact with nuclear hormone receptors, was not seen to be required for any of the activators, including estrogen receptor. Single-round transcription experiments revealed that the functionally important subregions of p300 contribute to its ability to promote the assembly of transcription initiation complexes. In addition, the acetyltransferase activity of p300 was observed to be distinct from the broadly essential activation function of the CH3 domain/E1A-binding region. These results indicate that specific regions of p300 possess distinct activation functions that are differentially required to enhance the assembly of transcription initiation complexes. Interestingly, with the estrogen receptor, four distinct regions of p300 each have an essential role in the transcription activation process. These data exemplify a situation in which a network of multiple activation functions is required to achieve gene transcription. PMID:10567538

  3. A mutation in the C31 subunit of Saccharomyces cerevisiae RNA polymerase III affects transcription initiation.

    PubMed Central

    Thuillier, V; Stettler, S; Sentenac, A; Thuriaux, P; Werner, M

    1995-01-01

    The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C-terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non-specific templates at wild-type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C-terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase. Images PMID:7835345

  4. 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.

  5. A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

    PubMed

    Bird, Jeremy G; Strobel, Eric J; Roberts, Jeffrey W

    2016-08-19

    The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. σ70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause. PMID:27098041

  6. 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

  7. 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. PMID:26779399

  8. 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

  9. Effects of Dispersal and Initial Diversity on the Composition and Functional Performance of Bacterial Communities

    PubMed Central

    Zha, Yinghua; Berga, Mercè; Comte, Jérôme; Langenheder, Silke

    2016-01-01

    Natural communities are open systems and consequently dispersal can play an important role for the diversity, composition and functioning of communities at the local scale. It is, however, still unclear how effects of dispersal differ depending on the initial diversity of local communities. Here we implemented an experiment where we manipulated the initial diversity of natural freshwater bacterioplankton communities using a dilution-to-extinction approach as well as dispersal from a regional species pool. The aim was further to test whether dispersal effects on bacterial abundance and functional parameters (average community growth rates, respiration rates, substrate utilisation ability) differ in dependence of the initial diversity of the communities. First of all, we found that both initial diversity and dispersal rates had an effect on the recruitment of taxa from a regional source, which was higher in communities with low initial diversity and at higher rates of dispersal. Higher initial diversity and dispersal also promoted higher levels of richness and evenness in local communities and affected, both, separately or interactively, the functional performance of communities. Our study therefore suggests that dispersal can influence the diversity, composition and functioning of bacterial communities and that this effect may be enhanced if the initial diversity of communities is depleted. PMID:27182596

  10. Effects of Dispersal and Initial Diversity on the Composition and Functional Performance of Bacterial Communities.

    PubMed

    Zha, Yinghua; Berga, Mercè; Comte, Jérôme; Langenheder, Silke

    2016-01-01

    Natural communities are open systems and consequently dispersal can play an important role for the diversity, composition and functioning of communities at the local scale. It is, however, still unclear how effects of dispersal differ depending on the initial diversity of local communities. Here we implemented an experiment where we manipulated the initial diversity of natural freshwater bacterioplankton communities using a dilution-to-extinction approach as well as dispersal from a regional species pool. The aim was further to test whether dispersal effects on bacterial abundance and functional parameters (average community growth rates, respiration rates, substrate utilisation ability) differ in dependence of the initial diversity of the communities. First of all, we found that both initial diversity and dispersal rates had an effect on the recruitment of taxa from a regional source, which was higher in communities with low initial diversity and at higher rates of dispersal. Higher initial diversity and dispersal also promoted higher levels of richness and evenness in local communities and affected, both, separately or interactively, the functional performance of communities. Our study therefore suggests that dispersal can influence the diversity, composition and functioning of bacterial communities and that this effect may be enhanced if the initial diversity of communities is depleted. PMID:27182596

  11. Transcriptional activation of c3 and hsp70 as part of the immune response of Acropora millepora to bacterial challenges.

    PubMed

    Brown, Tanya; Bourne, David; Rodriguez-Lanetty, Mauricio

    2013-01-01

    The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 10(6) cells ml(-1). Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges. PMID:23861754

  12. Transcriptional Activation of c3 and hsp70 as Part of the Immune Response of Acropora millepora to Bacterial Challenges

    PubMed Central

    Brown, Tanya; Bourne, David; Rodriguez-Lanetty, Mauricio

    2013-01-01

    The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 106 cells ml-1. Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges. PMID:23861754

  13. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

    PubMed Central

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A.

    2016-01-01

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  14. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism.

    PubMed

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A

    2016-04-12

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  15. The role of transcription initiation factor IIIB subunits in promoter opening probed by photochemical cross-linking.

    PubMed

    Kassavetis, George A; Han, Shulin; Naji, Souad; Geiduschek, E Peter

    2003-05-16

    The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening. TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter. Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as sigma subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes. Brf1, Bdp1, and several pol III subunits can be cross-linked to the nontranscribed strand of the U6 promoter at base pair -9/-8 and +2/+3 (relative to the transcriptional start as +1), respectively the upstream and downstream ends of the DNA segment that opens up into the transcription bubble. Cross-linking of Bdp1 and Brf1 is detected at 0 degrees C in closed preinitiation complexes and at 30 degrees C in complexes that are partly open, but also it is detected in mutant TFIIIB-pol III-DNA complexes that are unable to open the promoter. In contrast, promoter opening-defective TFIIIB mutants generate significant changes of cross-linking of polymerase subunits. The weight of this evidence argues in favor of an indirect mode of action of TFIIIB in promoter opening. PMID:12637540

  16. Redundant cooperative interactions for assembly of a human U6 transcription initiation complex.

    PubMed

    Ma, Beicong; Hernandez, Nouria

    2002-11-01

    The core human U6 promoter consists of a proximal sequence element (PSE) located upstream of a TATA box. The PSE is recognized by the snRNA-activating protein complex (SNAP(c)), which consists of five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. The TATA box is recognized by TATA box binding protein (TBP). In addition, basal U6 transcription requires the SANT domain protein Bdp1 and the transcription factor IIB-related factor Brf2. SNAP(c) and mini-SNAP(c), which consists of just SNAP43, SNAP50, and the N-terminal third of SNAP190, bind cooperatively with TBP to the core U6 promoter. By generating complexes smaller than mini-SNAP(c), we have identified a 50-amino-acid region within SNAP190 that is (i) required for cooperative binding with TBP in the context of mini-SNAP(c) and (ii) sufficient for cooperative binding with TBP when fused to a heterologous DNA binding domain. We show that derivatives of mini-SNAP(c) lacking this region are active for transcription and that with such complexes, TBP can still be recruited to the U6 promoter through cooperative interactions with Brf2. Our results identify complexes smaller than mini-SNAP(c) that are transcriptionally active and show that there are at least two redundant mechanisms to stably recruit TBP to the U6 transcription initiation complex. PMID:12391172

  17. Redundant Cooperative Interactions for Assembly of a Human U6 Transcription Initiation Complex

    PubMed Central

    Ma, Beicong; Hernandez, Nouria

    2002-01-01

    The core human U6 promoter consists of a proximal sequence element (PSE) located upstream of a TATA box. The PSE is recognized by the snRNA-activating protein complex (SNAPc), which consists of five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. The TATA box is recognized by TATA box binding protein (TBP). In addition, basal U6 transcription requires the SANT domain protein Bdp1 and the transcription factor IIB-related factor Brf2. SNAPc and mini-SNAPc, which consists of just SNAP43, SNAP50, and the N-terminal third of SNAP190, bind cooperatively with TBP to the core U6 promoter. By generating complexes smaller than mini-SNAPc, we have identified a 50-amino-acid region within SNAP190 that is (i) required for cooperative binding with TBP in the context of mini-SNAPc and (ii) sufficient for cooperative binding with TBP when fused to a heterologous DNA binding domain. We show that derivatives of mini-SNAPc lacking this region are active for transcription and that with such complexes, TBP can still be recruited to the U6 promoter through cooperative interactions with Brf2. Our results identify complexes smaller than mini-SNAPc that are transcriptionally active and show that there are at least two redundant mechanisms to stably recruit TBP to the U6 transcription initiation complex. PMID:12391172

  18. 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

  19. Regulation of Axillary Meristem Initiation by Transcription Factors and Plant Hormones

    PubMed Central

    Yang, Minglei; Jiao, Yuling

    2016-01-01

    One distinctive feature of plant post-embryonic development is that plants can undergo reiterative growth and continuous organogenesis throughout their lifetimes. Axillary meristems (AMs) in leaf axils play a central role in this growth and differences in meristem initiation and development produce the diversity of plant architecture. Studies in the past 15 years have shown that several transcription factors (TFs) and phytohormones affect AM initiation. In this review, we highlight recent research using systems biology approaches to examine the regulatory hierarchies underlying AM initiation and the role of auxins and cytokinins in AM initiation and development. This research revealed a developmental mechanism in which phytohormone signals act with a gene regulatory network containing multiple TFs to contribute to the initiation of AMs. PMID:26925087

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

    PubMed

    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

  1. Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism.

    PubMed

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

    2010-01-01

    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. PMID:19965383

  2. 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.

  3. Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.

    PubMed

    Murakami, Kenji; Gibbons, Brian J; Davis, Ralph E; Nagai, Shigeki; Liu, Xin; Robinson, Philip J J; Wu, Tinghe; Kaplan, Craig D; Kornberg, Roger D

    2012-03-27

    General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes. PMID:22411836

  4. 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

  5. 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. PMID:27257541

  6. The availability of the primer activation signal (PAS) affects the efficiency of HIV-1 reverse transcription initiation

    PubMed Central

    Ooms, Marcel; Cupac, Daniel; Abbink, Truus E. M.; Huthoff, Hendrik; Berkhout, Ben

    2007-01-01

    Initiation of reverse transcription of a retroviral RNA genome is strictly regulated. The tRNA primer binds to the primer binding site (PBS), and subsequent priming is triggered by the primer activation signal (PAS) that also pairs with the tRNA. We observed that in vitro reverse transcription initiation of the HIV-1 leader RNA varies in efficiency among 3′-end truncated transcripts, despite the presence of both PBS and PAS motifs. As the HIV-1 leader RNA can adopt two different foldings, we investigated if the conformational state of the transcripts did influence the efficiency of reverse transcription initiation. However, mutant transcripts that exclusively fold one or the other structure were similarly active, thereby excluding the possibility of regulation of reverse transcription initiation by the structure riboswitch. We next set out to determine the availability of the PAS element. This sequence motif enhances the efficiency of reverse transcription initiation, but its activity is regulated because the PAS motif is initially base paired within the wild-type template. We measured that the initiation efficiency on different templates correlates directly with accessibility of the PAS motif. Furthermore, changes in PAS are critical to facilitate a primer-switch to a new tRNA species, demonstrating the importance of this enhancer element. PMID:17308346

  7. 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.

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

    PubMed

    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

  9. 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

  10. Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

    PubMed Central

    Orihuela, Carlos J.; Mahdavi, Jafar; Thornton, Justin; Mann, Beth; Wooldridge, Karl G.; Abouseada, Noha; Oldfield, Neil J.; Self, Tim; Ala’Aldeen, Dlawer A.A.; Tuomanen, Elaine I.

    2009-01-01

    A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease. PMID:19436113

  11. 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

  12. 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

  13. 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

  14. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    PubMed

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms. PMID:27281207

  15. 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

  16. 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

  17. The little elongation complex functions at initiation and elongation phases of snRNA gene transcription.

    PubMed

    Hu, Deqing; Smith, Edwin R; Garruss, Alexander S; Mohaghegh, Nima; Varberg, Joseph M; Lin, Chengqi; Jackson, Jessica; Gao, Xin; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Eissenberg, Joel C; Shilatifard, Ali

    2013-08-22

    The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL. PMID:23932780

  18. The Little Elongation Complex functions at initiation and elongation phases of snRNA gene transcription

    PubMed Central

    Hu, Deqing; Smith, Edwin R.; Garruss, Alexander S.; Mohaghegh, Nima; Varberg, Joseph M.; Lin, Chengqi; Jackson, Jessica; Gao, Xin; Saraf, Anita; Florens, Laurence; Washburn, Michael P.; Eissenberg, Joel C.; Shilatifard, Ali

    2014-01-01

    SUMMARY The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA Polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the Little Elongation Complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL. PMID:23932780

  19. The relationship between transcription initiation RNAs and CCCTC-binding factor (CTCF) localization

    PubMed Central

    2011-01-01

    Background Transcription initiation RNAs (tiRNAs) are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII) transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation. Results In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF) binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A). Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21 (p21alt) transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis. Conclusions Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization. PMID:21813016

  20. 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.

  1. 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

  2. Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit.

    PubMed Central

    Buratowski, S; Sharp, P A

    1990-01-01

    RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro. Images PMID:2398901

  3. Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

    PubMed Central

    Mukhopadhyay, Suman; Chattoraj, Dhruba K.

    2000-01-01

    The replication origin of plasmid P1 contains an array of five repeats (iterons) that bind the plasmid-encoded initiator RepA. Within the array lies the repA promoter, which becomes largely repressed on RepA binding (autorepression). One might expect that extra iterons produced on plasmid replication would titrate RepA and release the repression. The promoter, however, is induced poorly by extra iterons. The P1 copy number is reduced by extra iterons in the presence of the autorepressed repA gene but not when additional RepA is provided from constitutive sources. It has been proposed that the iteron-bound RepA couples with the promoter-bound RepA and thereby maintains repression. Although not the product of replication, we find that the act of replication itself can renew RepA synthesis. Replication apparently cleans the promoter of bound RepA and provides a window of opportunity for repA transcription. We propose that replication-induced transcription is required to ensure initiator availability in a system that is induced poorly when challenged with additional initiator binding sites. PMID:10840063

  4. Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content

    PubMed Central

    Crawford, Matthew A.; Tapscott, Timothy; Fitzsimmons, Liam F.; Liu, Lin; Reyes, Aníbal M.; Libby, Stephen J.; Trujillo, Madia; Fang, Ferric C.; Radi, Rafael

    2016-01-01

    ABSTRACT The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. PMID:27094335

  5. Co-operative roles for DNA supercoiling and nucleoid-associated proteins in the regulation of bacterial transcription.

    PubMed

    Dorman, Charles J

    2013-04-01

    DNA supercoiling and NAPs (nucleoid-associated proteins) contribute to the regulation of transcription of many bacterial genes. The horizontally acquired SPI (Salmonella pathogenicity island) genes respond positively to DNA relaxation, they are activated and repressed by the Fis (factor for inversion stimulation) and H-NS (histone-like nucleoid-structuring) NAPs respectively, and are positively controlled by the OmpR global regulatory protein. The ompR gene is autoregulated and responds positively to DNA relaxation. Binding of the Fis and OmpR proteins to their targets in DNA is differentially sensitive to its topological state, whereas H-NS binds regardless of the topological state of the DNA. These data illustrate the overlapping and complex nature of NAP and DNA topological contributions to transcription control in bacteria. PMID:23514151

  6. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    PubMed

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. PMID:23178502

  7. Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis.

    PubMed

    Mueller, R F; Characklis, W G; Jones, W L; Sears, J T

    1992-05-01

    The processes leading to bacterial colonization on solid-water interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 microm (for silicon) to 0.015 microm (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varied by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented. PMID:18600919

  8. Nature of bacterial colonization influences transcription of mucin genes in mice during the first week of life

    PubMed Central

    2012-01-01

    Background Postnatal regulation of the small intestinal mucus layer is potentially important in the development of adult gut functionality. We hypothesized that the nature of bacterial colonization affects mucus gene regulation in early life. We thus analyzed the influence of the presence of a conventional microbiota as well as two selected monocolonizing bacterial strains on the transcription of murine genes involved in mucus layer development during the first week of life. Mouse pups (N = 8/group) from differently colonized dams: Germ-free (GF), conventional specific pathogen free (SPF), monocolonized with either Lactobacillus acidophilus NCFM (Lb) or Escherichia coli Nissle (Ec) were analyzed by qPCR on isolated ileal tissue sections from postnatal days 1 and 6 (PND1, PND6) after birth with respect to: (i) transcription of specific genes involved in mucus production (Muc1-4, Tff3) and (ii) amounts of 16S rRNA of Lactobacillus and E. coli. Quantification of 16S rRNA genes was performed to obtain a measure for amounts of colonized bacteria. Results We found a microbiota-independent transcriptional increase of all five mucus genes from PND1 to PND6. Furthermore, the relative level of transcription of certain mucus genes on PND1 was increased by the presence of bacteria. This was observed for Tff3 in the SPF, Ec, and Lb groups; for Muc2 in SPF; and for Muc3 and Muc4 in Ec and Lb, respectively. Detection of bacterial 16S rRNA genes levels above the qPCR detection level occurred only on PND6 and only for some of the colonized animals. On PND6, we found significantly lower levels of Muc1, Muc2 and Muc4 gene transcription for Lb animals with detectable Lactobacillus levels as compared to animals with Lactobacillus levels below the detection limit. Conclusions In summary, our data show that development of the expression of genes encoding secreted (Muc2/Tff3) and membrane-bound (Muc1/Muc3/Muc4) mucus regulatory proteins, respectively, is distinct and that the onset of

  9. Global transcriptional repression: An initial and essential step for Plasmodium sexual development.

    PubMed

    Yuda, Masao; Iwanaga, Shiroh; Kaneko, Izumi; Kato, Tomomi

    2015-10-13

    Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2-depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development. PMID:26417110

  10. Genome-wide survey of transcriptional initiation in the pathogenic fungus, Candida glabrata.

    PubMed

    Aoyama, Toshihiro; Nakayama, Hironobu; Ueno, Keigo; Inukai, Tatsuya; Tanabe, Koichi; Nagi, Minoru; Bard, Martin; Chibana, Hiroji

    2014-06-01

    DNA sequencing of the 5'-flanking region of the transcriptome effectively identifies transcription initiation sites and also aids in identifying unknown genes. This study describes a comprehensive polling of transcription start sites and an analysis of full-length complementary DNAs derived from the genome of the pathogenic fungus Candida glabrata. A comparison of the sequence reads derived from a cDNA library prepared from cells grown under different culture conditions against the reference genomic sequence of the Candida Genome Database (CGD: http://www.candidagenome.org/) revealed the expression of 4316 genes and their acknowledged transcription start sites (TSSs). In addition this analysis also predicted 59 new genes including 22 that showed no homology to the genome of Saccharomyces cerevisiae, a genetically close relative of C. glabrata. Furthermore, comparison of the 5'-untranslated regions (5'-UTRs) and core promoters of C. glabrata to those of S. cerevisiae showed various global similarities and differences among orthologous genes. Thus, the C. glabrata transcriptome can complement the annotation of the genome database and should provide new insights into the organization, regulation, and function of genes of this important human pathogen. PMID:24725256

  11. Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules.

    PubMed

    Sanchez, Alvaro; Osborne, Melisa L; Friedman, Larry J; Kondev, Jane; Gelles, Jeff

    2011-10-01

    The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled. PMID:21829165

  12. Symbiosis initiation in the bacterially luminous sea urchin cardinalfish Siphamia versicolor.

    PubMed

    Dunlap, P V; Gould, A L; Wittenrich, M L; Nakamura, M

    2012-09-01

    To determine how each new generation of the sea urchin cardinalfish Siphamia versicolor acquires the symbiotic luminous bacterium Photobacterium mandapamensis, and when in its development the S. versicolor initiates the symbiosis, procedures were established for rearing S. versicolor larvae in an aposymbiotic state. Under the conditions provided, larvae survived and developed for 28 days after their release from the mouths of males. Notochord flexion began at 8 days post release (dpr). By 28 dpr, squamation was evident and the caudal complex was complete. The light organ remained free of bacteria but increased in size and complexity during development of the larvae. Thus, aposymbiotic larvae of the fish can survive and develop for extended periods, major components of the luminescence system develop in the absence of the bacteria and the bacteria are not acquired directly from a parent, via the egg or during mouth brooding. Presentation of the symbiotic bacteria to aposymbiotic larvae at 8-10 dpr, but not earlier, led to initiation of the symbiosis. Upon colonization of the light organ, the bacterial population increased rapidly and cells forming the light-organ chambers exhibited a differentiated appearance. Therefore, the light organ apparently first becomes receptive to colonization after 1 week post-release development, the symbiosis is initiated by bacteria acquired from the environment and bacterial colonization induces morphological changes in the nascent light organ. The abilities to culture larvae of S. versicolor for extended periods and to initiate the symbiosis in aposymbiotic larvae are key steps in establishing the experimental tractability of this highly specific vertebrate and microbe mutualism. PMID:22957874

  13. 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.

  14. Patterns of Transcriptional Response to 1,25-Dihydroxyvitamin D3 and Bacterial Lipopolysaccharide in Primary Human Monocytes.

    PubMed

    Kariuki, Silvia N; Blischak, John D; Nakagome, Shigeki; Witonsky, David B; Di Rienzo, Anna

    2016-01-01

    The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), plays an important immunomodulatory role, regulating transcription of genes in the innate and adaptive immune system. The present study examines patterns of transcriptome-wide response to 1,25D, and the bacterial lipopolysaccharide (LPS) in primary human monocytes, to elucidate pathways underlying the effects of 1,25D on the immune system. Monocytes obtained from healthy individuals of African-American and European-American ancestry were treated with 1,25D, LPS, or both, simultaneously. The addition of 1,25D during stimulation with LPS induced significant upregulation of genes in the antimicrobial and autophagy pathways, and downregulation of proinflammatory response genes compared to LPS treatment alone. A joint Bayesian analysis enabled clustering of genes into patterns of shared transcriptional response across treatments. The biological pathways enriched within these expression patterns highlighted several mechanisms through which 1,25D could exert its immunomodulatory role. Pathways such as mTOR signaling, EIF2 signaling, IL-8 signaling, and Tec Kinase signaling were enriched among genes with opposite transcriptional responses to 1,25D and LPS, respectively, highlighting the important roles of these pathways in mediating the immunomodulatory activity of 1,25D. Furthermore, a subset of genes with evidence of interethnic differences in transcriptional response was also identified, suggesting that in addition to the well-established interethnic variation in circulating levels of vitamin D, the intensity of transcriptional response to 1,25D and LPS also varies between ethnic groups. We propose that dysregulation of the pathways identified in this study could contribute to immune-mediated disease risk. PMID:26976439

  15. Patterns of Transcriptional Response to 1,25-Dihydroxyvitamin D3 and Bacterial Lipopolysaccharide in Primary Human Monocytes

    PubMed Central

    Kariuki, Silvia N.; Blischak, John D.; Nakagome, Shigeki; Witonsky, David B.; Di Rienzo, Anna

    2016-01-01

    The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), plays an important immunomodulatory role, regulating transcription of genes in the innate and adaptive immune system. The present study examines patterns of transcriptome-wide response to 1,25D, and the bacterial lipopolysaccharide (LPS) in primary human monocytes, to elucidate pathways underlying the effects of 1,25D on the immune system. Monocytes obtained from healthy individuals of African-American and European-American ancestry were treated with 1,25D, LPS, or both, simultaneously. The addition of 1,25D during stimulation with LPS induced significant upregulation of genes in the antimicrobial and autophagy pathways, and downregulation of proinflammatory response genes compared to LPS treatment alone. A joint Bayesian analysis enabled clustering of genes into patterns of shared transcriptional response across treatments. The biological pathways enriched within these expression patterns highlighted several mechanisms through which 1,25D could exert its immunomodulatory role. Pathways such as mTOR signaling, EIF2 signaling, IL-8 signaling, and Tec Kinase signaling were enriched among genes with opposite transcriptional responses to 1,25D and LPS, respectively, highlighting the important roles of these pathways in mediating the immunomodulatory activity of 1,25D. Furthermore, a subset of genes with evidence of interethnic differences in transcriptional response was also identified, suggesting that in addition to the well-established interethnic variation in circulating levels of vitamin D, the intensity of transcriptional response to 1,25D and LPS also varies between ethnic groups. We propose that dysregulation of the pathways identified in this study could contribute to immune-mediated disease risk. PMID:26976439

  16. 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

    2016-01-01

    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

  17. 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

  18. 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...

  19. 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

  20. The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation.

    PubMed

    Grohmann, Dina; Nagy, Julia; Chakraborty, Anirban; Klose, Daniel; Fielden, Daniel; Ebright, Richard H; Michaelis, Jens; Werner, Finn

    2011-07-22

    TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation. PMID:21777815

  1. 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.

  2. 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.

  3. 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

  4. Core Promoter Plasticity Between Maize Tissues and Genotypes Contrasts with Predominance of Sharp Transcription Initiation Sites.

    PubMed

    Mejía-Guerra, María Katherine; Li, Wei; Galeano, Narmer F; Vidal, Mabel; Gray, John; Doseff, Andrea I; Grotewold, Erich

    2015-12-01

    Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop. PMID:26628745

  5. Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming

    PubMed Central

    Soufi, Abdenour; Garcia, Meilin Fernandez; Jaroszewicz, Artur; Osman, Nebiyu; Pellegrini, Matteo; Zaret, Kenneth S.

    2015-01-01

    SUMMARY Pioneer transcription factors (TFs) access silent chromatin and initiate cell fate changes, using diverse types of DNA binding domains (DBDs). FoxA, the paradigm pioneer TF, has a winged helix DBD that resembles linker histone and thereby binds its target sites on nucleosomes and in compacted chromatin. Herein we compare the nucleosome and chromatin targeting activities of Oct4 (POU DBD), Sox2 (HMG box DBD), Klf4 (zinc finger DBD), and c-Myc (bHLH DBD), which together reprogram somatic cells to pluripotency. Purified Oct4, Sox2, and Klf4 proteins can bind nucleosomes in vitro, and in vivo they preferentially target silent sites enriched for nucleosomes. Pioneer activity relates simply to the ability of a given DBD to target partial motifs displayed on the nucleosome surface. Such partial motif recognition can occur by coordinate binding between factors. Our findings provide insight into how pioneer factors can target naïve chromatin sites. PMID:25892221

  6. Transcription of Azotobacter phage deoxyribonucleic acid. Salt-dependent equilibrium between steps in initiation.

    PubMed

    Domingo, E; Escarmis, C; Warner, R C

    1975-04-25

    The transcription of Azotobacter phage A21 DNA by Escherichia coli or Azotobacter vinelandii RNA polymerase differs from that of some other DNAs in its inhibition by moderate concentrations of KCl. This characteristic results in an apparent low template activity for this DNA as compared with T4 DNA under standard assay conditions. From an analysis of the dependence of the various steps in initiation on KCl it is concluded that the effect is exerted on an equilibrium between an inactive polymerase-DNA complex and an active preintitiation complex. This salt-sensitive equilibrium favors the inactive complex at a lower KCl concentration than with other templates. It can be approached from other low or high salt concentrations at a measurably slow rate. PMID:1091643

  7. Preferential use of RNA leader sequences during influenza A transcription initiation in vivo.

    PubMed

    Geerts-Dimitriadou, Christina; Goldbach, Rob; Kormelink, Richard

    2011-01-01

    In vitro transcription initiation studies revealed a preference of influenza A virus for capped RNA leader sequences with base complementarity to the viral RNA template. Here, these results were verified during an influenza infection in MDCK cells. Alfalfa mosaic virus RNA3 leader sequences mutated in their base complementarity to the viral template, or the nucleotides 5' of potential base-pairing residues, were tested for their use either singly or in competition. These analyses revealed that influenza transcriptase is able to use leaders from an exogenous mRNA source with a preference for leaders harboring base complementarity to the 3'-ultimate residues of the viral template, as previously observed during in vitro studies. Internal priming at the 3'-penultimate residue, as well as "prime-and-realign" was observed. The finding that multiple base-pairing promotes cap donor selection in vivo, and the earlier observed competitiveness of such molecules in vitro, offers new possibilities for antiviral drug design. PMID:21030059

  8. Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice yield is most sensitive to salinity imposed during panicle initiation stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during panicle initiation. The genotypes selected included a pair of indicas (IR63731 and ...

  9. A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

    PubMed

    Nechaev, Sergei; Yuzenkova, Yulia; Niedziela-Majka, Anita; Heyduk, Tomasz; Severinov, Konstantin

    2002-06-28

    Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination. PMID:12079331

  10. Cefepime compared with ceftazidime as initial therapy for serious bacterial infections and sepsis syndrome.

    PubMed Central

    Kieft, H; Hoepelman, A I; Rozenberg-Arska, M; Branger, J M; Voskuil, J H; Geers, A B; Kluyver, M; Hart, H C; Poest-Clement, E; van Beugen, L

    1994-01-01

    In an open randomized multicenter comparative study, we evaluated the safety and efficacy of cefepime (CP; 2.0 g given intravenously every 12 h) and ceftazidime (CZ; 2.0 g given intravenously every 8 h) as initial treatment for adult patients with suspected serious bacterial infections. A total of 133 patients entered the study, of whom 114 were evaluable for clinical and microbiological response assessment: 56 received CP and 58 received CZ. About 50% (30 who received CP and 25 who received CZ) fulfilled the criteria of the sepsis syndrome. The treatment groups were comparable with respect to sex distribution, mean age, underlying diseases, treatment duration, APACHE II score, and type of infection. The most commonly cultured microorganisms were members of the family Enterobacteriaceae, Streptococcus pneumoniae, and Staphylococcus aureus. The causative microorganisms were eradicated from 92% (37 of 40) of patients with a microbiologically documented infection who underwent treatment with CP; they were eradicated from 86% (42 to 49) of patients who received CZ. The responses of only clinically documented infections in the CP group were 90% (27 of 30 patients); in the CZ group they were 87% (26 of 30 patients). When patients fulfilled the criteria of the sepsis syndrome (septic shock excluded), the causative microorganisms were eradicated from 89% (16 of 18) of CP-treated patients and 86% (12 of 14) of CZ-treated patients. None of these differences was statistically significant. Mortality was the same in both groups (four patients in each group) and was not attributable to the study medication. In conclusion, CP is at least as effective and as safe as CZ, as initial antimicrobial therapy for suspected serious bacterial infections in nonneutropenic patients with or without the sepsis syndrome. CP has the additional advantage in that it can be given twice daily, which may lead to a decrease in hospital costs. PMID:8203833

  11. Early canine plaque biofilms: characterization of key bacterial interactions involved in initial colonization of enamel.

    PubMed

    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

  12. 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

  13. [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. PMID:27215030

  14. 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

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

    PubMed

    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

  16. COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

    PubMed Central

    Marban, Céline; Redel, Laetitia; Suzanne, Stella; Van Lint, Carine; Lecestre, Dominique; Chasserot-Golaz, Sylvette; Leid, Mark; Aunis, Dominique; Schaeffer, Evelyne; Rohr, Olivier

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region. PMID:15849318

  17. Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

    PubMed

    Laman, A G; Lathe, R; Savinov, G V; Shepelyakovskaya, A O; Boziev, Kh M; Baidakova, L K; Chulin, A N; Brovko, F A; Svirshchevskaya, E V; Kotelevtsev, Y; Eliseeva, I A; Guryanov, S G; Lyabin, D N; Ovchinnikov, L P; Ivanov, V T

    2015-07-01

    The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling. PMID:26026270

  18. Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

    PubMed

    Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

    2004-01-30

    A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape. PMID:14597619

  19. Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation.

    PubMed

    Machado, António; Jefferson, Kimberly Kay; Cerca, Nuno

    2013-01-01

    Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder. PMID:23739678

  20. CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

    PubMed Central

    Yoshinari, S; Nagy, P D; Simon, A E; Dreher, T W

    2000-01-01

    It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible. PMID:10836791

  1. 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.

  2. 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

  3. 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

  4. A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft.

    PubMed

    Hu, Hui-Lan; Wu, Chih-Chien; Lee, Jin-Cheng; Chen, Hung-Ta

    2015-08-01

    The RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced into Saccharomyces cerevisiae Bdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf1, consistent with the findings of previous studies. In addition, we identified an essential N-terminal region that cross-linked with the Pol III catalytic subunit C128 as well as Brf1. Closer examination revealed that this region interacted with the C128 N-terminal region, the N-terminal half of Brf1, and the C-terminal domain of the C37 subunit, together positioning this region within the active site cleft of the preinitiation complex. With our functional data, our analyses identified an essential region of Bdp1 that is positioned within the active site cleft of Pol III and necessary for transcription initiation. PMID:26055328

  5. A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft

    PubMed Central

    Hu, Hui-Lan; Wu, Chih-Chien; Lee, Jin-Cheng

    2015-01-01

    The RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced into Saccharomyces cerevisiae Bdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf1, consistent with the findings of previous studies. In addition, we identified an essential N-terminal region that cross-linked with the Pol III catalytic subunit C128 as well as Brf1. Closer examination revealed that this region interacted with the C128 N-terminal region, the N-terminal half of Brf1, and the C-terminal domain of the C37 subunit, together positioning this region within the active site cleft of the preinitiation complex. With our functional data, our analyses identified an essential region of Bdp1 that is positioned within the active site cleft of Pol III and necessary for transcription initiation. PMID:26055328

  6. Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

    PubMed

    Busam, Klaus J; Vilain, Ricardo E; Lum, Trina; Busam, Jonathan A; Hollmann, Travis J; Saw, Robyn P M; Coit, Daniel C; Scolyer, Richard A; Wiesner, Thomas

    2016-06-01

    A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors. PMID:26872010

  7. An archaebacterial RNA polymerase binding site and transcription initiation of the hisA gene in Methanococcus vannielii.

    PubMed Central

    Brown, J W; Thomm, M; Beckler, G S; Frey, G; Stetter, K O; Reeve, J N

    1988-01-01

    Transcription initiation of the hisA gene in vivo in the archaebacterium Methanococcus vannielii, as determined by nuclease S1 and primer extension analyses, occurs 73 base pairs (bp) upstream of the translation initiation site. Binding of M. vannielii RNA polymerase protects 43 bp of DNA, from 35 bp upstream (-35) to 8 bp downstream (+8) of the hisA mRNA initiation site, from digestion by DNase I and exonuclease III. An A + T rich region, with a sequence which conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location (-25) upstream of the polypeptide-encoding hisA gene. It appears therefore that a TATA-like sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium. Images PMID:2829115

  8. ATF4 (activating transcription factor 4) from grass carp (Ctenopharyngodon idella) modulates the transcription initiation of GRP78 and GRP94 in CIK cells.

    PubMed

    Fan, Qidi; Mao, Huiling; Wu, Chuxin; Liu, Yong; Hu, Yousheng; Zhong, Bin; Mi, Yichuan; Hu, Chengyu

    2014-05-01

    GRP78 and GRP94, belong to GRP (glucose-regulated protein) family of endoplasmatic reticulum (ER) chaperone superfamily, are essential for cell survival under ER stress. ATF4 is a protective protein which regulates the adaptation of cells to ER stress by modulating the transcription of UPR (Unfolded Protein Response) target genes, including GRP78 and GRP94. To understand the molecular mechanism of ATF4 modulates the transcription initiation of CiGRP78 and CiGRP94, we cloned ATF4 ORF cDNA sequences (CiATF4) by homologous cloning techniques. The expression trend of CiATF4 was similar to CiGRP78 and CiGRP94 did under 37 °C thermal stress, namely, the expression of CiATF4 was up-regulated twice at 2 h post-thermal stress and at 18 h post recovery from thermal stress. In this paper, CiATF4 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. On the basis of the cloned CiGRP78 and CiGRP94 cDNA in our laboratory previously, we cloned their promoter sequences by genomic walking approach. In vitro, gel mobility shift assays revealed that CiATF4 could bind to CiGRP78 and CiGRP94 promoter with high affinity. Subsequently, the recombinant plasmid of pGL3-CiGRPs and pcDNA3.1-CiATF4 were constructed and transiently co-transfected into Ctenopharyngodon idella kidney (CIK) cells. The impact of CiATF4 on CiGRP promoter sequences were measured by luciferase assays. These results demonstrated that CiATF4 could activate the transcription of CiGRP78 and CiGRP94. What's more, for better understanding the molecular mechanism of CiATF4 modulate the transcription initiation of CiGRP, three mutant fragments of CiGRP78 promoter recombinant plasmids (called CARE-mut/LUC, CRE1-mut/LUC and CRE2-mut/LUC) were constructed and transiently co-transfected with CiATF4 into CIK cells. The results indicated that CRE or CARE elements were the regulatory element for transcription initiation of CiGRP78. Between them, CRE

  9. Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    PubMed Central

    Allgayer, Julia; Kitsera, Nataliya; Bartelt, Solveig; Epe, Bernd; Khobta, Andriy

    2016-01-01

    DNA damage can significantly modulate expression of the affected genes either by direct structural interference with transcription components or as a collateral outcome of cellular repair attempts. Thus, DNA glycosylases of the base excision repair (BER) pathway have been implicated in negative transcriptional response to several spontaneously generated DNA base modifications, including a common oxidative DNA base modification 8-oxoguanine (8-oxoG). Here, we report that single 8-oxoG situated in the non-transcribed DNA strand of a reporter gene has a pronounced negative effect on transcription, driven by promoters of various strength and with different structural properties, including viral, human, and artificial promoters. We further show that the magnitude of the negative effect on the gene expression correlates with excision of the modified base by OGG1 in all promoter constructs tested. Moreover, by using expression vectors with nuclease resistant backbone modifications, we demonstrate that OGG1 does not catalyse DNA strand cleavage in vivo. Rather, cleavage of the phosphate bond 5′ to 8-oxodG (catalysed by APE1) is essential and universally required for the onset of transcriptional silencing, regardless of the promoter structure. Hence, induction of transcriptional silencing emerges as a ubiquitous mode of biological response to 8-oxoG in DNA. PMID:27220469

  10. 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. PMID:25487088

  11. Species sorting and neutral processes are both important during the initial assembly of bacterial communities

    PubMed Central

    Langenheder, Silke; Székely, Anna J

    2011-01-01

    Many studies have shown that species sorting, that is, the selection by local environmental conditions is important for the composition and assembly of bacterial communities. On the other hand, there are other studies that could show that bacterial communities are neutrally assembled. In this study, we implemented a microcosm experiment with the aim to determine, at the same time, the importance of species sorting and neutral processes for bacterial community assembly during the colonisation of new, that is, sterile, habitats, by atmospheric bacteria. For this we used outdoor microcosms, which contained sterile medium from three different rock pools representing different environmental conditions, which were seeded by rainwater bacteria. We found some evidence for neutral assembly processes, as almost every 4th taxon growing in the microcosms was also detectable in the rainwater sample irrespective of the medium. Most of these taxa belonged to widespread families with opportunistic growth strategies, such as the Pseudomonadaceae and Comamonadaceae, indicating that neutrally assembled taxa may primarily be generalists. On the other hand, we also found evidence for species sorting, as one out of three media selected a differently composed bacterial community. Species sorting effects were relatively weak and established themselves via differences in relative abundance of generalists among the different media, as well as media-specific occurrences of a few specific taxa. In summary, our results suggest that neutral and species sorting processes interact during the assembly of bacterial communities and that their importance may differ depending on how many generalists and specialists are present in a community. PMID:21270841

  12. Convergent transcription initiates from oppositely oriented promoters within the 5 prime end regions of Drosophila melanogaster F elements

    SciTech Connect

    Minchiotti, G. ); Di Nocera, P.P. )

    1991-10-01

    Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, F{sub in} and F{sub out}, that transcribe in opposite orientations. Analysis of the template activity of 3{prime} deletion derivatives indicates that the level of accumulation of F{sub in}RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The F{sub out} promoter drives transcription in the opposite orientation with respect to F{sub in}, F{sub out} transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the F{sub out} promoter. Deletions knocking out the F{sub in} promoter do not impair F{sub out} transcription; conversely, initiation at the F{sub in} promoter still takes place in templates that lack the F{sub out} promoter. At a low level, both promoters are active in cultured cells.

  13. A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes.

    PubMed

    Gamper, H B; Hearst, J E

    1982-05-01

    DNA unwinding induced by Escherichia coli RNA polymerase is measured for binary, initiation and ternary complexes formed from a unique promoter sequence on simian virus 40 DNA. At 37 degrees C the complexes all have an unwinding angle of 17 +/- 1 base pairs (580 degrees +/- 30 degrees). This unwinding is attributed to an enzyme-stabilized separation of the double helix at the promoter site, which is maintained throughout initiation and elongation. There is no heterogeneity in the unwinding angle of the ternary complex as it progresses down the helical template. The constant DNA unwinding during all phases of transcription leads us to propose the existence of unwindase and rewindase activities on the enzyme that allow it to travel down the helix like a nut on a DNA bolt. During elongation, the unwindase unwinds the DNA helix while the rewindase, lagging by 17 base pairs, displaces the RNA transcript and reseals the helix. Both activities induce a rotation in the DNA double helix relative to the polymerase. The RNA-DNA hybrid also rotates, maintaining both ends of that helix fixed relative to the catalytic and windase sites. Formation of an RNA-DNA hybrid which spans the distal end of the DNA unwound region is proposed as a possible mechanism for polymerase pausing and termination. This model requires that the polymerase direct the transcript past the noncoding DNA strand. Pausing occurs 16-20 nucleotides downstream from the centers of appropriately sized dyad symmetry elements. PMID:6286146

  14. NAIPs: building an innate immune barrier against bacterial pathogens. NAIPs function as sensors that initiate innate immunity by detection of bacterial proteins in the host cell cytosol.

    PubMed

    Kofoed, Eric M; Vance, Russell E

    2012-07-01

    The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens. PMID:22513803

  15. Structural basis of transcription activation.

    PubMed

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. PMID:27284196

  16. Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces.

    PubMed

    Lee, Jin-Woo; Nam, Ji-Hyun; Kim, Yang-Hoon; Lee, Kyu-Ho; Lee, Dong-Hun

    2008-04-01

    Succession of bacterial communities during the first 36 h of biofilm formation in coastal water was investigated at 3 approximately 15 h intervals. Three kinds of surfaces (i.e., acryl, glass, and steel substratum) were submerged in situ at Sacheon harbor, Korea. Biofilms were harvested by scraping the surfaces, and the compositions of bacterial communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of 16S rRNA genes. While community structure based on T-RFLP analysis showed slight differences by substratum, dramatic changes were commonly observed for all substrata between 9 and 24 h. Identification of major populations by 16S rRNA gene sequences indicated that gamma-Proteobacteria (Pseudomonas, Acinetobacter, Alteromonas, and uncultured gamma-Proteobacteria) were predominant in the community during 0 approximately 9 h, while the ratio of alpha-Proteobacteria (Loktanella, Methylobacterium, Pelagibacter, and uncultured alpha-Proteobacteria) increased 2.6 approximately 4.8 folds during 24 approximately 36 h of the biofilm formation, emerging as the most predominant group. Previously, alpha-Proteobacteria were recognized as the pioneering organisms in marine biofilm formation. However, results of this study, which revealed the bacterial succession with finer temporal resolution, indicated some species of gamma-Proteobacteria were more important as the pioneering population. Measures to control pioneering activities of these species can be useful in prevention of marine biofilm formation. PMID:18545967

  17. Do Milk Samples Stored for 12 Days after Collection Exhibit a Change in Composition Related to the Initial Bacterial Load?

    PubMed

    de Freitas, Larissa Nazareth; Cassoli, Laerte Dagher; da Silva, Janielen; de Figueiredo Pantoja, José Carlos; Machado, Paulo Fernando

    2016-05-01

    Total bacterial count (TBC) is a tool used to assess milk quality and is associated with not only the initial sample contamination but also the sample storage time and temperature. Several countries have reported milk samples with a high TBC, and the influence of TBC on milk preservation remains unclear. Thus, the aim of this study was to evaluate the impact of the initial bacterial contamination level on the macrocomponents and somatic cell count (SCC) of raw milk samples preserved with bronopol and maintained at two storage temperatures (7 and 25°C) for up to 12 days. Thus, we collected milk samples from 51 dairy farms, which were divided into two groups according to the initial bacterial load: low TBC (<100,000 CFU/ml) and high TBC (≥100,000 CFU/ml). We analyzed the sample composition for protein, fat, total solids, lactose, milk urea nitrogen, and the SCC. We did not observe an effect from TBC and storage time and temperature on the concentration of protein, fat, total solids, and lactose. SCC changes were not observed for samples maintained under refrigeration (7°C); however, samples maintained at room temperature (25°C) exhibited a decrease in the SCC beginning on day 6 of storage. For milk urea nitrogen, values increased when the samples were maintained at room temperature, beginning on the ninth storage day. Samples with the preservative bronopol added and maintained under refrigeration may be analyzed up to 12 days after collection, regardless of the milk microbial load. PMID:27296431

  18. An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition

    PubMed Central

    Li, Jingfeng; Kannan, Manoj; Trivett, Anna L.; Liao, Hongling; Wu, Xiaolin; Akagi, Keiko; Symer, David E.

    2014-01-01

    Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition. PMID:24493738

  19. 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

  20. 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

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

    PubMed

    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

  2. Crl Activates Transcription Initiation of RpoS-Regulated Genes Involved in the Multicellular Behavior of Salmonella enterica Serovar Typhimurium

    PubMed Central

    Robbe-Saule, Véronique; Jaumouillé, Valentin; Prévost, Marie-Christine; Guadagnini, Stéphanie; Talhouarne, Christelle; Mathout, Hayette; Kolb, Annie; Norel, Françoise

    2006-01-01

    In Salmonella enterica serovar Typhimurium, the stationary-phase sigma factor σS (RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein of Escherichia coli interacts with σS and activates expression of σS-regulated genes, such as the csgBAC operon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein of Salmonella serovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of the csgD, csgB, adrA, and bcsA genes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His6-Crl showed that Crl directly activated σS-dependent transcription initiation at the csgD and adrA promoters. We observed no effect of Crl on σ70-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28°C. We obtained complementation of the crl mutation by increasing σS levels. This suggests that Crl has a major physiological impact at low concentrations of σS. PMID:16707690

  3. Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage

    PubMed Central

    Wilson, Clyde; Zeng, Linghe; Ismail, Abdelbagi M.; Condamine, Pascal; Close, Timothy J.

    2006-01-01

    Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9112-0 and is accessible for authorized users. PMID:17160619

  4. Field experimental evidence that stochastic processes predominate in the initial assembly of bacterial communities.

    PubMed

    Hao, Yi-Qi; Zhao, Xin-Feng; Zhang, Da-Yong

    2016-06-01

    To assess the relative importance of environmental selection, dispersal and stochastic processes in structuring ecological communities, we conducted a bacterial community assembly experiment using microcosms filled with sterile liquid medium under field conditions in the Inner Mongolian grasslands. Multiple replicate microcosms containing different carbon substrates were placed at nine locations across three spatial scales (10, 300 and 10 000 m distance between locations) in such a way that the environment of microcosms varies independently of the geographical distance. The operational taxonomic units within the experimental communities were assessed via the terminal restriction fragment length polymorphism techniques on the 10th and 17th days after the onset of the experiment. We found no evidence of distance decay in community similarity, and communities within a given location were more similar to each other regardless of environment than communities at other locations within the same spatial scale. Variance partitioning indicated that location explained more compositional variation in microbial communities than environment, particularly on the 17th day, despite that environment and location in combination could only explain less than half of the total variation. These results suggest that bacterial dispersal is not limited by distance in this experiment, and community assembly in microcosms is not environmentally determined but governed by stochastic processes. PMID:25809418

  5. Modeling the Transcriptional Consequences of Epidermal Growth Factor Receptor Ablation in Ras-Initiated Squamous Cancer

    PubMed Central

    Wright, Lisa Nolan; Ryscavage, Andrew; Merlino, Glenn; Yuspa, Stuart H.

    2011-01-01

    Purpose EGFR targeted therapy is in clinical use to treat squamous cell carcinoma of the head and neck and other cancers of lining epithelium. Ras mutations in these tumors are a negative prognostic factor for response and skin inflammation is an adverse reaction to therapy. We investigated transcriptional and biochemical changes that could account for the confounding effects of RAS activation and inflammation in a squamous tissue. Experimental Design We performed gene expression profiling on oncogenic Ras transformed and wildtype mouse and human keratinocytes with EGFR ablated chronically by genetic deletion or acutely by drug treatment and followed leads provided by pathway analysis with biochemical studies. Results We identified a 25 gene signature specific to the Ras-EGFR ablation interaction and a distinct 19 gene EGFR ablation signature on normal keratinocytes. EGFR ablation in the context of wildtype Ras reduces ontologies favoring cell cycle control and transcription while oncogenic Ras enriches ontologies for ion channels and membrane transporters, particularly focused on calcium homeostasis. Ontologies between chronic EGFR ablation and acute pharmacological ablation were unique, both with and without Ras activation. p38α is activated in response to abrogation of EGFR signaling under conditions of Ras activation in both mouse and human keratinocytes and in RAS transformed tumor orthografts of EGFR ablated mouse keratinocytes. EGFR ablation in the absence of oncogenic Ras revealed Erk and IL-1β related pathways. Conclusion These findings reveal unrecognized interactions between Ras and EGFR signaling in squamous tumor cells that could influence the therapeutic response to EGFR ablation therapy. PMID:22068661

  6. Antibacterial activity of Eisenia fetida andrei coelomic fluid: transcription and translation regulation of lysozyme and proteins evidenced after bacterial infestation.

    PubMed

    Hirigoyenberry, F; Lassalle, F; Lassegues, M

    1990-01-01

    1. After bacterial infestation lysozyme and antibacterial activities are enhanced, peaking at 4 hr and 3 days, respectively. 2. Both humoral defenses require RNA and protein de novo synthesis in response to pathogenic bacteria injection (actinomycin D and cycloheximide experiments). 3. Antibacterial activity exists naturally at some basic level, involving regular translation of stable RNAs. 4. When antibacterial activity reaches its maximum after bacterial injection, proteins responsible for it undergo a turn-over. 5. Lysozyme and antibacterial proteins cannot account for the whole response to bacterial infestation; some cellular defense mechanisms like phagocytosis are involved at the same time. PMID:2331874

  7. Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system.

    PubMed Central

    Arias, J A; Dixon, R A; Lamb, C J

    1993-01-01

    CHS15 is one of a family of bean genes encoding chalcone synthase, which catalyzes the first reaction in a branch pathway of phenylpropanoid biosynthesis for the production of flavonoid pigments and UV protectants and isoflavonoid-derived phytoalexins. The functional architecture of the CHS15 promoter was dissected by a novel homologous plant in vitro transcription initiation system in which whole-cell and nuclear extracts from suspension-cultured soybean cells direct accurate and efficient RNA polymerase II-mediated transcription from an immobilized promoter template. Authentic transcription from the CHS15 promoter template was also observed with whole-cell extracts from suspension-cultured cells of bean, tobacco, and the monocot rice, and the soybean whole-cell extract transcribed several other immobilized promoter templates. Hence, this procedure may be of general use in the study of plant gene regulation mechanisms in vitro. Assay of the effects of depletion of the soybean whole-cell extract by preincubation with small regions of the CHS15 promoter or defined cis elements showed that trans factors that bind to G-box (CACGTG, -74 to -69) and H-box (CCTACC, -61 to -56 and -121 to -126) cis elements, respectively, make major contributions to the transcription of the CHS15 promoter in vitro. Both cis element/trans factor interactions in combination are required for maximal activity. Delineation of these functional cis element/trans factor interactions in vitro provides the basis for study of the mechanisms underlying developmental expression of CHS15 in pigmented petal cells established by G-box and H-box combinatorial interactions, and for characterization of the terminal steps of the signal pathway for stress induction of the phytoalexin defense response. PMID:8485404

  8. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed Central

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-01-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. Images PMID:7529017

  9. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-12-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. PMID:7529017

  10. Transcription Factors and Medium Suitable for Initiating the Differentiation of Human-Induced Pluripotent Stem Cells to the Hepatocyte Lineage.

    PubMed

    Tomizawa, Minoru; Shinozaki, Fuminobu; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Ishige, Naoki

    2016-09-01

    Transcription factors and culture media were investigated to determine the condition to initiate the differentiation of human-induced pluripotent stem (iPS) cells most efficiently. The expression of genes in human adult liver was compared with that in 201B7 cells (iPS cells) using cDNA microarray analysis. Episomal plasmids expressing transcription factors were constructed. 201B7 cells were transfected with the episomal plasmids and cultured in ReproFF (feeder-free media maintaining pluripotency), Leibovitz-15 (L15), William's E (WE), or Dulbecco's modified Eagle medium/Nutrient F-12 Ham (DF12) for 7 days. RNA was isolated and subjected to real-time quantitative PCR to analyze the expression of alpha-feto protein (AFP) and albumin. cDNA microarray analysis revealed 16 transcription factors that were upregulated in human adult liver relative to that in 201B7 cells. Episomal plasmids expressing these 16 genes were transfected into 201B7 cells. CCAAT/enhancer-binding protein alpha (CEBPA), CCAAT/enhancer-binding protein beta (CEBPB), forkhead box A1 (FOXA1), and forkhead box A3 (FOXA3) up-regulated AFP and down-regulated Nanog. These four genes were further analyzed. The expression of AFP and albumin was the highest in 201B7 cells transfected with the combination of CEBPA, CEBPB, FOXA1, and FOXA3 and cultured in WE. The combination of CEBPA, CEBPB, FOXA1, and FOXA3 was suitable for 201B7 cells to initiate differentiation to the hepatocyte lineage and WE was the most suitable medium for culture after transfection. J. Cell. Biochem. 117: 2001-2009, 2016. © 2016 Wiley Periodicals, Inc. PMID:26773721

  11. Three different proteins recognize a multifunctional determinant that controls replication initiation, fork arrest and transcription in Tetrahymena.

    PubMed

    Mohammad, M; Saha, S; Kapler, G M

    2000-02-01

    Type I elements regulate the initiation of DNA replication, elongation of replication forks and transcription of the Tetrahymena thermophila rDNA minichromosome. Previous studies identified a 24 kDa protein, ssA-TIBF, which binds the A-rich strand of type I elements. Here we describe two additional type I element binding activities (native mol. wt approximately 65 and approximately 250 kDa) that interact with DNA via previously unidentified 32 and 110 kDa polypeptides. The 65 kDa activity was purified to homogeneity and consists of a homodimer of a 32 kDa polypeptide. In contrast to the other type I element binding factors, the 65 kDa activity partitions preferentially to the nuclear fraction during isolation. Levels of the 65 kDa activity increase dramatically in starved cells, raising the possibility that it might negatively regulate replication or transcription. By comparison, the other two binding activities were elevated slightly during macronuclear development, when the rDNA was undergoing DNA replication. Previous studies indicate that the initiation of rDNA replication is regulated by long range interactions between dispersed type I elements. Competitive DNA binding or cooperative protein-protein interactions between the factors described here may play a regulatory role in replication or expression of the rDNA minichromosome. PMID:10637338

  12. Activation of the prophenoloxidase cascade and initiation of nodule formation in locusts by bacterial lipopolysaccharides.

    PubMed

    Ratcliffe, N A; Brookman, J L; Rowley, A F

    1991-01-01

    The activation of the prophenoloxidase (proPO) system of the locusts, Schistocerca gregaria and Locusta migratoria, by several bacterial lipopolysaccharides (LPS) is described. Activation of proPO by LPS occurred only in the presence of whole blood homogenates and not with hemocyte lysate preparations alone. Levels of phenoloxidase generated by the different LPSs in vitro were also correlated with numbers of nodules formed in vivo by injection of these LPSs. This further strengthens the evidence for the involvement of proPO activation in the insect cellular defenses. Finally, the wisdom in using anticoagulants in order to stabilize fragile hemocytes in studies on the proPO system is discussed. PMID:1904828

  13. Regulation of bacterial methane oxidation: transcription of the soluble methane mono-oxygenase operon of Methylococcus capsulatus (Bath) is repressed by copper ions.

    PubMed

    Nielsen, A K; Gerdes, K; Degn, H; Murrell, J C

    1996-05-01

    Methane is oxidized to methanol by the enzyme methane mono-oxygenase (MMO) in methanotrophic bacteria. In previous work, this multicomponent enzyme system has been extensively characterized at the biochemical and molecular level. Copper ions have been shown to irreversibly inhibit MMO activity in vivo and in vitro, but the effect of copper ions on transcription of the genes encoding the soluble (cytoplasmic) MMO (sMMO) has not previously been investigated. To examine more closely the regulation of bacterial methane oxidation and to determine the role of copper in this process, we have investigated transcriptional regulation of the sMMO gene cluster in the methanotrophic bacterium Methylococcus capsulatus (Bath). Using Northern blot analysis and primer extension experiments, it was shown that the six ORFs of the sMMO gene cluster are organized as an operon and the transcripts produced upon expression of this operon have been identified. The synthesis of these transcripts was under control of a single copper-regulated promoter, which is as yet not precisely defined. PMID:8704968

  14. 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. PMID:27510971

  15. Initial design of a bacterial actuated microrobot for operations in an aqueous medium.

    PubMed

    Andre, Walder; Martel, Sylvain

    2006-01-01

    The initial design of a 500 micromx200 microm untethered microrobot for future operations in an aqueous medium is briefly described. Electrical energy requirement is minimized by exploiting the motility of magnetotactic bacteria embedded in special reservoirs and used to propel the microrobot. An embedded control microcircuit powered through photovoltaic cells is developed to control the swimming directions of the bacteria and hence, the direction of the robot. The work presented is an initial step towards the development of platforms capable of relatively complex tasks being executed by a swarm of such microrobots pre-programmed with various behaviors. PMID:17946532

  16. Resistance to Streptomyces turgidiscabies in potato involves an early and sustained transcriptional reprogramming at initial stages of tuber formation.

    PubMed

    Dees, Merete Wiken; Lysøe, Erik; Alsheikh, Muath; Davik, Jahn; Brurberg, May Bente

    2016-06-01

    Common scab, caused by species from the bacterial genus Streptomyces, is an important disease of potato (Solanum tuberosum) crops worldwide. Early tuberization is a critical period for pathogen infection; hence, studies of host gene expression responses during this developmental stage can be important to expand our understanding of the infection process and to identify putative resistance genes. In an infection experiment with the highly susceptible potato cultivar Saturna and the relatively resistant cultivar Beate, transcription profiles were obtained by RNA sequencing at two developmental stages: the early hook stage and the early tuber formation stage. Our results indicate that 'Beate' mounts an early and sustained response to infection by S. turgidiscabies, whereas the defence response by 'Saturna' ceases before the early tuber formation stage. Most pronounced were the putative candidate defence-associated genes uniquely expressed in 'Beate'. We observed an increase in alternative splicing on pathogen infection at the early hook stage for both cultivars. A significant down-regulation of genes involved in the highly energy-demanding process of ribosome biogenesis was observed for the infected 'Beate' plants at the early hook stage, which may indicate an allocation of resources that favours the expression of defence-related genes. PMID:26416294

  17. The question of dispersive kinetics for the initial phase of charge separation in bacterial reaction centers

    SciTech Connect

    Small, G.J.; Hayes, J.M.; Silbey, R.J.

    1992-09-17

    Recently, femtosecond time domain data have raised the possibility that the kinetics of the initial phase of charge separation could be dispersive due to the glasslike structural heterogeneity of proteins. Guided by spectral hole burning data, we have derived simple theoretical rate expressions which allow the question posed in the title to be explored. 27 refs., 1 fig., 2 tabs.

  18. An intersubunit contact stimulating transcription initiation by E. coli RNA polymerase: interaction of the α C-terminal domain and σ region 4

    PubMed Central

    Ross, Wilma; Schneider, David A.; Paul, Brian J.; Mertens, Aaron; Gourse, Richard L.

    2003-01-01

    The C-terminal domain of the Escherichia coli RNA polymerase (RNAP) alpha subunit (αCTD) stimulates transcription initiation by interacting with upstream (UP) element DNA and a variety of transcription activators. Here we identify specific substitutions in region 4.2 of sigma 70 (σ70) and in αCTD that decrease transcription initiation from promoters containing some, but not all, UP elements. This decrease in transcription derives from a decrease in the initial equilibrium constant for RNAP binding (KB). The open complexes formed by the mutant and wild-type RNAPs differ in DNAse I sensitivity at the junction of the αCTD and σ DNA binding sites, correlating with the differences in transcription. A model of the DNA–αCTD–σ region 4.2 ternary complex, constructed from the previously determined X-ray structures of the Thermus aquaticus σ region 4.2–DNA complex and the E. coli αCTD–DNA complex, indicates that the residues identified by mutation in σ region 4.2 and in αCTD are in very close proximity. Our results strongly suggest that αCTD, when bound to an UP element proximal subsite, contacts the RNAP σ70 subunit, increasing transcription. Previous data from the literature suggest that this same σ–αCTD interaction also plays a role in transcription factor-mediated activation. PMID:12756230

  19. Core Promoter Plasticity Between Maize Tissues and Genotypes Contrasts with Predominance of Sharp Transcription Initiation Sites[OPEN

    PubMed Central

    Li, Wei; Vidal, Mabel; Gray, John; Doseff, Andrea I.; Grotewold, Erich

    2015-01-01

    Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop. PMID:26628745

  20. 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...

  1. The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.

    PubMed

    López-Carrasco, Amparo; Gago-Zachert, Selma; Mileti, Giuseppe; Minoia, Sofia; Flores, Ricardo; Delgado, Sonia

    2016-01-01

    Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5' ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (-) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae. PMID:26618399

  2. 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. PMID:22178024

  3. Expression and Functional Roles of the Pepper Pathogen-Induced bZIP Transcription Factor CabZIP2 in Enhanced Disease Resistance to Bacterial Pathogen Infection.

    PubMed

    Lim, Chae Woo; Baek, Woonhee; Lim, Sohee; Han, Sang-Wook; Lee, Sung Chul

    2015-07-01

    A pepper bZIP transcription factor gene, CabZIP2, was isolated from pepper leaves infected with a virulent strain of Xanthomonas campestris pv. vesicatoria. Transient expression analysis of the CabZIP2-GFP fusion protein in Nicotiana benthamiana revealed that the CabZIP2 protein is localized in the cytoplasm as well as the nucleus. The acidic domain in the N-terminal region of CabZIP2 that is fused to the GAL4 DNA-binding domain is required to activate the transcription of reporter genes in yeast. Transcription of CabZIP2 is induced in pepper plants inoculated with virulent or avirulent strains of X. campestris pv. vesicatoria. The CabZIP2 gene is also induced by defense-related hormones such as salicylic acid, methyl jasmonate, and ethylene. To elucidate the in vivo function of the CabZIP2 gene in plant defense, virus-induced gene silencing in pepper and overexpression in Arabidopsis were used. CabZIP2-silenced pepper plants were susceptible to infection by the virulent strain of X. campestris pv. vesicatoria, which was accompanied by reduced expression of defense-related genes such as CaBPR1 and CaAMP1. CabZIP2 overexpression in transgenic Arabidopsis plants conferred enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Together, these results suggest that CabZIP2 is involved in bacterial disease resistance. PMID:25738319

  4. Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.

    PubMed Central

    Zenzie-Gregory, B; Khachi, A; Garraway, I P; Smale, S T

    1993-01-01

    Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex. Images PMID:8321191

  5. Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

    PubMed Central

    Chang, D D; Clayton, D A

    1986-01-01

    Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species. Images PMID:3785226

  6. 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

  7. Yeast RNA polymerase II initiates transcription in vitro at TATA sequences proximal to potential non-B forms of the DNA template.

    PubMed Central

    Lescure, B; Arcangioli, B

    1984-01-01

    Pure yeast RNA polymerase II selectively initiates an abortive in vitro transcript within a TATA box of the yeast iso-1 cytochrome c gene promoter. Using a series of promoter deletions we show that a DNA sequence located upstream of the TATA box is needed for an efficient in vitro transcription. Supercoiling of the DNA template is an absolute requirement for the specific in vitro transcription. Examination of the DNA structure near several in vitro initiation sites shows that the common features observed are the presence of a TATA sequence in which RNA synthesis is initiated, and which is proximal to a potential non-B form of the DNA (a B to Z transition or a cruciform structure). Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6376116

  8. Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation.

    PubMed

    Klauser, Benedikt; Saragliadis, Athanasios; Ausländer, Simon; Wieland, Markus; Berthold, Michael R; Hartig, Jörg S

    2012-09-01

    In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors. PMID:22777205

  9. Transcription Initiation in a Single-subunit RNA Polymerase proceeds through DNA Scrunching and Rotation of the N-terminal subdomains†

    PubMed Central

    Tang, Guo-Qing; Roy, Rahul; Ha, Taekjip; Patel, Smita S.

    2008-01-01

    Summary Elucidating the mechanism of transcription initiation by RNA polymerases (RNAP) is essential for understanding gene transcription and regulation. Although several models such as DNA scrunching, RNAP translation, and RNAP rotation have been proposed, the mechanism of initiation by T7 RNAP has remained unclear. Using ensemble and single molecule Förster resonance energy transfer (FRET) studies, we provide evidence for concerted DNA scrunching and rotation during initiation by T7 RNAP. A constant spatial distance between the upstream and downstream edges of initiation complexes making 4-7 nt RNA supports the DNA scrunching model, but not the RNAP translation or the pure rotation model. DNA scrunching is accompanied by moderate hinging motion (18 ± 4°) of the promoter towards the downstream DNA. The observed step-wise conformational changes provide a basis to understand abortive RNA synthesis during early stages of initiation and promoter escape during the later stages that allows transition to processive elongation. PMID:18538655

  10. 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

    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

  11. 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

  12. Specialization versus conservation: How Pol I and Pol III use the conserved architecture of the pre-initiation complex for specialized transcription

    PubMed Central

    Hoffmann, Niklas A.; Sadian, Yashar; Tafur, Lucas; Kosinski, Jan; Müller, Christoph W.

    2016-01-01

    ABSTRACT Here, we discuss the overall architecture of the RNA polymerase I (Pol I) and III (Pol III) core enzymes and their associated general transcription factors in the context of models of the Pol I and Pol III pre-initiation complexes, thereby highlighting potential functional adaptations of the Pol I and Pol III enzymes to their respective transcription tasks. Several new insights demonstrate the great degree of specialization of each of the eukaryotic RNA polymerases that is only beginning to be revealed as the structural and functional characterization of all eukaryotic RNA polymerases and their pre-initiation complexes progresses. PMID:27327079

  13. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea.

    PubMed

    Wang, Ji; Xu, Ziqian; Niu, Peihua; Zhang, Chen; Zhang, Jingyun; Guan, Li; Kan, Biao; Duan, Zhaojun; Ma, Xuejun

    2014-01-01

    Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 10(2)-10(3) CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus. PMID:24711998

  14. Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid

    PubMed Central

    Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

    2015-01-01

    Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5′-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5′-TGTAAGCCCTAACA-3′) upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network. PMID:26581248

  15. 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

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

    PubMed

    Milano, Teresa; Angelaccio, Sebastiana; Tramonti, Angela; Di Salvo, Martino Luigi; Contestabile, Roberto; Pascarella, Stefano

    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

  17. 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. PMID:26949087

  18. Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

    SciTech Connect

    Stagno, Jason R.; Altieri, Amanda S.; Bubunenko, Mikhail; Tarasov, Sergey G.; Li, Jess; Court, Donald L.; Byrd, R. Andrew; Ji, Xinhua

    2012-03-26

    Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB-NusE-BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB-NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein-protein and protein-RNA interactions. Further crystallographic investigation of a NusB-NusE-dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

  19. Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA

    PubMed Central

    Resch, Marcus; Schiltz, Emile; Titgemeyer, Fritz; Muller, Yves A.

    2010-01-01

    YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-Å crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix–turn–helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion. PMID:20047956

  20. Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

    PubMed Central

    Chesterton, C. James; Coupar, Barbara E. H.; Butterworth, Peter H. W.

    1974-01-01

    The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA. PMID:4464858

  1. The Landscape of Host Transcriptional Response Programs Commonly Perturbed by Bacterial Pathogens: Towards Host-Oriented Broad-Spectrum Drug Targets

    PubMed Central

    Kidane, Yared H.; Lawrence, Christopher; Murali, T. M.

    2013-01-01

    Background The emergence of drug-resistant pathogen strains and new infectious agents pose major challenges to public health. A promising approach to combat these problems is to target the host’s genes or proteins, especially to discover targets that are effective against multiple pathogens, i.e., host-oriented broad-spectrum (HOBS) drug targets. An important first step in the discovery of such drug targets is the identification of host responses that are commonly perturbed by multiple pathogens. Results In this paper, we present a methodology to identify common host responses elicited by multiple pathogens. First, we identified host responses perturbed by each pathogen using a gene set enrichment analysis of publicly available genome-wide transcriptional datasets. Then, we used biclustering to identify groups of host pathways and biological processes that were perturbed only by a subset of the analyzed pathogens. Finally, we tested the enrichment of each bicluster in human genes that are known drug targets, on the basis of which we elicited putative HOBS targets for specific groups of bacterial pathogens. We identified 84 up-regulated and three down-regulated statistically significant biclusters. Each bicluster contained a group of pathogens that commonly dysregulated a group of biological processes. We validated our approach by checking whether these biclusters correspond to known hallmarks of bacterial infection. Indeed, these biclusters contained biological process such as inflammation, activation of dendritic cells, pro- and anti- apoptotic responses and other innate immune responses. Next, we identified biclusters containing pathogens that infected the same tissue. After a literature-based analysis of the drug targets contained in these biclusters, we suggested new uses of the drugs Anakinra, Etanercept, and Infliximab for gastrointestinal pathogens Yersinia enterocolitica, Helicobacter pylori kx2 strain, and enterohemorrhagic Escherichia coli and the drug

  2. 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.

  3. A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription.

    PubMed Central

    Zhang, X; Lai, M M

    1996-01-01

    Coronavirus transcription is a discontinuous process, involving interactions between a trans-acting leader and the intergenic transcription initiation sequences. A 9-nucleotide (nt) sequence (UUUAUAAAC), which is located immediately downstream of the leader at the 5' terminus of the mouse hepatitis virus (MHV) genomic RNA, contains a sequence resembling the consensus intergenic sequence (UCUAAAC). It has been shown previously that the presence of the 9-nt sequence facilitates leader RNA switching and may enhance subgenomic mRNA transcription. It is unclear how the 9-nt sequence exerts these functions. In this study, we inserted the 9-nt sequence into a defective interfering (DI) RNA reporter system and demonstrated that mRNA transcription could be initiated from the 9-nt sequence almost as efficiently as from the intergenic sequence between genes 6 and 7. Sequence analysis of the mRNAs showed that the 9-nt sequence served as a site of fusion between the leaders and mRNA. The transcription initiation function of the 9-nt sequence could not be substituted by other 5'-terminal sequences. When the entire 5'-terminal sequence, including four copies of the UCUAA sequence plus the 9-nt sequence, was present, transcription could be initiated from any of the UCUAA copies or the 9-nt sequence, resulting in different copy numbers of the UCUAA sequence and the deletion of the 9-nt sequence in some mRNAs. All of these heterogeneous RNA species were also detected from the 5'-terminal region of the viral genomic-length RNA in MHV-infected cells. These results thus suggest tha the heterogeneity of the copy number of UCUAA sequences at the 5' end, the deletion of the 9-nt sequence in viral and DI RNAs, and the leader RNA switching are the results of transcriptional initiation from the 9-nt site. They also show that an mRNA species (mRNA 1) that lacks the 9-nt sequence can be synthesized during MHV infection. Therefore, MHV genomic RNA replication and mRNA 1 transcription may be

  4. Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

    PubMed Central

    Koppstein, David; Ashour, Joseph; Bartel, David P.

    2015-01-01

    The influenza polymerase cleaves host RNAs ∼10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs. PMID:25901029

  5. Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites

    SciTech Connect

    Rossi, M.; Anderson, C.; Demidov, O. N.; Appella, E.; Mazur, S. J.

    2008-12-01

    PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

  6. Epigenetic Regulation of Polymerase II Transcription Initiation in Trypanosoma cruzi: Modulation of Nucleosome Abundance, Histone Modification, and Polymerase Occupancy by O-Linked Thymine DNA Glucosylation▿ †

    PubMed Central

    Ekanayake, Dilrukshi; Sabatini, Robert

    2011-01-01

    Very little is understood regarding how transcription is initiated/regulated in the early-diverging eukaryote Trypanosoma cruzi. Unusually for a eukaryote, genes transcribed by RNA polymerase (Pol) II in T. cruzi are arranged in polycistronic transcription units (PTUs). On the basis of this gene organization, it was previously thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. We recently localized a novel glucosylated thymine DNA base, called base J, to potential promoter regions of PTUs throughout the trypanosome genome. Loss of base J, following the deletion of JBP1, a thymidine hydroxylase involved with synthesis, led to a global increase in the Pol II transcription rate and gene expression. In order to determine the mechanism by which base J regulates transcription, we have characterized changes in chromatin structure and Pol II recruitment to promoter regions following the loss of base J. The loss of base J coincides with a decrease in nucleosome abundance, increased histone H3/H4 acetylation, and increased Pol II occupancy at promoter regions, including the well-characterized spliced leader RNA gene promoter. These studies present the first direct evidence for epigenetic regulation of Pol II transcription initiation via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for regulation of trypanosome gene expression via the novel hypermodified base J. PMID:21926332

  7. The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

    PubMed

    Clavijo, Fernando; Diedhiou, Issa; Vaissayre, Virginie; Brottier, Laurent; Acolatse, Jennifer; Moukouanga, Daniel; Crabos, Amandine; Auguy, Florence; Franche, Claudine; Gherbi, Hassen; Champion, Antony; Hocher, Valerie; Barker, David; Bogusz, Didier; Tisa, Louis S; Svistoonoff, Sergio

    2015-11-01

    Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways. PMID:26096779

  8. Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Jayasinghe, J D H E; Godahewa, G I; Park, Hae-Chul; Lee, Jehee

    2016-09-01

    The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qDC3 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor-joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different

  9. Functions of the N- and C-Terminal Domains of Human RAP74 in Transcriptional Initiation, Elongation, and Recycling of RNA Polymerase II

    PubMed Central

    Lei, Lei; Ren, Delin; Finkelstein, Ann; Burton, Zachary F.

    1998-01-01

    Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (pol II) during multiple stages of the transcription cycle including preinitiation complex assembly, initiation, elongation, and possibly termination and recycling. Human TFIIF appears to be an α2β2 heterotetramer of RNA polymerase II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30). From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N- and C-terminal domains connected by a flexible loop. In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle. The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TATA-binding protein, TFIIB, and RAP30. A more complete N-terminal domain fragment (aa 1 to 217) strongly stimulates both accurate initiation and elongation by pol II. The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both accurate initiation and elongation, and mutations in these regions have similar effects on initiation and elongation. Based on these observations, RAP74 appears to have similar functions in initiation and elongation. The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system. PMID:9528785

  10. The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses

    PubMed Central

    Gundogdu, Ozan; da Silva, Daiani T.; Mohammad, Banaz; Elmi, Abdi; Mills, Dominic C.; Wren, Brendan W.; Dorrell, Nick

    2015-01-01

    The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment. PMID:26257713