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Sample records for rna polymerase subunit

  1. Molecular Evolution of Multi-subunit RNA Polymerases: Sequence Analysis

    PubMed Central

    Lane, William J.; Darst, Seth A.

    2009-01-01

    Transcription in all cellular organisms is performed by multi-subunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multi-subunit RNA polymerases. In addition RNA polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. We have created comprehensive multiple sequence alignments using all available sequence data for the multi-subunit RNA polymerase large subunits, including the bacterial β and β′ subunits and their homologues from archaebacterial RNA polymerases, the eukaryotic RNA polymerases I, II, and III, the nuclear-cytoplasmic large double-stranded DNA Virus RNA polymerases, and plant plastid RNA polymerases. In order to overcome technical difficulties inherent to the large subunit sequences, including large sequence length, small and large lineage-specific insertions, split subunits, and fused proteins, we created an automated and customizable sequence retrieval and processing system. In addition, we used our alignments to create a more expansive set of shared sequence regions and bacterial lineage-specific domain insertions. We also analyzed the intergenic gap between the bacterial β and β′ genes. PMID:19895820

  2. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

    PubMed Central

    Sweetser, D; Nonet, M; Young, R A

    1987-01-01

    Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases. PMID:3547406

  3. RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

    PubMed Central

    Kolodziej, P A; Woychik, N; Liao, S M; Young, R A

    1990-01-01

    RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. Images PMID:2183013

  4. Chromosomal localization of human RNA polymerase II subunit genes

    SciTech Connect

    Acker, J.; Wintzerith, M.; Vigneron, M.; Kedinger, C. ); Mattei, M.G.; Roeckel, N.; Depetris, D. )

    1994-04-01

    The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, the authors have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, they show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist. 20 refs., 2 figs., 1 tab.

  5. Genetic exploration of interactive domains in RNA polymerase II subunits.

    PubMed Central

    Martin, C; Okamura, S; Young, R

    1990-01-01

    The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed. Images PMID:2183012

  6. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases

    PubMed Central

    Iyer, Lakshminarayan M; Koonin, Eugene V; Aravind, L

    2003-01-01

    Background The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. Results Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the β' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi β-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP β' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases

  7. Characterization of a 7-kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic RNA polymerase II.

    PubMed

    Amegadzie, B Y; Ahn, B Y; Moss, B

    1992-05-01

    A previously unrecognized 7-kDa polypeptide copurified with the DNA-dependent RNA polymerase of vaccinia virus virions. Internal amino acid sequences of the small protein matched a viral genomic open reading frame of 63 codons. Antipeptide antiserum was used to confirm the specific and complete association of the 7-kDa protein with RNA polymerase. The amino acid sequence predicted from the viral gene, named rpo7, was 23% identical to that of the smallest subunit of Saccharomyces cerevisiae RNA polymerase II, and a metal-binding motif, Cys-X-X-Cys-Gly, was located at precisely the same location near the N terminus in the two proteins. RNA analyses demonstrated early transcriptional initiation and termination signals in the rpo7 gene sequence. The viral RNA polymerase subunit was synthesized during the early phase of infection and continued to accumulate during the late phase.

  8. Structure and sequence of the gene for the largest subunit of trypanosomal RNA polymerase III.

    PubMed Central

    Köck, J; Evers, R; Cornelissen, A W

    1988-01-01

    As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes. Images PMID:3174432

  9. The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA.

    PubMed Central

    Perales, B; Ortín, J

    1997-01-01

    The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA. PMID:8995663

  10. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  11. A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

    PubMed Central

    Guilfoyle, T J

    1989-01-01

    A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. PMID:2535525

  12. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

    PubMed

    Jin, J; Dong, W; Guarino, L A

    1998-12-01

    The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity. PMID:9811739

  13. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  14. Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

    PubMed Central

    Kolodziej, P A; Young, R A

    1991-01-01

    Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II. Images PMID:1715023

  15. RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.

    PubMed Central

    Kolodziej, P; Young, R A

    1989-01-01

    To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III. Images PMID:2685562

  16. Functional Consequences of Subunit Diversity in RNA Polymerases II and V

    SciTech Connect

    Tan, Ek Han; Blevins, Todd; Ream, Thomas S.; Pikaard, Craig S.

    2012-03-01

    Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.

  17. Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits.

    PubMed Central

    Scafe, C; Martin, C; Nonet, M; Podos, S; Okamura, S; Young, R A

    1990-01-01

    Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues. Images PMID:2406567

  18. RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

    PubMed Central

    Woychik, N A; Young, R A

    1989-01-01

    RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range. Images PMID:2674672

  19. Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

    SciTech Connect

    Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.; Nicora, Carrie D.; Norbeck, Angela D.; Zhu, J. K.; Hagen, G.; Guilfoyle, T. J.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2009-01-30

    In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.

  20. Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

    PubMed Central

    Weiss, Andy; Shaw, Lindsey N.

    2015-01-01

    The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

  1. Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

    PubMed Central

    Chung, H M; Lee, M G; Dietrich, P; Huang, J; Van der Ploeg, L H

    1993-01-01

    Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I. Images PMID:8497277

  2. Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

    PubMed Central

    McKune, K; Woychik, N A

    1994-01-01

    We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6. Images PMID:8196653

  3. RNA polymerase I remains intact without subunit exchange through multiple rounds of transcription in Saccharomyces cerevisiae

    PubMed Central

    Schneider, David A.; Nomura, Masayasu

    2004-01-01

    Previous experiments using mammalian cells suggested that after each round of transcription, RNA polymerase I (Pol I) dissociates into subunits that leave and reenter the nucleolus as individual subunits, before formation of a new initiation complex. In this study, we show that the size and subunit composition of Pol I did not change significantly when Pol I was not engaged in rRNA transcription, brought about by either the absence of Pol I-specific rDNA template or specific inhibition of the transcription initiation step that requires Rrn3p. In fact, Pol I purified from cells completely lacking rDNA repeats was more active than when purified from wild-type cells in an in vitro transcription system designed to assay active Pol I–Rrn3p complexes. Furthermore, measurements of the exchange of A135 and A190 subunits between preexistent Pol I and newly synthesized Pol I showed that these two largest subunits of Pol I do not disassociate through many rounds of transcription in vivo. Thus, Pol I is not a dynamic protein complex but rather a stable enzyme. PMID:15477604

  4. In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin.

    PubMed Central

    Nguyen, V T; Giannoni, F; Dubois, M F; Seo, S J; Vigneron, M; Kédinger, C; Bensaude, O

    1996-01-01

    Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII. PMID:8760875

  5. Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

    SciTech Connect

    Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

    2010-07-01

    The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution. The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2{sub 1} and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R{sub free} = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

  6. Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I.

    PubMed

    Vonwirth, H; Frank, P; Kedinger, C; Büsen, W

    1990-09-10

    It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49, the putative yeast equivalent of bovine ribonuclease H IIb. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H IIb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H IIb epitopes. This observation suggests that a protein displaying ribonuclease H IIb antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.

  7. Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

    SciTech Connect

    Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.; Nicora, Carrie D.; Norbeck, Angela D.; Zhu, Jian-Kang; Hagen, Gretchen; Guilfoyle, Thomas J.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2009-01-30

    In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

  8. The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

    PubMed Central

    Weiss, Andy; Ibarra, J. Antonio; Paoletti, Jessica; Carroll, Ronan K.

    2014-01-01

    In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β′ subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage ϕSA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. PMID:24491578

  9. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  10. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  11. The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

    PubMed Central

    Harrison, D A; Mortin, M A; Corces, V G

    1992-01-01

    A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The gene is expressed at all developmental stages and in all tissues. Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar. Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development. The RpII15 gene product is thus necessary for viability of D. melanogaster. Images PMID:1545824

  12. Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

    PubMed

    Xie, Lili; Wartchow, Charles; Shia, Steven; Uehara, Kyoko; Steffek, Micah; Warne, Robert; Sutton, James; Muiru, Gladys T; Leonard, Vincent H J; Bussiere, Dirksen E; Ma, Xiaolei

    2016-01-01

    Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

  13. Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II

    SciTech Connect

    Rappaport, J.; Cho, K.; Saltzman, A.; Prenger, J.; Golomb, M.; Weinmann, R.

    1988-08-01

    Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the ..beta..-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of ..beta..-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while ..beta..-galactosidase had no effect. The effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that anitibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

  14. CBR antimicrobials alter coupling between the bridge helix and the β subunit in RNA polymerase

    PubMed Central

    Malinen, Anssi M.; NandyMazumdar, Monali; Turtola, Matti; Malmi, Henri; Grocholski, Thadee; Artsimovitch, Irina; Belogurov, Georgiy A

    2014-01-01

    Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit. PMID:24598909

  15. A human RNA polymerase II subunit is encoded by a recently generated multigene family

    PubMed Central

    Grandemange, Sylvie; Schaller, Sophie; Yamano, Shigeru; Du Manoir, Stanislas; Shpakovski, George V; Mattei, Marie-Geneviève; Kedinger, Claude; Vigneron, Marc

    2001-01-01

    Background The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. Results While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a. Conclusions The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus. PMID:11747469

  16. Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II.

    PubMed Central

    Kontermann, R. E.; Kobor, M.; Bautz, E. K.

    1993-01-01

    The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases. PMID:8443600

  17. Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

    PubMed Central

    Yano, R; Nomura, M

    1991-01-01

    The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit. Images PMID:1990281

  18. Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT.

    PubMed

    Fislová, Tatiana; Thomas, Benjamin; Graef, Katy M; Fodor, Ervin

    2010-09-01

    The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

  19. A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

    PubMed Central

    Archambault, J; Schappert, K T; Friesen, J D

    1990-01-01

    RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact. Images PMID:2247052

  20. Cloning and sequence determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest subunit of RNA polymerase II.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ueshima, R; Ishihama, A

    1991-01-01

    The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined. Images PMID:2011520

  1. Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

    PubMed

    Bridier-Nahmias, Antoine; Tchalikian-Cosson, Aurélie; Baller, Joshua A; Menouni, Rachid; Fayol, Hélène; Flores, Amando; Saïb, Ali; Werner, Michel; Voytas, Daniel F; Lesage, Pascale

    2015-05-01

    Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host. PMID:25931562

  2. Structure of the Escherichia coli RNA polymerase a Subunit C-terminal Domain

    SciTech Connect

    Lara-Gonzalez, S.; Birktoft, J; Lawson, C

    2010-01-01

    The {alpha} subunit C-terminal domain ({alpha}CTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli {alpha}CTD ({alpha} subunit residues 245-329) determined to 2.0 {angstrom} resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2{sub 1} and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R{sub free} = 0.236) has improved geometry compared with prior lower resolution determinations of the {alpha}CTD structure [Jeon et al. (1995), Science, 270, 1495-1497; Benoff et al. (2002), Science, 297, 1562-1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of {alpha}CTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

  3. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

    PubMed Central

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-01-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars. PMID:26273261

  4. Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

    PubMed Central

    Acker, J; Wintzerith, M; Vigneron, M; Kedinger, C

    1993-01-01

    The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity. Images PMID:8265347

  5. Structural Basis for Promoter ;#8722;10 Element Recognition by the Bacterial RNA Polymerase [sigma] Subunit

    SciTech Connect

    Feklistov, Andrey; Darst, Seth A.

    2011-12-15

    The key step in bacterial promoter opening is recognition of the -10 promoter element (T-{sub 12}A-{sub 11}T-{sub 10}A-{sub 9}A-{sub 8}T{sub -7} consensus sequence) by the RNA polymerase {alpha} subunit. We determined crystal structures of {alpha} domain 2 bound to single-stranded DNA bearing -10 element sequences. Extensive interactions occur between the protein and the DNA backbone of every -10 element nucleotide. Base-specific interactions occur primarily with A{sub -11} and T{sub -7}, which are flipped out of the single-stranded DNA base stack and buried deep in protein pockets. The structures, along with biochemical data, support a model where the recognition of the -10 element sequence drives initial promoter opening as the bases of the nontemplate strand are extruded from the DNA double-helix and captured by {alpha}. These results provide a detailed structural basis for the critical roles of A{sub -11} and T{sub -7} in promoter melting and reveal important insights into the initiation of transcription bubble formation.

  6. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement

    PubMed Central

    Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.

    2016-01-01

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA. PMID:27087667

  7. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement.

    PubMed

    Blazier, J Chris; Ruhlman, Tracey A; Weng, Mao-Lun; Rehman, Sumaiyah K; Sabir, Jamal S M; Jansen, Robert K

    2016-01-01

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA. PMID:27087667

  8. 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

  9. Mutations in the alpha-amanitin conserved domain of the largest subunit of yeast RNA polymerase III affect pausing, RNA cleavage and transcriptional transitions.

    PubMed Central

    Thuillier, V; Brun, I; Sentenac, A; Werner, M

    1996-01-01

    The alpha-amanitin domain or domain f of the largest subunit of RNA polymerases is one of the most conserved of these enzymes. We have found that the C-terminal part of domain f can be swapped between yeast RNA polymerase II and III. An extensive mutagenesis of domain f of C160, the largest subunit of RNA polymerase III, was carried out to better define its role and understand the mechanism through which C160 participates in transcription. One mutant enzyme, C160-270, showed much reduced transcription of a non-specific template at low DNA concentrations. Abortive synthesis of trinucleotides in a dinucleotide-primed reaction proceeded at roughly wild-type levels, indicating that the mutation did not affect the formation of the first phosphodiester bond, but rather the transition from abortive initiation to processive elongation. In specific transcription assays, on the SUP4 tRNA gene, pausing was extended but the rate of RNA elongation between pause sites was not affected. Finally, the rate of cleavage of nascent RNA transcripts by halted mutant RNA polymerase was increased approximately 10-fold. We propose that the domain f mutation affects the transition between two transcriptional modes, one being adopted during abortive transcription and at pause sites, the other during elongation between pause sites. Images PMID:8599945

  10. Repression and activation of transcription by Gal and Lac repressors: involvement of alpha subunit of RNA polymerase.

    PubMed Central

    Choy, H E; Park, S W; Aki, T; Parrack, P; Fujita, N; Ishihama, A; Adhya, S

    1995-01-01

    Gal or Lac repressor binding to an upstream DNA segment, in the absence of DNA looping, represses the P1 promoter located on the same face and activates the P2 promoter situated on the opposite face of the DNA helix in the gal operon. Both inhibition and stimulation of transcription requires the physical presence of the C-terminal domain of the alpha subunit of RNA polymerase although the latter is not required for transcription itself. We propose that Gal and Lac repressors inhibit or stimulate transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the C-terminal alpha domain to an unfavorable state at P1 or a more favorable state at P2, respectively. Images PMID:7556095

  11. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

    PubMed Central

    Lee, J M; Greenleaf, A L

    1989-01-01

    The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts. Images PMID:2657724

  12. DNA-dependent RNA polymerase II from Acanthamoeba castellanii. Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes.

    PubMed

    Detke, S; Paule, M R

    1978-09-27

    The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 21 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and 16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase. PMID:708741

  13. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    PubMed Central

    Nemchinov, Lev G.; Boutanaev, Alexander M.; Postnikova, Olga A.

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  14. A partial loss-of-function mutation in an Arabidopsis RNA polymerase III subunit leads to pleiotropic defects

    PubMed Central

    Johnson, Kaeli C. M.; Yu, Yu; Gao, Lei; Eng, Ryan C.; Wasteneys, Geoffrey O.; Chen, Xuemei; Li, Xin

    2016-01-01

    Plants employ five DNA-dependent RNA polymerases (Pols) in transcription. One of these polymerases, Pol III, has previously been reported to transcribe 5S rRNA, tRNAs, and a number of small RNAs. However, in-depth functional analysis is complicated by the fact that knockout mutations in Pol subunits are typically lethal. Here, we report the characterization of the first known viable Pol III subunit mutant, nrpc7-1. This mutant was originally isolated from a forward genetic screen designed to identify enhancers of the autoimmune mutant snc1, which contains a gain-of-function mutation in a nucleotide-binding leucine-rich repeat (NLR) immune receptor-encoding gene. The nrpc7-1 mutation occurs in an intron–exon splice site and results in intron retention in some NRPC7 transcripts. There is a global disruption in RNA equilibrium in nrpc7-1, exemplified by the altered expression of a number of RNA molecules, some of which are not reported to be transcribed by Pol III. There are developmental defects associated with the mutation, as homozygous mutant plants are dwarf, have stunted roots and siliques, and possess serrated leaves. These defects are possibly due to altered small RNA stability or activity. Additionally, the nrpc7-1 mutation confers an NLR-specific alternative splicing defect that correlates with enhanced disease resistance, highlighting the importance of alternative splicing in regulating NLR activity. Altogether, these results reveal novel roles for Pol III in maintaining RNA homeostasis, adjusting the expression of a diverse suite of genes, and indirectly modulating gene splicing. Future analyses using the nrpc7-1 mutant will be instrumental in examining other unknown Pol III functions. PMID:26865731

  15. Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

    PubMed Central

    Bartolomei, M S; Corden, J L

    1987-01-01

    RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene. Images PMID:3821724

  16. Specific Residues of PB2 and PA Influenza Virus Polymerase Subunits Confer the Ability for RNA Polymerase II Degradation and Virus Pathogenicity in Mice

    PubMed Central

    Llompart, C. M.

    2014-01-01

    ABSTRACT Influenza virus transcription requires functional coupling with cellular transcription for the cap-snatching process. Despite this fact, RNA polymerase II (RNAP II) is degraded during infection in a process triggered by the viral polymerase. Reassortant viruses from the A/PR/8/34 (PR8) strain that induce (hvPR8) or do not induce (lvPR8) RNAP II degradation led to the identification of PA and PB2 subunits as responsible for the degradation process. Three changes in the PB2 sequence (I105M, N456D, and I504V) and two in PA (Q193H and I550L) differentiate PA and PB2 of lvPR8 from those of hvPR8. Using recombinant viruses, we observed that changes at position 504 of PB2, together with 550 of PA, confer the ability on lvPR8 for RNAP II degradation and, conversely, abolish hvPR8 degradation capacity. Since hvPR8 is more pathogenic than lvPR8 in mice, we tested the potential contribution of RNAP II degradation in a distant viral strain, the 2009 pandemic A/California/04/09 (CAL) virus, whose PA and PB2 subunits are of avian origin. As in the hvPR8 virus, mutations at positions 504 of PB2 and 550 of PA in CAL virus abolished its RNAP II degradation capacity. Moreover, in an in vivo model, the CAL-infected mice lost more body weight, and 75% lethality was observed in this situation compared with 100% survival in mutant-CAL- or mock-infected animals. These results confirm the involvement of specific PB2 and PA residues in RNAP II degradation, which correlates with pathogenicity in mice of viruses containing human or avian polymerase PB2 and PA subunits. IMPORTANCE The influenza virus polymerase induces the degradation of RNAP II, which probably cooperates to avoid the antiviral response. Here, we have characterized two specific residues located in the PA and PB2 polymerase subunits that mediate this degradation in different influenza viruses. Moreover, a clear correlation between RNAP II degradation and in vivo pathogenicity in mice was observed, indicating that the

  17. Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development1[C][W][OA

    PubMed Central

    Steiner, Sebastian; Schröter, Yvonne; Pfalz, Jeannette; Pfannschmidt, Thomas

    2011-01-01

    The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the “prokaryotic” subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants. PMID:21949211

  18. Conditional Mutants of Rpc160, the Gene Encoding the Largest Subunit of RNA Polymerase C in Saccharomyces Cerevisiae

    PubMed Central

    Gudenus, R.; Mariotte, S.; Moenne, A.; Ruet, A.; Memet, S.; Buhler, J. M.; Sentenac, A.; Thuriaux, P.

    1988-01-01

    A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO(3) and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pci1-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation. PMID:2841184

  19. The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes.

    PubMed Central

    Vincent, M; Lauriault, P; Dubois, M F; Lavoie, S; Bensaude, O; Chabot, B

    1996-01-01

    The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix protein (p255) recently shown to associate with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Chabot et al. (1995) Nucleic Acids Res. 23, 3206-3213]. In mouse and Drosophila cultured cells the electrophoretic mobility of p255, faster in the latter species, was identical to that of the hyperphosphorylated form of RNA polymerase II largest subunit (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which is known to cause the dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery of RNA polymerase II largest subunit from HeLa splicing mixtures was compromised by EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a highly phosphorylated form of RNA polymerase II largest subunit physically associated with spliceosomes and possibly involved in coupling transcription to RNA processing. PMID:8972849

  20. NRPB3, the third largest subunit of RNA polymerase II, is essential for stomatal patterning and differentiation in Arabidopsis

    PubMed Central

    Chen, Liang; Guan, Liping; Qian, Pingping; Xu, Fan; Wu, Zhongliang; Wu, Yujun; He, Kai; Gou, Xiaoping; Li, Jia; Hou, Suiwen

    2016-01-01

    ABSTRACT Stomata are highly specialized epidermal structures that control transpiration and gas exchange between plants and the environment. Signal networks underlying stomatal development have been previously uncovered but much less is known about how signals involved in stomatal development are transmitted to RNA polymerase II (Pol II or RPB), which plays a central role in the transcription of mRNA coding genes. Here, we identify a partial loss-of-function mutation of the third largest subunit of nuclear DNA-dependent Pol II (NRPB3) that exhibits an increased number of stomatal lineage cells and paired stomata. Phenotypic and genetic analyses indicated that NRPB3 is not only required for correct stomatal patterning, but is also essential for stomatal differentiation. Protein-protein interaction assays showed that NRPB3 directly interacts with two basic helix-loop-helix (bHLH) transcription factors, FAMA and INDUCER OF CBF EXPRESSION1 (ICE1), indicating that NRPB3 serves as an acceptor for signals from transcription factors involved in stomatal development. Our findings highlight the surprisingly conserved activating mechanisms mediated by the third largest subunit of Pol II in eukaryotes. PMID:26989174

  1. NRPB3, the third largest subunit of RNA polymerase II, is essential for stomatal patterning and differentiation in Arabidopsis.

    PubMed

    Chen, Liang; Guan, Liping; Qian, Pingping; Xu, Fan; Wu, Zhongliang; Wu, Yujun; He, Kai; Gou, Xiaoping; Li, Jia; Hou, Suiwen

    2016-05-01

    Stomata are highly specialized epidermal structures that control transpiration and gas exchange between plants and the environment. Signal networks underlying stomatal development have been previously uncovered but much less is known about how signals involved in stomatal development are transmitted to RNA polymerase II (Pol II or RPB), which plays a central role in the transcription of mRNA coding genes. Here, we identify a partial loss-of-function mutation of the third largest subunit of nuclear DNA-dependent Pol II (NRPB3) that exhibits an increased number of stomatal lineage cells and paired stomata. Phenotypic and genetic analyses indicated that NRPB3 is not only required for correct stomatal patterning, but is also essential for stomatal differentiation. Protein-protein interaction assays showed that NRPB3 directly interacts with two basic helix-loop-helix (bHLH) transcription factors, FAMA and INDUCER OF CBF EXPRESSION1 (ICE1), indicating that NRPB3 serves as an acceptor for signals from transcription factors involved in stomatal development. Our findings highlight the surprisingly conserved activating mechanisms mediated by the third largest subunit of Pol II in eukaryotes. PMID:26989174

  2. Modulating the level of the Rpb7 subunit of RNA polymerase II affects cell separation in Schizosaccharomyces pombe.

    PubMed

    Kumar, Deepak; Sharma, Nimisha

    2015-01-01

    The rpb7(+) gene encodes the seventh largest subunit of RNA polymerase II and is essential for survival of yeast cells. To gain insight into its functions, we expressed rpb7(+) under the control of the nmt1 promoter and investigated its role in regulating multiple phenotypes in Schizosaccharomyces pombe. We observed that low rpb7(+) levels resulted in slow growth of cells under optimum growth conditions. However, no growth defect was observed under different stress conditions tested in this study. Our results also showed that the most prominent phenotype of cells expressing reduced rpb7(+) is a defect in cell separation. Quantitative real-time PCR analysis further revealed that the transcription of specific cell septation genes was significantly reduced in these cells. Collectively, results presented in this study highlight the distinct role of Rpb7p in regulating cell separation in S. pombe.

  3. Microsporidia are related to Fungi: Evidence from the largest subunit of RNA polymerase II and other proteins

    PubMed Central

    Hirt, Robert P.; Logsdon, John M.; Healy, Bryan; Dorey, Michael W.; Doolittle, W. Ford; Embley, T. Martin

    1999-01-01

    We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1α and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination. PMID:9892676

  4. Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins.

    PubMed

    Hirt, R P; Logsdon, J M; Healy, B; Dorey, M W; Doolittle, W F; Embley, T M

    1999-01-19

    We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.

  5. The Euglena gracilis chloroplast rpoB gene. Novel gene organization and transcription of the RNA polymerase subunit operon.

    PubMed Central

    Yepiz-Plascencia, G M; Radebaugh, C A; Hallick, R B

    1990-01-01

    The rpoB gene coding for a beta-like subunit of the chloroplast DNA-dependent RNA polymerase has been located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. We have determined 5760 base-pairs of DNA sequence, including 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resembles the E. coli rpoB-rpoC and higher plant chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with a predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp respectively and a Group II intron of 309 bp. All other known rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNA, as well as rpoB-rpoC1 and rpoB1-rpoC1-rpoC2 mRNAs were identified. Images PMID:2110656

  6. BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei.

    PubMed

    Vélez-Ramírez, D E; Florencio-Martínez, L E; Romero-Meza, G; Rojas-Sánchez, S; Moreno-Campos, R; Arroyo, R; Ortega-López, J; Manning-Cela, R; Martínez-Calvillo, S

    2015-11-01

    RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

  7. The PB2 Subunit of the Influenza A Virus RNA Polymerase Is Imported into the Mitochondrial Matrix

    PubMed Central

    Long, Joshua C. D.

    2016-01-01

    ABSTRACT The polymerase basic 2 (PB2) subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localize to the mitochondria. Various roles, including the regulation of apoptosis and innate immune responses to viral infection, have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signaling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we used enhanced ascorbate peroxidase (APEX)-tagged PB2 proteins and electron microscopy to study the localization of PB2 in mitochondria. We found that PB2 is imported into mitochondria, where it localizes to the mitochondrial matrix. We also demonstrated that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we found that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localization of PB2 of human from that of avian influenza A virus strains. We also showed that a virus encoding nonmitochondrial PB2 is attenuated in mouse embryonic fibroblasts (MEFs) compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria. IMPORTANCE The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial

  8. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  9. Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1.

    PubMed

    Badjatia, Nitika; Ambrósio, Daniela L; Lee, Ju Huck; Günzl, Arthur

    2013-05-01

    Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m(7)G capping enzymes. The first critical step, Ser(5) phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m(7)G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

  10. African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

    PubMed Central

    Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

    1993-01-01

    A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

  11. Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis.

    PubMed

    Yu, Wenxi; Zhang, Zhichang; Min, Daliu; Yang, Qingcheng; Du, Xuefei; Tang, Lina; Lin, Feng; Sun, Yuanjue; Zhao, Hui; Zheng, Shuier; He, Aina; Li, Hongtao; Yao, Yang; Shen, Zan

    2014-04-01

    Osteosarcoma (OS) is the most common primary malignant tumour of bone. Nearly 30-40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumourigenesis and progression of OS is crucial for developing new therapies. Here, we found that the expression of Mediator of RNA polymerase II transcription subunit 19 (Med19) was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis (P<0.05) and poor prognosis (P<0.01). Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP). In addition, Med19 knockdown significantly suppressed tumour growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 downregulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumour samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumour metastasis in a model of OS metastatic spreading. Taken together, our data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway.

  12. Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis.

    PubMed

    Yu, Wenxi; Zhang, Zhichang; Min, Daliu; Yang, Qingcheng; Du, Xuefei; Tang, Lina; Lin, Feng; Sun, Yuanjue; Zhao, Hui; Zheng, Shuier; He, Aina; Li, Hongtao; Yao, Yang; Shen, Zan

    2014-04-01

    Osteosarcoma (OS) is the most common primary malignant tumour of bone. Nearly 30-40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumourigenesis and progression of OS is crucial for developing new therapies. Here, we found that the expression of Mediator of RNA polymerase II transcription subunit 19 (Med19) was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis (P<0.05) and poor prognosis (P<0.01). Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP). In addition, Med19 knockdown significantly suppressed tumour growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 downregulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumour samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumour metastasis in a model of OS metastatic spreading. Taken together, our data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway. PMID:24565852

  13. Helicobacter pylori RNA polymerase α-subunit C-terminal domain shows features unique to ɛ-proteobacteria and binds NikR/DNA complexes

    PubMed Central

    Borin, Brendan N; Tang, Wei; Krezel, Andrzej M

    2014-01-01

    Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the α subunit of RNA polymerase (αCTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although αCTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori αCTD. It is larger than other structurally determined αCTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among ɛ-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to αCTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori αCTD to NikR:DNA complexes. We also produced αCTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori αCTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators. PMID:24442709

  14. DNA-dependent RNA polymerase III from the fungus Podospora comata. Purification, subunit structure and comparison with the homologous enzyme of a related species.

    PubMed

    Barreau, C; Begueret, J

    1982-12-15

    DNA-dependent RNA polymerase III has been purified to homogeneity from the filamentous fungus Podospora comata. The enzyme was extracted at low ionic strength, separated from the polymerases I and II by DEAE-Sephadex chromatography and purified by heparin-Sepharose and phosphocellulose chromatography; 0.1-0.2 mg highly purified homogeneous enzyme with a specific activity of 220 units/mg could be obtained from 2 kg wet mycelium. The subunit composition of the enzyme was determined after sodium dodecyl sulphate/polyacrylamide gel electrophoresis; thirteen putative subunits of molecular weight 174000 (a), 129000 b), 87000 (c), 50000 (d), 39000 (e), 23500 (f), 21000 (g), 19000 (h), 17000 (i), 16500 (j), 13500 (k), 11000 (l) and 10000 (m) were identified. All of the polypeptide components of the enzyme are present in about integral stoichiometric amounts as judged by dye binding. The presence of subunit Mr = 87000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were observed in a preparation of RNA polymerase III from Podospora anserina, which is a related species. Only subunit i is different in the two species. PMID:7151805

  15. Structure-Based Drug Design Targeting a Subunit Interaction of Influenza Virus RNA Polymerase

    NASA Astrophysics Data System (ADS)

    Sugiyama, Kanako; Obayashi, Eiji; Yoshida, Hisashi; Park, Sam-Yong

    Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here, we describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.

  16. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

    PubMed Central

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-01-01

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same ‘double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs. PMID:27548043

  17. NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation

    SciTech Connect

    He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga; Zhu, Jianhua; Lu, Jian; Bressan, Ray A.; Pikaard, Craig S.; Wang, Co-Shine; Zhu, Jian-Kang

    2009-01-01

    RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

  18. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    SciTech Connect

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.

  19. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    DOE PAGES

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Polmore » III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.« less

  20. [Structural-functional characteristics of the Schizosaccharomyces pombe rpb8+ gene, coding the subunit of RNA polymerase I-III, specific only for eukaryotes].

    PubMed

    Shpakovskiĭ, G V; Proshkin, S A; Kaiushin, A L; Korosteleva, M D; Lebedenko, E N

    1998-02-01

    A full-length cDNA of the rpb8+ gene encoding a common subunit Rpb8 of nuclear RNA polymerases I-III only specific for Eucarya was isolated from an expression library of the fission yeast Schizosaccharomyces pombe. The primary structure of the corresponding fragment of the Sz. pombe genome was also established. The rpb8+ gene contains two short introns, 59 and 48 bp long. Only short segments of homology were found upon comparing the Rpb8 subunit homologs from various eukaryotic species, and substantial differences exist between the corresponding proteins of unicellular and multicellular organisms. Subunit Rpb8 of Sz. pombe proved to be the smallest one among the known related proteins: it lacks the 21-aa fragment corresponding to amino acids residues 68-88 of the central part of the homologous subunit ABC14.5 of Saccharomyces cerevisiae. Accordingly, subunit Rpb8 of the fission yeast was not capable of substituting in vivo subunit ABC14.5 in nuclear RNA polymerases of the baker's yeast. PMID:10335407

  1. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: A conserved structure with an essential function

    SciTech Connect

    Allison, L.A.; Wong, J.K.C.; Fitzpatrick, V.D.; Moyle, M.; Ingles, C.J.

    1988-01-01

    Using DNA encoding the largest subunit of Drosophila melanogaster RNA polymerase II, the authors isolated the homologous hamster RPO21 gene. Nucleotide sequencing of both the hamster and D. melanogaster RPO21 DNAs confirmed that the RPO21 polypeptides of these two species, like the Saccharomyces cerevisiae RPO21 polypeptide, contain both an N-terminal region homologous to the Escherichia coli RNA polymerase subunit ..beta..' and a unique polymerase II-specific C-terminal domain. This C-terminal domain, encoded by separate exons in the D. melanogaster and hamster genes, consists of a tandemly repeated heptapeptide sequence. By constructing a series of deletions in DNA encoding the 26 heptapeptide repeats normally present in the S. cerevisiae RPO21 polypeptide, the authors have established that a minimum of between 9 and 11 repeats is necessary for RPO21 function in yeast cells. Replacement of the yeast RPO21 heptapeptide repeats by the longer hamster repetitive domain resulted in viable yeast cells with no detectable mutant phenotype, while a similar replacement of the yeast repeats by the more divergent D. melanogaster repeats was a recessive lethal mutation. The authors suggest that this novel repetitive domain is essential for proper initiation of transcription by RNA polymerase II and that it may mediate the functions of TATA boxes, upstream activating sequences, and enhancers.

  2. Interaction of Escherichia coli RNA polymerase σ70 subunit with promoter elements in the context of free σ70, RNA polymerase holoenzyme, and the β'-σ70 complex.

    PubMed

    Mekler, Vladimir; Pavlova, Olga; Severinov, Konstantin

    2011-01-01

    Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

  3. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    PubMed

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  4. Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

    PubMed Central

    Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

    1995-01-01

    Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

  5. RNA Polymerases of Maize: Nuclear RNA Polymerases*

    PubMed Central

    Strain, Gustave C.; Mullinix, Kathleen P.; Bogorad, Lawrence

    1971-01-01

    Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH4)2SO4, respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg++ than Mn++ and are inhibited by (NH4)2-SO4 or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by α-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA. PMID:5288239

  6. Reverse Genetics of Drosophila RNA Polymerase II: Identification and Characterization of Rpii140, the Genomic Locus for the Second-Largest Subunit

    PubMed Central

    Hamilton, B. J.; Mortin, M. A.; Greenleaf, A. L.

    1993-01-01

    We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus. PMID:8325487

  7. The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome.

    PubMed

    Noack Watt, Kristin E; Achilleos, Annita; Neben, Cynthia L; Merrill, Amy E; Trainor, Paul A

    2016-07-01

    Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention. PMID:27448281

  8. The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome.

    PubMed

    Noack Watt, Kristin E; Achilleos, Annita; Neben, Cynthia L; Merrill, Amy E; Trainor, Paul A

    2016-07-01

    Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention.

  9. The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome

    PubMed Central

    Achilleos, Annita; Neben, Cynthia L.; Merrill, Amy E.; Trainor, Paul A.

    2016-01-01

    Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention. PMID:27448281

  10. Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase β′ Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins

    PubMed Central

    MacGregor, Barbara J.

    2015-01-01

    The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. “Maribeggiatoa” filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. “Maribeggiatoa” Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in

  11. The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability

    PubMed Central

    Chapman, Rob D.; Palancade, Benoit; Lang, Andreas; Bensaude, Olivier; Eick, Dirk

    2004-01-01

    The phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) has been shown to affect the initiation, and transition to elongation of the Pol II complex. The differential phosphorylation of serines within this domain coincides with the recruitment of factors important for pre-mRNA processing and transcriptional elongation. A role for tyrosine and threonine phosphorylation has yet to be described. The discovery of kinases that express a preference for specific residues within this sequence suggests a mechanism for the controlled recruitment and displacement of CTD-interacting partners during the transcription cycle. The last CTD repeat (CTD52) contains unique interaction sites for the only known CTD tyrosine kinases, Abl1/c-Abl and Abl2/Arg, and the serine/threonine kinase casein kinase II (CKII). Here, we show that removal or severe disruption of the last CTD repeat, but not point mutation of its CKII sites, results in its proteolytic degradation to the Pol IIb form in vivo, but does not appear to affect the specific transcription of genes. These results suggest a possible mechanism of transcription control through the proteolytic removal of the Pol II CTD. PMID:14704341

  12. Fine structure of E. coli RNA polymerase-promoter interactions: α subunit binding to the UP element minor groove

    PubMed Central

    Ross, Wilma; Ernst, Alexander; Gourse, Richard L.

    2001-01-01

    The α subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the ς subunit, the −35 and −10 hexamers. We examined DNA–RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that α interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to α binding. On the basis of these and previous results, we propose a model in which α contacts UP element DNA through amino acid residues located in a pair of helix–hairpin–helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by ς70 by identifying functional groups in the major grooves of the −35 and −10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter–RNAP interaction. PMID:11238372

  13. In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements

    PubMed Central

    Maciąg, Anna; Peano, Clelia; Pietrelli, Alessandro; Egli, Thomas; De Bellis, Gianluca; Landini, Paolo

    2011-01-01

    Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by EσS. PMID:21398637

  14. Evasion of the Innate Immune Response: the Old World Alphavirus nsP2 Protein Induces Rapid Degradation of Rpb1, a Catalytic Subunit of RNA Polymerase II

    PubMed Central

    Akhrymuk, Ivan; Kulemzin, Sergey V.

    2012-01-01

    The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response. PMID:22514352

  15. Involvement of region 4 of the sigma70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing.

    PubMed

    Johnson, Deborah C; Ishihama, Akira; Stevens, Ann M

    2003-11-21

    Quorum sensing-dependent activation of the luminescence (lux) genes of Vibrio fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl)-L-homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. In its active conformation, LuxR binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site and is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma70 subunit of RNAP in LuxR-dependent activation of the luxI promoter has been investigated. Single-round transcription assays were performed in the presence of purified LuxRDeltaN, the autoinducer-independent C-terminal domain of LuxR, and a variant RNAP which contained a C-terminally truncated sigma70 subunit devoid of region 4. Results indicated that region 4 is essential for LuxRDeltaN-dependent luxI transcription, therefore 16 single and two triple alanine substitutions in region 4.2 of sigma70 between amino acid residues 590 and 613 were examined for their effects on LuxR- and LuxRDeltaN-dependent transcription at the luxI promoter. Taken together, the analyses performed on these variants of RpoD suggest that some individual residues in region 4.2 are important to the mechanism of activator-dependent transcription initiation under investigation.

  16. Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of mediator subunit Med19.

    PubMed

    Boube, Muriel; Hudry, Bruno; Immarigeon, Clément; Carrier, Yannick; Bernat-Fabre, Sandra; Merabet, Samir; Graba, Yacine; Bourbon, Henri-Marc; Cribbs, David L

    2014-05-01

    Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded "co-factor" that acts together with Hox proteins through their homeodomains in regulated developmental transcription.

  17. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    SciTech Connect

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  18. The largest subunit of RNA polymerase II as a new marker gene to study assemblages of arbuscular mycorrhizal fungi in the field.

    PubMed

    Stockinger, Herbert; Peyret-Guzzon, Marine; Koegel, Sally; Bouffaud, Marie-Lara; Redecker, Dirk

    2014-01-01

    Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.

  19. A mutation of RNA polymerase β' subunit (RpoC) converts heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) into "slow VISA".

    PubMed

    Matsuo, Miki; Hishinuma, Tomomi; Katayama, Yuki; Hiramatsu, Keiichi

    2015-07-01

    Various mutations in the rpoB gene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found "slow VISA" (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024-5035, 2014, http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of the rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, in rpoC. Introduction of the wild-type (WT) rpoC gene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that the rpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: the rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ, which is involved in the stringent response. It appears that the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

  20. NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

    PubMed Central

    Nguyen, Bao D.; Abbott, Karen L.; Potempa, Krzysztof; Kobor, Michael S.; Archambault, Jacques; Greenblatt, Jack; Legault, Pascale; Omichinski, James G.

    2003-01-01

    FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II). The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436–517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879–961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74/cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an α-helix (H1′; E945–M961) in the complex. The cterRAP74/cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1′ helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1′ of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1′ helix. The cterRAP74/cterFCP complex is the first high-resolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in protein/protein complexes involving acidic residue-rich domains in transcription regulatory proteins. PMID:12732728

  1. NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1.

    PubMed

    Nguyen, Bao D; Abbott, Karen L; Potempa, Krzysztof; Kobor, Michael S; Archambault, Jacques; Greenblatt, Jack; Legault, Pascale; Omichinski, James G

    2003-05-13

    FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II). The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436-517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879-961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an alpha-helix (H1'; E945-M961) in the complex. The cterRAP74cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1' helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1' of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1' helix. The cterRAP74cterFCP complex is the first high-resolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in proteinprotein complexes involving acidic residue-rich domains in transcription regulatory proteins. PMID:12732728

  2. Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

    PubMed

    Esyunina, D M; Kulbachinskiy, A V

    2015-10-01

    The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

  3. Reticulocyte RNA-Dependent RNA Polymerase

    PubMed Central

    Downey, Kathleen M.; Byrnes, John J.; Jurmark, Bonnie S.; So, Antero G.

    1973-01-01

    A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes. PMID:4519633

  4. RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processive mode of action of avian myeloblastosis virus polymerase.

    PubMed Central

    Leis, J P

    1976-01-01

    Purified avian myeloblastosis virus (AMV) polymerase consisting of alpha,beta subunits has been shown to act processively in catalyzing DNA synthesis primed with 34S AMV RNA oligo(dT), poly(A)-poly(dT), and poly(I)-poly(dC). DNA transcripts prepared with 34S AMV RNA-oligo(dT)14 and AMV polymerase (alphabeta) have been shown to have a molecular weight of 1.05 X 10(6), or approximately one-third the size of the 34S RNA genome. Polymerase subunit alpha acts nonprocessively with the above templates. PMID:61286

  5. Mutation of RNA polymerase beta subunit (rpoB) promotes hVISA-to-VISA phenotypic conversion of strain Mu3.

    PubMed

    Matsuo, Miki; Hishinuma, Tomomi; Katayama, Yuki; Cui, Longzhu; Kapi, Maria; Hiramatsu, Keiichi

    2011-09-01

    The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR, carrying the mutated two-component response regulator graR, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.

  6. The three subunits of the polymerase and the nucleoprotein of influenza B virus are the minimum set of viral proteins required for expression of a model RNA template.

    PubMed

    Jambrina, E; Bárcena, J; Uez, O; Portela, A

    1997-09-01

    The genes encoding the nucleoprotein, PB1, PB2, and PA proteins of the influenza virus strain B/Panamá/45/90 have been cloned under control of the T7 RNA polymerase promoter of plasmid pGEM-3. Transfection of the recombinant plasmids obtained into mammalian cells, which had been infected with a vaccinia virus encoding the T7 RNA polymerase, resulted in expression of the expected influenza B virus polypeptides. Moreover, it is shown that coexpression of the four recombinant core proteins in COS-1 cells reconstituted a functional polymerase capable of expressing a synthetic influenza B virus-like CAT RNA. By using the influenza B virus recombinant plasmids and a set of pGEM-derived plasmids encoding the homologous core proteins of the influenza A virus A/Victoria/3/75 (I. Mena et al. (1994). J. Gen. Virol. 75, 2109-2114), the capabilities of homo- and heterotypic mixtures of the four core proteins to express synthetic type A and B CAT RNAs were analyzed. Both the influenza A and B virus polymerases were active in expressing, albeit with reduced efficiencies, the heterotypic model CAT RNAs. However, none of all possible heterotypic mixtures of the core proteins reconstituted a functional polymerase. In order to fully characterize the recombinant plasmids obtained, the nucleotide sequences of the cloned genes were determined and compared to sequences of other type B virus isolates. The results obtained from these latter analyses are discussed in terms of the conservation and evolution of the influenza B virus core genes.

  7. Solving the RNA polymerase I structural puzzle

    SciTech Connect

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  8. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

    PubMed Central

    Yuryev, A; Patturajan, M; Litingtung, Y; Joshi, R V; Gentile, C; Gebara, M; Corden, J L

    1996-01-01

    Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692929

  9. Glycosylation of RNA polymerase II from wheat germ.

    PubMed

    Cervoni, L; Turano, C; Ferraro, A; Ciavatta, P; Marmocchi, F; Eufemi, M

    1997-11-10

    RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N-acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416-10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N-acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes. PMID:9395301

  10. Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

    PubMed

    Du, Yu-Jie; Hou, Yi-Ling; Hou, Wan-Ru

    2013-02-01

    The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.

  11. Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

    PubMed

    Du, Yu-Jie; Hou, Yi-Ling; Hou, Wan-Ru

    2013-02-01

    The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance. PMID:23070920

  12. The RNA Polymerase of Marine Cyanophage Syn5*

    PubMed Central

    Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava A.; Hernandez, Alfredo; King, Jonathan A.; Richardson, Charles C.

    2013-01-01

    A single subunit DNA-dependent RNA polymerase was identified and purified to apparent homogeneity from cyanophage Syn5 that infects the marine cyanobacteria Synechococcus. Syn5 is homologous to bacteriophage T7 that infects Escherichia coli. Using the purified enzyme its promoter has been identified by examining transcription of segments of Syn5 DNA and sequencing the 5′-termini of the transcripts. Only two Syn5 RNAP promoters, having the sequence 5′-ATTGGGCACCCGTAA-3′, are found within the Syn5 genome. One promoter is located within the Syn5 RNA polymerase gene and the other is located close to the right genetic end of the genome. The purified enzyme and its promoter have enabled a determination of the requirements for transcription. Unlike the salt-sensitive bacteriophage T7 RNA polymerase, this marine RNA polymerase requires 160 mm potassium for maximal activity. The optimal temperature for Syn5 RNA polymerase is 24 °C, much lower than that for T7 RNA polymerase. Magnesium is required as a cofactor although some activity is observed with ferrous ions. Syn5 RNA polymerase is more efficient in utilizing low concentrations of ribonucleotides than T7 RNA polymerase. PMID:23258537

  13. A third mitochondrial RNA polymerase in the moss Physcomitrella patens.

    PubMed

    Richter, Uwe; Richter, Björn; Weihe, Andreas; Börner, Thomas

    2014-02-01

    In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.

  14. Recruitment of RNA polymerase III in vivo.

    PubMed

    Kenneth, Niall S; Marshall, Lynne; White, Robert J

    2008-06-01

    RNA polymerase (pol) III contains a dissociable subcomplex that is required for initiation, but not for elongation or termination of transcription. This subcomplex is composed of subunits RPC3, RPC6 and RPC7, and interacts with TFIIIB, a factor that is necessary and sufficient to support accurate pol III transcription in vitro. Direct binding of TFIIIB to RPC6 is believed to recruit pol III to its genetic templates. However, this has never been tested in vivo. Here we combine chromatin immunoprecipitation with RNA interference to demonstrate that the RPC3/6/7 subcomplex is required for pol III recruitment in mammalian cells. Specific knockdown of RPC6 by RNAi results in post-transcriptional depletion of the other components of the subcomplex, RPC3 and RPC7, without destabilizing core pol III subunits or TFIIIB. The resultant core enzyme is defective in associating with TFIIIB and target genes in vivo. Promoter occupancy by pol II is unaffected, despite sharing five subunits with the pol III core. These observations provide evidence for the validity in vivo of the model for pol III recruitment that was built on biochemical data.

  15. RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II*

    PubMed Central

    Mullinix, Kathleen P.; Strain, Gustave C.; Bogorad, Lawrence

    1973-01-01

    Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000. Images PMID:4525172

  16. Binding interface between the Salmonella σS/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl

    PubMed Central

    Cavaliere, Paola; Sizun, Christina; Levi-Acobas, Fabienne; Nowakowski, Mireille; Monteil, Véronique; Bontems, François; Bellalou, Jacques; Mayer, Claudine; Norel, Françoise

    2015-01-01

    In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σS accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σS binding to the Crl protein increases σS activity by favouring its association to E. Taking advantage of evolution of the σS sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σS to the S. Typhimurium σS-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σS and to generate in silico models of the σS-Crl complex constrained by mutational analysis. The σS-Crl models suggest that the identified arginine in σS interacts with an aspartate of Crl that is required for σS binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σS-Crl complex. PMID:26338235

  17. Genetics of eukaryotic RNA polymerases I, II, and III.

    PubMed Central

    Archambault, J; Friesen, J D

    1993-01-01

    The transcription of nucleus-encoded genes in eukaryotes is performed by three distinct RNA polymerases termed I, II, and III, each of which is a complex enzyme composed of more than 10 subunits. The isolation of genes encoding subunits of eukaryotic RNA polymerases from a wide spectrum of organisms has confirmed previous biochemical and immunological data indicating that all three enzymes are closely related in structures that have been conserved in evolution. Each RNA polymerase is an enzyme complex composed of two large subunits that are homologous to the two largest subunits of prokaryotic RNA polymerases and are associated with smaller polypeptides, some of which are common to two or to all three eukaryotic enzymes. This remarkable conservation of structure most probably underlies a conservation of function and emphasizes the likelihood that information gained from the study of RNA polymerases from one organism will be applicable to others. The recent isolation of many mutations affecting the structure and/or function of eukaryotic and prokaryotic RNA polymerases now makes it feasible to begin integrating genetic and biochemical information from various species in order to develop a picture of these enzymes. The picture of eukaryotic RNA polymerases depicted in this article emphasizes the role(s) of different polypeptide regions in interaction with other subunits, cofactors, substrates, inhibitors, or accessory transcription factors, as well as the requirement for these interactions in transcription initiation, elongation, pausing, termination, and/or enzyme assembly. Most mutations described here have been isolated in eukaryotic organisms that have well-developed experimental genetic systems as well as amenable biochemistry, such as Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. When relevant, mutations affecting regions of Escherichia coli RNA polymerase that are conserved among eukaryotes and prokaryotes are also presented

  18. Crystallographic analysis of an RNA polymerase σ-subunit fragment complexed with -10 promoter element ssDNA: quadruplex formation as a possible tool for engineering crystal contacts in protein-ssDNA complexes.

    PubMed

    Feklistov, Andrey; Darst, Seth A

    2013-09-01

    Structural studies of -10 promoter element recognition by domain 2 of the RNA polymerase σ subunit [Feklistov & Darst (2011), Cell, 147, 1257-1269] reveal an unusual crystal-packing arrangement dominated by G-quartets. The 3'-terminal GGG motif of the oligonucleotide used in crystallization participates in G-quadruplex formation with GGG motifs from symmetry-related complexes. Stacking between neighboring G-quadruplexes results in the formation of pseudo-continuous four-stranded columns running throughout the length of the crystal (G-columns). Here, a new crystal form is presented with a different arrangement of G-columns and it is proposed that the fortuitous finding of G-quartet packing could be useful in engineering crystal contacts in protein-ssDNA complexes. PMID:23989139

  19. The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations.

    PubMed Central

    Berroteran, R W; Ware, D E; Hampsey, M

    1994-01-01

    Mutations in the Saccharomyces cerevisiae sua8 gene were found to be suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons. This effect is not cyc1 gene specific since initiation at other genes, including ADH1, CYC7, and HIS4, was similarly affected, although initiation at HIS3 and SPT15 was unaffected. The SUA8 gene was cloned and partially sequenced, revealing identity to RPB1, which encodes the largest subunit of RNA polymerase II. The sua8 suppressors are the result of single amino acid replacements of highly conserved residues. Three replacements were found either within or immediately preceding homology block D, and a fourth was found adjacent to homology block H, indicating that these regions play a role in defining start sites in vivo. Nearly identical effects on start site selection were observed for sua7 suppressors, which encode altered forms of TFIIB. Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation). These data indicate that the largest subunit of RNA polymerase II and TFIIB are important determinants of transcription start site selection in S. cerevisiae and suggest that this function might be conferred by interaction between these two proteins. Images PMID:8264591

  20. Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

  1. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

    PubMed Central

    Nonet, M; Scafe, C; Sexton, J; Young, R

    1987-01-01

    We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis. Images PMID:3299050

  2. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  3. Post-Transcriptional Regulation of RNA Polymerase II Levels in Caenorhabditis Elegans

    PubMed Central

    Dalley, B. K.; Rogalski, T. M.; Tullis, G. E.; Riddle, D. L.; Golomb, M.

    1993-01-01

    To investigate the regulation of RNA polymerase II levels in Caenorhabditis elegans, we have constructed nematode strains having one, two, or three copies of ama-1, the gene for the largest subunit of RNA polymerase II. Steady-state levels of RNA polymerase II polypeptides and solubilized enzyme activity are invariant with gene dosage, indicating regulatory compensation. However, steady-state levels of ama-1 mRNA are directly proportional to gene dosage. These results imply that RNA polymerase II levels in C. elegans are regulated post-transcriptionally. PMID:8436272

  4. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  5. Alternative Splicing of Toll-Like Receptor 9 Transcript in Teleost Fish Grouper Is Regulated by NF-κB Signaling via Phosphorylation of the C-Terminal Domain of the RPB1 Subunit of RNA Polymerase II

    PubMed Central

    Lee, Frank Fang-Yao; Hui, Cho-Fat; Chang, Tien-Hsien; Chiou, Pinwen Peter

    2016-01-01

    Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb’s role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway. PMID:27658294

  6. Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

    PubMed Central

    Valenzuela, P; Hager, G L; Weinberg, F; Rutter, W J

    1976-01-01

    Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000, 82,000, 41,000, 40,500, 37,000, 34,000, 28,000, 24,000, 20,000, 14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits. As in other eukaryotes, yeast polymerase II is inhibited by relatively low concentrations of alpha-amanitin; however, contrary to what has been found in higher eukaryotes, yeast polymerase III is resistant (up to 2 mg/ml) to alpha-amanitin, while yeast polymerase I is sensitive to high concentrations of the drug (50% inhibition at 0.3 mg/ml). These results establish the existence of RNA polymerase III in yeast and provide a structural basis for the discrimination of the three functional polymerases in eukaryotes. Images PMID:772675

  7. Activation of P2 late transcription by P2 Ogr protein requires a discrete contact site on the C terminus of the alpha subunit of Escherichia coli RNA polymerase.

    PubMed

    Wood, L F; Tszine, N Y; Christie, G E

    1997-11-21

    Bacteriophage P2 late transcription requires the product of the P2 ogr gene. Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the alpha subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the alpha C-terminal domain (alphaCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters. In addition, the effects of alanine substitutions in the regions of the alphaCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined. Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, define a surface-exposed patch on the alphaCTD important for Ogr-dependent activation. These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified activation surface for protein:protein contact. Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation. This suggests that upstream DNA-alpha contacts, in addition to alpha-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of alpha with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the alphaCTD. PMID:9398509

  8. Norovirus Proteinase-Polymerase and Polymerase Are Both Active Forms of RNA-Dependent RNA Polymerase

    PubMed Central

    Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J.; Cameron, Craig E.; Green, Kim Y.

    2005-01-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro−Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro−Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis. PMID:15681440

  9. Structural and Functional Characterization of an Influenza Virus RNA Polymerase-Genomic RNA Complex ▿

    PubMed Central

    Resa-Infante, Patricia; Recuero-Checa, María Ángeles; Zamarreño, Noelia; Llorca, Óscar; Ortín, Juan

    2010-01-01

    The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negative-polarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex. PMID:20702645

  10. Transcription termination by the eukaryotic RNA polymerase III

    PubMed Central

    Arimbasseri, Aneeshkumar G.; Rijal, Keshab; Maraia, Richard J.

    2012-01-01

    RNA polymerase (pol) III transcribes a multitude of tRNA and 5S rRNA genes as well as other small RNA genes distributed through the genome. By being sequence-specific, precise and efficient, transcription termination by pol III not only defines the 3′ end of the nascent RNA which directs subsequent association with the stabilizing La protein, it also prevents transcription into downstream DNA and promotes efficient recycling. Each of the RNA polymerases appears to have evolved unique mechanisms to initiate the process of termination in response to different types of termination signals. However, in eukaryotes much less is known about the final stage of termination, destabilization of the elongation complex with release of the RNA and DNA from the polymerase active center. By comparison to pols I & II, pol III exhibits the most direct coupling of the initial and final stages of termination, both of which occur at a short oligo(dT) tract on the non-template strand (dA on the template) of the DNA. While pol III termination is autonomous involving the core subunits C2 and probably C1, it also involves subunits C11, C37 and C53, which act on the pol III catalytic center and exhibit homology to the pol II elongation factor TFIIS, and TFIIFα/β respectively. Here we compile knowledge of pol III termination and associate mutations that affect this process with structural elements of the polymerase that illustrate the importance of C53/37 both at its docking site on the pol III lobe and in the active center. The models suggest that some of these features may apply to the other eukaryotic pols. PMID:23099421

  11. The RNA polymerase II CTD coordinates transcription and RNA processing

    PubMed Central

    Hsin, Jing-Ping; Manley, James L.

    2012-01-01

    The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1–Ser2–Pro3–Thr4–Ser5–Pro6–Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity. PMID:23028141

  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. Amplification of RNA by an RNA polymerase ribozyme.

    PubMed

    Horning, David P; Joyce, Gerald F

    2016-08-30

    In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins. PMID:27528667

  14. Single-molecule Studies of RNA Polymerase: Motoring Along

    PubMed Central

    Herbert, Kristina M.; Greenleaf, William J.; Block, Steven M.

    2010-01-01

    Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates. PMID:18410247

  15. Mechanisms of backtrack recovery by RNA polymerases I and II

    PubMed Central

    Lisica, Ana; Engel, Christoph; Jahnel, Marcus; Roldán, Édgar; Galburt, Eric A.; Cramer, Patrick; Grill, Stephan W.

    2016-01-01

    During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes. PMID:26929337

  16. [RNA polymerase as a molecular machine].

    PubMed

    Spirin, A S

    2002-01-01

    Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. The use of mechanical energy and mechanical devices, such as "power-stroke motor", is supposed unlikely in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex thus providing the directionality of the conveyance ("Brownian ratchet mechanism"). The following sequence of events "substrate binding--fixation of a certain conformational state--chemical reaction--fixation of an alternative conformational state--translocation (dissociation and downstream reassociation) of product-template duplex" is proposed as the principal scheme of the forward movement of RNA polymerase along DNA template.

  17. Active site labeling of the RNA polymerases A, B, and C from yeast.

    PubMed

    Riva, M; Schäffner, A R; Sentenac, A; Hartmann, G R; Mustaev, A A; Zaychikov, E F; Grachev, M A

    1987-10-25

    RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.

  18. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

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

  19. Remodeling of Bacterial RNA Polymerase Supramolecular Complex in Response to Environmental Conditions

    SciTech Connect

    Verma, Seema; Xiong, Yijia; Mayer, M. Uljana; Squier, Thomas C.

    2007-03-20

    Directed binding of RNA polymerase to distinct promoter elements controls transcription and promotes adaptive responses to changing environmental conditions. To identify proteins that modulate transcription, we have expressed a tagged alpha-subunit of RNA polymerase in Shewanella oneidensis under controlled growth conditions, isolated the protein complex using newly developed multiuse affinity probes, and used LC-MS/MS to identify proteins in the complex. Complementary fluorescence correlation spectroscopy measurements were used to determine the average size of the RNA polymerase complex in cellular lysates. We find that RNA polymerase exists as a large supramolecular complex with an apparent mass in excess of 1.4 MDa, whose protein composition substantially changes in response to growth conditions. Enzymes that copurify with RNA polymerase include those associated with tRNA processing, nucleotide metabolism, and energy biosynthesis, which we propose to be necessary for optimal transcriptional rates.

  20. Purification and lipid-layer crystallization of yeast RNA polymerase II.

    PubMed Central

    Edwards, A M; Darst, S A; Feaver, W J; Thompson, N E; Burgess, R R; Kornberg, R D

    1990-01-01

    Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules. Images PMID:2179949

  1. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

  2. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    PubMed

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

  3. Modeling RNA polymerase interaction in mitochondria of chordates

    PubMed Central

    2012-01-01

    Background In previous work, we introduced a concept, a mathematical model and its computer realization that describe the interaction between bacterial and phage type RNA polymerases, protein factors, DNA and RNA secondary structures during transcription, including transcription initiation and termination. The model accurately reproduces changes of gene transcription level observed in polymerase sigma-subunit knockout and heat shock experiments in plant plastids. The corresponding computer program and a user guide are available at http://lab6.iitp.ru/en/rivals. Here we apply the model to the analysis of transcription and (partially) translation processes in the mitochondria of frog, rat and human. Notably, mitochondria possess only phage-type polymerases. We consider the entire mitochondrial genome so that our model allows RNA polymerases to complete more than one circle on the DNA strand. Results Our model of RNA polymerase interaction during transcription initiation and elongation accurately reproduces experimental data obtained for plastids. Moreover, it also reproduces evidence on bulk RNA concentrations and RNA half-lives in the mitochondria of frog, human with or without the MELAS mutation, and rat with normal (euthyroid) or hyposecretion of thyroid hormone (hypothyroid). The transcription characteristics predicted by the model include: (i) the fraction of polymerases terminating at a protein-dependent terminator in both directions (the terminator polarization), (ii) the binding intensities of the regulatory protein factor (mTERF) with the termination site and, (iii) the transcription initiation intensities (initiation frequencies) of all promoters in all five conditions (frog, healthy human, human with MELAS syndrome, healthy rat, and hypothyroid rat with aberrant mtDNA methylation). Using the model, absolute levels of all gene transcription can be inferred from an arbitrary array of the three transcription characteristics, whereas, for selected genes only

  4. Biogenesis of RNA polymerases II and III requires the conserved GPN small GTPases in Saccharomyces cerevisiae.

    PubMed

    Minaker, Sean W; Filiatrault, Megan C; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C

    2013-03-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport.

  5. Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

    PubMed Central

    Minaker, Sean W.; Filiatrault, Megan C.; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C.

    2013-01-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport. PMID:23267056

  6. Nucleolin Is Required for RNA Polymerase I Transcription In Vivo▿

    PubMed Central

    Rickards, Brenden; Flint, S. J.; Cole, Michael D.; LeRoy, Gary

    2007-01-01

    Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin. PMID:17130237

  7. Purification and Properties of Two RNA Polymerases from Physarum polycephalum

    PubMed Central

    Burgess, Ann Baker; Burgess, Richard R.

    1974-01-01

    Two RNA polymerases have been purified from the slime mold Physarum polycephalum, one sensitive and one resistant to α-amanitin. Both enzymes are more active with denatured DNA than native DNA as a template and prefer Mn++ rather than Mg++ as a divalent cation. The α-amanitin-sensitive enzyme shows maximum activity at 0.15 M KCl, whereas the resistant enzyme is most active at very low ionic strength. Analysis of the resistant enzyme on polyacrylamide gels containing sodium dodecyl sulfate shows two subunits present in a 1:1 ratio with molecular weights of 205,000 and 125,000. Images PMID:4524629

  8. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  9. An alternative RNA polymerase I structure reveals a dimer hinge.

    PubMed

    Kostrewa, Dirk; Kuhn, Claus-D; Engel, Christoph; Cramer, Patrick

    2015-09-01

    RNA polymerase I (Pol I) is the central, 14-subunit enzyme that synthesizes the ribosomal RNA (rRNA) precursor in eukaryotic cells. The recent crystal structure of Pol I at 2.8 Å resolution revealed two novel elements: the `expander' in the active-centre cleft and the `connector' that mediates Pol I dimerization [Engel et al. (2013), Nature (London), 502, 650-655]. Here, a Pol I structure in an alternative crystal form that was solved by molecular replacement using the original atomic Pol I structure is reported. The resulting alternative structure lacks the expander but still shows an expanded active-centre cleft. The neighbouring Pol I monomers form a homodimer with a relative orientation distinct from that observed previously, establishing the connector as a hinge between Pol I monomers.

  10. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA. PMID:27618490

  11. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

  12. Nascent transcription affected by RNA polymerase IV in Zea mays.

    PubMed

    Erhard, Karl F; Talbot, Joy-El R B; Deans, Natalie C; McClish, Allison E; Hollick, Jay B

    2015-04-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance.

  13. Nascent Transcription Affected by RNA Polymerase IV in Zea mays

    PubMed Central

    Erhard, Karl F.; Talbot, Joy-El R. B.; Deans, Natalie C.; McClish, Allison E.; Hollick, Jay B.

    2015-01-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3ʹ-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  14. Molecular structures of unbound and transcribing RNA polymerase III

    PubMed Central

    Hoffmann, Niklas A.; Jakobi, Arjen J.; Moreno-Morcillo, Maria; Glatt, Sebastian; Kosinski, Jan; Hagen, Wim J. H.; Sachse, Carsten; Müller, Christoph W.

    2015-01-01

    Transcription of genes encoding small structured RNAs such as tRNAs, spliceosomal U6 snRNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. The cryo-EM structures of the S. cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, allow for the first time to build a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82/C34/C31 heterotrimer in close proximity to the stalk. The C53/C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets. PMID:26605533

  15. Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells

    PubMed Central

    Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A.; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S.; Capra, John A.; Schnölzer, Martina; Cole, Philip A.; Geyer, Matthias; Bruneau, Benoit G.; Adelman, Karen; Ott, Melanie

    2014-01-01

    SUMMARY Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes. PMID:24207025

  16. RNA polymerase molecular beacon as tool for studies of RNA polymerase-promoter interactions.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-09-15

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ(70) holoenzyme and its regulation by bacterial and phage encoded factors.

  17. RNA polymerase molecular beacon as tool for studies of RNA polymerase - promoter interactions

    PubMed Central

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ70 holoenzyme and its regulation by bacterial and phage encoded factors. PMID:25956222

  18. ppGpp: magic beyond RNA polymerase.

    PubMed

    Dalebroux, Zachary D; Swanson, Michele S

    2012-02-16

    During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

  19. Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.

    PubMed

    Pilsl, Michael; Merkl, Philipp E; Milkereit, Philipp; Griesenbeck, Joachim; Tschochner, Herbert

    2016-01-01

    RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S. cerevisiae were performed using either complex in vitro systems reconstituted from more or less purified fractions or genetic analyses. While providing many valuable insights these strategies did not always discriminate between direct and indirect effects in transcription initiation and termination, when mutated forms of Pol I subunits or transcription factors were investigated. Therefore, a well-defined minimal system was developed which allows to reconstitute highly efficient promoter-dependent Pol I initiation and termination of transcription. Transcription can be initiated at a minimal promoter only in the presence of recombinant core factor and extensively purified initiation competent Pol I. Addition of recombinant termination factors triggers transcriptional pausing and release of the ternary transcription complex. This minimal system represents a valuable tool to investigate the direct impact of (lethal) mutations in components of the initiation and termination complexes on the mechanism and regulation of rRNA synthesis. PMID:27576713

  20. Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I

    PubMed Central

    Li, Weizhong; Chen, Hongjun; Sutton, Troy; Obadan, Adebimpe

    2014-01-01

    ABSTRACT The influenza A virus genome possesses eight negative-strand RNA segments in the form of viral ribonucleoprotein particles (vRNPs) in association with the three viral RNA polymerase subunits (PB2, PB1, and PA) and the nucleoprotein (NP). Through interactions with multiple host factors, the RNP subunits play vital roles in replication, host adaptation, interspecies transmission, and pathogenicity. In order to gain insight into the potential roles of RNP subunits in the modulation of the host's innate immune response, the interactions of each RNP subunit with retinoic acid-inducible gene I protein (RIG-I) from mammalian and avian species were investigated. Studies using coimmunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFc), and colocalization using confocal microscopy provided direct evidence for the RNA-independent binding of PB2, PB1, and PA with RIG-I from various hosts (human, swine, mouse, and duck). In contrast, the binding of NP with RIG-I was found to be RNA dependent. Expression of the viral NS1 protein, which interacts with RIG-I, did not interfere with the association of RNA polymerase subunits with RIG-I. The association of each individual virus polymerase component with RIG-I failed to significantly affect the interferon (IFN) induction elicited by RIG-I and 5′ triphosphate (5′ppp) RNA in reporter assays, quantitative reverse transcription-PCR (RT-PCR), and IRF3 phosphorylation tests. Taken together, these findings indicate that viral RNA polymerase components PB2, PB1, and PA directly target RIG-I, but the exact biological significance of these interactions in the replication and pathogenicity of influenza A virus needs to be further clarified. IMPORTANCE RIG-I is an important RNA sensor to elicit the innate immune response in mammals and some bird species (such as duck) upon influenza A virus infection. Although the 5′-triphosphate double-stranded RNA (dsRNA) panhandle structure at the end of viral genome RNA is

  1. Modification of RNA polymerase IIO subspecies after poliovirus infection.

    PubMed Central

    Rangel, L M; Fernandez-Tomas, C; Dahmus, M E; Gariglio, P

    1987-01-01

    Infection of HeLa cells with poliovirus results in a shutdown of host transcription. In an effort to understand the mechanism(s) that underlies this process, we analyzed the distribution of RNA polymerase IIO before and after viral infection. Analysis of free and chromatin-bound enzyme indicated that there is a significant reduction in RNA polymerase IIO following infection. This observation, together with increasing evidence that transcription is catalyzed by RNA polymerase IIO, supports the hypothesis that poliovirus-induced inhibition of host transcription occurs at the level of RNA chain initiation and involves the direct modification of RNA polymerase II. Images PMID:3029396

  2. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome.

    PubMed

    Van Driessche, Benoit; Rodari, Anthony; Delacourt, Nadège; Fauquenoy, Sylvain; Vanhulle, Caroline; Burny, Arsène; Rohr, Olivier; Van Lint, Carine

    2016-01-01

    Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3'LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5'LTR promoter. PMID:27545598

  3. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome

    PubMed Central

    Van Driessche, Benoit; Rodari, Anthony; Delacourt, Nadège; Fauquenoy, Sylvain; Vanhulle, Caroline; Burny, Arsène; Rohr, Olivier; Van Lint, Carine

    2016-01-01

    Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3′LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5′LTR promoter. PMID:27545598

  4. Inhibition of RNA polymerase by streptolydigin: no cycling allowed.

    PubMed

    Kyzer, Scotty; Zhang, Jinwei; Landick, Robert

    2005-08-26

    Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a "bridge" alpha helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site. PMID:16122417

  5. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  6. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Cheng, Jinkui; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2016-06-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  7. Transcriptional bypass of regioisomeric ethylated thymidine lesions by T7 RNA polymerase and human RNA polymerase II

    PubMed Central

    You, Changjun; Wang, Pengcheng; Dai, Xiaoxia; Wang, Yinsheng

    2014-01-01

    Alkylative damage to DNA can be induced by environmental chemicals, endogenous metabolites and some commonly prescribed chemotherapeutic agents. The regioisomeric N3-, O2- and O4-ethylthymidine (N3-, O2- and O4-EtdT, respectively) represent an important class of ethylated DNA lesions. Using nonreplicative double-stranded vectors containing an N3-EtdT, O2-EtdT or O4-EtdT at a defined site in the template strand, herein we examined the effects of these lesions on DNA transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. We found that O4-EtdT is highly mutagenic and exclusively induces the misincorporation of guanine opposite the lesion, whereas N3-EtdT and O2-EtdT display promiscuous miscoding properties during transcription. In addition, N3-EtdT and O2-EtdT were found to inhibit strongly DNA transcription in vitro and in certain human cells. Moreover, N3-EtdT, but not O2-EtdT or O4-EtdT, is an efficient substrate for transcription-coupled nucleotide excision repair. These findings provide new important insights into how these alkylated DNA lesions compromise the flow of genetic information, which may help to understand the risk of these lesions in living cells. PMID:25404131

  8. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  9. Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase.

    PubMed

    Beckman, M T; Kirkegaard, K

    1998-03-20

    The poliovirus RNA-dependent RNA polymerase binds cooperatively to single-stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wild-type polymerase to oligo(U)12 and oligo(U)24 RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)10 suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA-binding site.

  10. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  11. Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA

    PubMed Central

    Kim, Euikyung; Du, Lei; Bregman, David B.; Warren, Stephen L.

    1997-01-01

    The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the

  12. Unusual promoter-independent transcription reactions with bacteriophage RNA polymerases.

    PubMed Central

    Krupp, G

    1989-01-01

    Efficient transcription reactions of DNA-dependent RNA polymerases require the presence of a specific promoter sequence. This report shows that in the absence of their cognate promoter, two bacteriophage RNA polymerases are capable of performing unusual transcription reactions: (i) the DNA template serves also as a primer for RNA synthesis and this leads to hybrid DNA/RNA molecules, (ii) if the DNA template forms a hairpin structure, the linear DNA can be transcribed via the 'rolling circle' mechanism. Images PMID:2471146

  13. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  14. Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

    PubMed

    Thota, Swarna Gowri; Unnikannan, C P; Thampatty, Sitalakshmi R; Manorama, R; Bhandari, Rashna

    2015-02-15

    Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

  15. Stable RNA-DNA-RNA polymerase complexes can accompany formation of a single phosphodiester bond.

    PubMed

    Sylvester, J E; Cashel, M

    1980-03-18

    Incubation of RNA polymerase with poly[d(A-T)n] template results in a binary enzyme-DNA complex. Further addition of the dinucleotide UpA and [alpha-32P]UTP results in catalytic formation of the labeled trinucleotide UpApU until substrate exhaustion. In contrast, incubation of binary enzyme-DNA complexes with ApU and [alpha-32P]ATP results in labeled ApUpA formation to an extent that is stoichiometric with the amount of enzyme present despite an excess of substrates. The occurrence of ApUpA in a stable DNA-enzyme-RNA ternary complex is shown by gel exclusion chromatography, Millipore filtration, and the ability of ternary complexes to support subsequent RNA chain elongation. Radioactivity is not bound to Millipore filters when purified, labeled ApUpA is added to enzyme-DNA binary complexes. Hence, phosphodiester bond formation is required for stable ternary complex formation. The absence of the delta subunit of RNA polymerase or the addition of rifampicin to the reaction before ribonucleotide substrates results in catalytic ApUpA formation instead of stable ternary complexes.

  16. RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection.

    PubMed Central

    Rice, S A; Long, M C; Lam, V; Spencer, C A

    1994-01-01

    During lytic infection, herpes simplex virus subverts the host cell RNA polymerase II transcription machinery to efficiently express its own genome while repressing the expression of most cellular genes. The mechanism by which RNA polymerase II is directed to the viral delayed-early and late genes is still unresolved. We report here that RNA polymerase II is preferentially localized to viral replication compartments early after infection with herpes simplex virus type 1. Concurrent with recruitment of RNA polymerase II into viral compartments is a rapid and aberrant phosphorylation of the large subunit carboxy-terminal domain (CTD). Aberrant phosphorylation of the CTD requires early viral gene expression but is not dependent on viral DNA replication or on the formation of viral replication compartments. Localization of RNA polymerase II and modifications to the CTD may be instrumental in favoring transcription of viral genes and repressing specific transcription of cellular genes. Images PMID:8289400

  17. The Rapid Turnover of RNA Polymerase of Rat Liver Nucleolus, and of Its Messenger RNA

    PubMed Central

    Yu, Fu-Li; Feigelson, Philip

    1972-01-01

    Turnover rates of the components of systems for RNA synthesis of rat-liver nucleus, nucleolus, and nucleoplasm were investigated. Cycloheximide administered in vivo selectively diminished nucleolar RNA synthesis in vitro. In contrast to the relatively stable nucleoplasmic RNA polymerase, nucleolar RNA polymerase (polymerase I) from rat liver decays rapidly upon cycloheximide administration, following pseudo-first order kinetics with a half-life of about 1.3 hr. Cycloheximide elicits this effect not through direct interaction with nucleolar RNA polymerase itself, nor by alteration of template function, but rather by inhibition of de novo synthesis of one or more of the protein components of nucleolar RNA polymerase. Similarly, when actinomycin-D was administered in vivo to inhibit RNA synthesis, the rate of decay of nucleolar RNA polymerase, assayed in the presence of exogenous poly d(A-T) template, was similar to that observed after cycloheximide administration. Thus, the messenger RNA(s) that codes for one or more of the catalytically essential polypeptide components of this enzyme turn over very rapidly with a half-life considerably shorter than 1.3 hr. The rapidity of turnover of both the enzyme protein and its messenger RNA(s) renders nucleolar RNA polymerase highly responsive to altered transcriptional, translational, or post-translational modulation. The marked differences in turnover rates of nucleolar and nucleoplasmic RNA polymerases indicate that at least certain of the protomeric components of nucleolar RNA polymerase I are distinct from those of nucleoplasmic RNA polymerases II and III. PMID:4507607

  18. Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

    PubMed Central

    Biebricher, C K; Luce, R

    1996-01-01

    A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase. Images PMID:8670848

  19. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  20. The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

    PubMed Central

    Padilla-Mejía, Norma E.; Florencio-Martínez, Luis E.; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan C.; Cevallos, Ana M.; Hernández-Rivas, Rosaura; Manning-Cela, Rebeca

    2014-01-01

    Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes. PMID:25548151

  1. Mapping of Protein-Protein Interactions of E. coli RNA Polymerase with Microfluidic Mechanical Trapping

    PubMed Central

    Bates, Steven R.; Quake, Stephen R.

    2014-01-01

    The biophysical details of how transcription factors and other proteins interact with RNA polymerase are of great interest as they represent the nexus of how structure and function interact to regulate gene expression in the cell. We used an in vitro microfluidic approach to map interactions between a set of ninety proteins, over a third of which are transcription factors, and each of the four subunits of E. coli RNA polymerase, and we compared our results to those of previous large-scale studies. We detected interactions between RNA polymerase and transcription factors that earlier high-throughput screens missed; our results suggest that such interactions can occur without DNA mediation more commonly than previously appreciated. PMID:24643045

  2. In-ice evolution of RNA polymerase ribozyme activity

    PubMed Central

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2014-01-01

    Mechanisms of molecular self-replication have the potential to shed light upon the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA World’. However, existing polymerase ribozymes lack the capacity to synthesise RNAs approaching their own size. Here we report the in vitro evolution of such catalysts directly in the RNA-stabilising medium of water-ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesise RNA in ices at temperatures as low as −19°C. Combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication. PMID:24256864

  3. In-ice evolution of RNA polymerase ribozyme activity

    NASA Astrophysics Data System (ADS)

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2013-12-01

    Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA world’. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5‧ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

  4. In-ice evolution of RNA polymerase ribozyme activity.

    PubMed

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2013-12-01

    Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial 'RNA world'. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5' extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication. PMID:24256864

  5. Dysregulation of RNA polymerase I transcription during disease.

    PubMed

    Hannan, K M; Sanij, E; Rothblum, L I; Hannan, R D; Pearson, R B

    2013-01-01

    Transcription of the ribosomal RNA genes by the dedicated RNA polymerase I enzyme and subsequent processing of the ribosomal RNA are fundamental control steps in the synthesis of functional ribosomes. Dysregulation of Pol I transcription and ribosome biogenesis is linked to the etiology of a broad range of human diseases. Diseases caused by loss of function mutations in the molecular constituents of the ribosome, or factors intimately associated with RNA polymerase I transcription and processing are collectively termed ribosomopathies. Ribosomopathies are generally rare and treatment options are extremely limited tending to be more palliative than curative. Other more common diseases are associated with profound changes in cellular growth such as cardiac hypertrophy, atrophy or cancer. In contrast to ribosomopathies, altered RNA polymerase I transcriptional activity in these diseases largely results from dysregulated upstream oncogenic pathways or by direct modulation by oncogenes or tumor suppressors at the level of the RNA polymerase I transcription apparatus itself. Ribosomopathies associated with mutations in ribosomal proteins and ribosomal RNA processing or assembly factors have been covered by recent excellent reviews. In contrast, here we review our current knowledge of human diseases specifically associated with dysregulation of RNA polymerase I transcription and its associated regulatory apparatus, including some cases where this dysregulation is directly causative in disease. We will also provide insight into and discussion of possible therapeutic approaches to treat patients with dysregulated RNA polymerase I transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols. PMID:23153826

  6. RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

    PubMed Central

    Martínez-Alonso, Mónica; Hengrung, Narin

    2016-01-01

    ABSTRACT Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation. IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for

  7. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

    PubMed

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  8. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    PubMed Central

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  9. Molecular Structures of DNA-Dependent RNA Polymerases (II) from Calf Thymus and Rat Liver

    PubMed Central

    Weaver, R. F.; Blatti, S. P.; Rutter, W. J.

    1971-01-01

    DNA-dependent RNA polymerase II has been purified to high specific activity and apparent homogeneity from both calf thymus and rat liver. Two form II enzymes are present in rat-liver preparations, one with the molecular structure [(190,000)1(150,000)1(35,000)1(25,000)1], the other with a molecular structure of [(170,000)1(150,000)1(35,000)1(25,000)1] (molecular weights are within ±5% but the absolute values are approximate). Inclusion of a proteolytic inhibitor during the isolation procedure decreases the proportion of the molecule containing the 170,000 subunit. Calf-thymus RNA polymerase preparations typically exhibit four components on polyacrylamide gels that contain sodium dodecyl sulfate, with an apparent molecular structure of [(190,000)1(150,000)1(35,000)1(25,000)1]. In addition, some calf-thymus polymerase II preparations contain small quantities of the [(170,000)1(150,000)1(35,000)1(25,000)1] species; the quantity of this species may also be increased from less than 5% in the normal preparation to at least 40% in an “aged” preparation. Thus, the 170,000 subunit may be derived from the 190,000 subunit in both tissues. Until unequivocal evidence is obtained on this point, however, the possibility that the large subunits are unique species should not be eliminated. The general structural similarity of the eukaryotic RNA polymerase II with that of the prokaryotic polymerase suggests that the modes of action and regulation may be analogous. Images PMID:5289245

  10. The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome

    SciTech Connect

    Greco-Stewart, Valerie S.; Miron, Paul; Abrahem, Abrahem; Pelchat, Martin . E-mail: mpelchat@uottawa.ca

    2007-01-05

    The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.

  11. Mechanism of release of active alpha subunit from dimeric alpha beta avian myeloblastosis virus DNA polymerase.

    PubMed Central

    Papas, T S; Marciani, D J; Samuel, K; Chirikjian, J G

    1976-01-01

    Storage of the dimeric (alphabeta) form of avian myeloblastosis virus (AMV) DNA polymerase in glycerol resulted in the release of the smaller alpha subunit, as detected by glycerol gradient sedimentation. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme stored in glycerol showed the concomitant appearance of several polypeptides and a lowering in the level of both beta and alpha components. This reduction appears to be the result of cleavages introduced by traces of hydrolytic activity present in glycerol samples. An enhancement of alpha subunit released, as detected by activity profile, was also achieved upon direct but limited exposure of purified avian myeloblastosis virus DNA polymerase to carboxymethyl-cellulose-bound trypsin matrix. Electrophoretic analysis of digested enzyme revealed a progressive fragmentation, with simultaneous increase in the alpha subunit and decrease in the beta subunit. PMID:58080

  12. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  13. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

    NASA Astrophysics Data System (ADS)

    Zhang, Xing; Ding, Ke; Yu, Xuekui; Chang, Winston; Sun, Jingchen; Hong Zhou, Z.

    2015-11-01

    Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

  14. Catching RNA Polymerase in the act of Binding: Intermediates in Transcription Illuminated by Synchrotron Footprinting

    SciTech Connect

    Brenowitz,M.; Erie, D.; Chance, M.

    2005-01-01

    The article by Sclavi et al. in this issue of PNAS addresses 'initiation, ' the first step in transcription. Gene transcription is catalyzed in cells by large multisubunit proteins called RNA polymerases (RNAP). The eubacteria holoenzyme of RNAP is composed of five core subunits ({alpha}, {alpha}2, {beta}, {beta}', and {omega}) that contain the amino acid residues required for the enzyme's catalytic activity. A sixth subunit ({sigma}) guides RNAP to specific sequences on the genomic DNA (promoters) that mark the beginning of a gene or group of genes.

  15. [Role of RNA-polymerase in gene activity regulation of E. coli RNA-polymerase mutants with a pleiotropic effect. I. Physiological and biochemical studies].

    PubMed

    Kamzolova, S G; Arutiunian, A V; Ozolin', O N; Oganesian, M G

    1979-01-01

    Four Rifr-mutants of E. coli B/r (rpo B401, rpo B402, rpo B403, rpo B409) which differ from the wild strain in one or more phenotypic properties besides rifampicin resistance were obtained. Transfer of the mutant Rifr-alleles into the parent strain gives the latter all the properties of the mutant. This indicates that the new properties are due to the pleiotropic effect of Rifr-mutations. Biochemical studies of the properties of RNA-polymerases from the mutants and the parent showed that some new properties of the mutants could not be explained by the appearance of analogous properties in the mutant RNA-polymerase itself. They seem to be caused by alteration in functional activity of the mutant enzyme, particulary, alteration of its control properties during transcription. The function of the beta-subunit in genetic transcription is discussed.

  16. Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

    2016-05-01

    The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

  17. Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

    2016-05-01

    The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression. PMID:27416361

  18. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding.

    PubMed

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1.

  19. Differential binding of ppGpp and pppGpp to E. coli RNA polymerase: photo-labeling and mass spectral studies.

    PubMed

    Syal, Kirtimaan; Chatterji, Dipankar

    2015-12-01

    (p)ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the β-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of β'/ω-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra- and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on β'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the β-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the β'-subunit. However, we were unable to identify any binding site of pppGpp on the β-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay.

  20. Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells

    SciTech Connect

    Fromaget, Maud; Cook, Peter R. . E-mail: peter.cook@path.ox.ac.uk

    2007-08-15

    RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors - some of which are used therapeutically - affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.

  1. Recombinant Thermus aquaticus RNA Polymerase for Structural Studies

    SciTech Connect

    Juznedelov,K.; Lamour, V.; Patikoglou, G.; Chlenov, M.; Darst, S.; Severinov, K.

    2006-01-01

    Advances in the structural biology of bacterial transcription have come from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural studies have been limited by the fact that only endogenous Taq or Tth RNAP, laboriously purified from large quantities of Taq or Tth cell paste and offering few options for genetic modification, is suitable for structural studies. Recombinant systems for the preparation of Taq RNAP by co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for crystallographic studies. Here we describe recombinant systems for the preparation of Taq RNAP harboring full or partial deletions of the Taq {beta}' non-conserved domain (NCD), yielding enzyme suitable for crystallographic studies. This opens the way for structural studies of genetically manipulated enzymes, allowing the preparation of more crystallizable enzymes and facilitating detailed structure/function analysis. Characterization of the Taq{beta}'NCD deletion mutants generated in this study showed that the {beta}'NCD is important for the efficient binding of the s subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1 Angstroms resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the {beta}-flap, further defining the range of conformations accessible to this flexible structural element.

  2. Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection.

    PubMed Central

    Hekmatpanah, D S; Young, R A

    1991-01-01

    A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation. Images PMID:1922077

  3. Euglena gracilis DNA dependent RNA polymerase II: a zinc metalloenzyme.

    PubMed

    Falchuk, K H; Mazus, B; Ulpino, L; Vallee, B L

    1976-10-01

    Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.

  4. Purification of bacterial RNA polymerase: tools and protocols

    PubMed Central

    Svetlov, Vladimir; Artsimovitch, Irina

    2014-01-01

    Summary Bacterial RNA polymerase is the first point of gene expression and a validated target for antibiotics. Studied for several decades, the Escherichia coli transcriptional apparatus is by far the best characterized, with numerous RNA polymerase mutants and auxiliary factors isolated and analyzed in great detail. Since the E. coli enzyme was refractory to crystallization, structural studies have been focused on Thermus RNA polymerase s, revealing atomic details of the catalytic center and RNA polymerase interactions with nucleic acids, antibiotics, and regulatory proteins. However, numerous differences between these enzymes, including resistance of Thermus RNA polymerases to some antibiotics, underscored the importance of the E. coli enzyme structures. Three groups published the se long awaited structures in 2013, enabling functional and structural studies of the same model system. This progress was made possible, in large part, by the use of multicistronic vectors for expression of the E. coli enzyme in large quantities and in a highly active form. Here we describe the commonly used vectors and procedures for purification of the E. coli RNA polymerase. PMID:25665556

  5. DNA-dependent RNA polymerase from Crithidia oncopelti kinetoplasts

    SciTech Connect

    Zaitseva, G.N.; Levchenko, I.V.; Tarasov, I.A.; Kuz'min, E.V.

    1986-03-10

    Mitochondrial DNA-dependent RNA polymerase was isolated from Crithidia oncopelti kinetoplasts, and its properties were studied. RNA polymerase was solubilized from the structures with 2% digitonin in 0.25 M KCl. The enzyme was purified 550-fold according to activity by gel filtration through Sephadex 4B, followed by chromatography on heparin-Sepharose 4B, phosphocellulose, and DEAE-Sephadex A-50. The optimum conditions of the RNA polymerase reaction (time of incubation, temperature, Mg/sup 2 +/, K/sup +/ concentrations, etc) were determined. It was established that the activity of the enzyme is not inhibited by ..cap alpha..-amanitin, rifampicin, and streptolidigin, but is strongly suppressed by Mn/sup 2 +/ ions, a high KCl concentrations, as well as ethidium bromide. The RNA polymerase isolated transcribes denatured DNA substantially better than the native form. The enzyme utilizes mtDNA (in hybrid plasmids) as a substrate appreciably more actively than the nuclear form. Among the substrates used, the greatest template activity is possessed by single-stranded poly(dAT). In all the properties studied, DNA-dependent RNA polymerase from C. oncopelti kinetoplasts is similar to the mitochondrial enzymes of other eukaryotes but differs from the nuclear enzymes of this organism and from bacterial RNA polymerases.

  6. Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization

    PubMed Central

    Chen, Hung-Kai; Pai, Chi-Yun; Huang, Jing-Yi; Yeh, Ning-Hsing

    1999-01-01

    Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity. PMID:10567578

  7. Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate.

    PubMed

    Woody, A Y; Vader, C R; Woody, R W; Haley, B E

    1984-06-19

    A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

  8. Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2.

    PubMed

    Forget, Diane; Lacombe, Andrée-Anne; Cloutier, Philippe; Lavallée-Adam, Mathieu; Blanchette, Mathieu; Coulombe, Benoit

    2013-08-01

    The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1-170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156-612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.

  9. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    PubMed Central

    Černý, Jiří; Černá Bolfíková, Barbora; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach. The resulting phylogram of vRdPs demonstrates that RNA-dependent DNA polymerases (RdDPs) of viruses within Retroviridae family cluster in a clearly separated group of vRdPs, while RNA-dependent RNA polymerases (RdRPs) of dsRNA and +ssRNA viruses are mixed together. This evidence supports the hypothesis that RdRPs replicating +ssRNA viruses evolved multiple times from RdRPs replicating +dsRNA viruses, and vice versa. Moreover, our phylogram may be presented as a scheme for RNA virus evolution. The results are in concordance with the actual concept of RNA virus evolution. Finally, the methods used in our work provide a new direction for studying ancient virus evolution. PMID:24816789

  10. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    PubMed

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  11. Continuous in vitro evolution of bacteriophage RNA polymerase promoters

    NASA Technical Reports Server (NTRS)

    Breaker, R. R.; Banerji, A.; Joyce, G. F.

    1994-01-01

    Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

  12. Inhibitory effect of modified 5'-capped short RNA fragments on influenza virus RNA polymerase gene expression.

    PubMed

    Tado, M; Abe, T; Hatta, T; Ishikawa, M; Nakada, S; Yokota, T; Takaku, H

    2001-11-01

    We have shown previously that the 5'-capped short phosphodiester RNA fragments, Cap decoy, (Gm 12 nt) are potent inhibitors of influenza virus RNA polymerase gene expression. Here we investigate the modified capped RNA derivative containing phosphorothioate oligonucleotides (Cap decoy) as a potential influenza virus RNA polymerase inhibitor. The modified 5'-capped short phosphorothioate RNA fragments (Gms 12-15 nt) with the 5'-capped structure (m7GpppGm) were synthesized by T7 RNA polymerase. The 5'-capped short RNA fragments (Gms 12-15 nt) were encapsulated in liposome particulates and tested for their inhibitory effects on influenza virus RNA polymerase gene expression in the clone 76 cells. The 12-15 nt long Gms RNA fragments showed highly inhibitory effects. By contrast, the inhibitory effects of the 13 nt long short RNA fragments (Gm 13 nt) were considerably less in comparison with the 5'-capped short phosphorothioate RNA fragments (Gms 12-15 nt). In particular, the various Gms RNA chain lengths showed no significant differences in the inhibition of influenza virus RNA polymerase gene expression. Furthermore, the capped RNA with a phosphorothioate backbone was resistant to nuclease activity. These phosphorothioate RNA fragments exhibited higher inhibitory activity than the 5'-capped short RNA fragments (Gm 12 nt). These decoys may prove to be useful in anti-influenza virus therapeutics. PMID:12018680

  13. A multistep process gave rise to RNA polymerase IV of land plants.

    PubMed

    Luo, Jie; Hall, Benjamin D

    2007-01-01

    Since their discovery in Metazoa, the three nuclear RNA polymerases (RNAPs) have been found in fungi, plants, and diverse protists. In all eukaryotes studied to date, RNAPs I, II, and III collectively transcribe all major RNAs made in the nucleus. We have found genes for the largest subunit (RPD1/RPE1) of a new DNA-dependent RNAP, RNAP IV, in all major land plant taxa and in closely related green algae. Genes for the second-largest subunit (RPD2) of this enzyme were found in all land plants. Phylogenetic study indicates that RNAP IV genes are sister to the corresponding RNAP II genes. Our results show the genesis of RNAP IV to be a multistep process in which the largest and second-largest subunit genes evolved by independent duplication events in the ancestors of Charales and land plants. These findings provide insights into evolutionary mechanisms that can explain the origin of multiple RNAPs in the eukaryotic nucleus.

  14. Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

    PubMed

    Brannan, Kris; Bentley, David L

    2012-01-01

    The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression. PMID:22567377

  15. Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

    PubMed Central

    Qiu, Shihong; Ogino, Minako; Luo, Ming

    2015-01-01

    ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the

  16. Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7.

    PubMed

    Jensen, G J; Meredith, G; Bushnell, D A; Kornberg, R D

    1998-04-15

    The three-dimensional structure of wild-type yeast RNA polymerase II has been determined at a nominal resolution of 24 A. A difference map between this structure and that of the polymerase lacking subunits Rpb4 and Rpb7 showed these two subunits forming part of the floor of the DNA-binding (active center) cleft, and revealed a slight inward movement of the protein domain surrounding the cleft. Surface plasmon resonance measurements showed that Rpb4 and Rpb7 stabilize a minimal pre-initiation complex containing promoter DNA, TATA box-binding protein (TBP), transcription factor TFIIB and the polymerase. These findings suggest that Rpb4 and Rpb7 play a role in coupling the entry of DNA into the active center cleft to closure of the cleft. Such a role can explain why these subunits are necessary for promoter-specific transcription in vitro and for a normal stress response in vivo.

  17. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  18. Direct Characterization of Transcription Elongation by RNA Polymerase I.

    PubMed

    Ucuncuoglu, Suleyman; Engel, Krysta L; Purohit, Prashant K; Dunlap, David D; Schneider, David A; Finzi, Laura

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  19. Transcription inactivation through local refolding of the RNA polymerase structure

    SciTech Connect

    Belogurov, Georgiy A.; Vassylyeva, Marina N.; Sevostyanova, Anastasiya; Appleman, James R.; Xiang, Alan X.; Lira, Ricardo; Webber, Stephen E.; Klyuyev, Sergiy; Nudler, Evgeny; Artsimovitch, Irina; Vassylyev, Dmitry G.

    2009-02-12

    Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx - a desmethyl derivative of myxopyronin B - complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the {beta}'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex - the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

  20. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD) simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15, and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS) P-test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein. PMID:26594643

  1. Rbs1, a new protein implicated in RNA polymerase III biogenesis in yeast Saccharomyces cerevisiae.

    PubMed

    Cieśla, Małgorzata; Makała, Ewa; Płonka, Marta; Bazan, Rafał; Gewartowski, Kamil; Dziembowski, Andrzej; Boguta, Magdalena

    2015-04-01

    Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1Δ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1Δ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.

  2. ALFALFA MOSAIC VIRUS COAT PROTEIN BRIDGES RNA AND RNA-DEPENDENT RNA POLYMERASE IN VITRO

    PubMed Central

    Reichert, Vienna L.; Choi, Mehee; Petrillo, Jessica E.; Gehrke, Lee

    2007-01-01

    Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3' termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution (“far-northwestern blotting”). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals. PMID:17400272

  3. A dynamic model for PC4 coactivator function in RNA polymerase II transcription

    PubMed Central

    Malik, Sohail; Guermah, Mohamed; Roeder, Robert G.

    1998-01-01

    Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state. PMID:9482861

  4. Stochastic resetting in backtrack recovery by RNA polymerases

    NASA Astrophysics Data System (ADS)

    Roldán, Édgar; Lisica, Ana; Sánchez-Taltavull, Daniel; Grill, Stephan W.

    2016-06-01

    Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.

  5. Stochastic resetting in backtrack recovery by RNA polymerases.

    PubMed

    Roldán, Édgar; Lisica, Ana; Sánchez-Taltavull, Daniel; Grill, Stephan W

    2016-06-01

    Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage. PMID:27415302

  6. Back-priming mode of phi6 RNA-dependent RNA polymerase.

    PubMed

    Laurila, Minni R L; Salgado, Paula S; Stuart, David I; Grimes, Jonathan M; Bamford, Dennis H

    2005-02-01

    The RNA-dependent RNA polymerase of the double-stranded RNA bacteriophage phi6 is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain (QYKW, aa 629-632), that was essential for de novo initiation. A similar element has been identified in hepatitis C virus RNA-dependent RNA polymerase. Biochemical studies have addressed the role of this platform, revealing that a mutant version can utilize a back-priming initiation mechanism, where the 3' terminus of the template adopts a hairpin-like conformation. Here, the mechanism of back-primed initiation is studied further by biochemical and structural methods.

  7. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  8. Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

    PubMed

    Reisbig, R R; Woody, A Y; Woody, R W

    1979-11-25

    We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.

  9. The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

    PubMed

    Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-01-01

    Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression

  10. Cloning of the gene for the 73 kD subunit of the DNA polymerase alpha primase of Drosophila melanogaster.

    PubMed Central

    Cotterill, S; Lehman, I R; McLachlan, P

    1992-01-01

    We have isolated both cDNA and genomic clones for the 73 kDa subunit of the DNA polymerase alpha primase of Drosophila melanogaster. Analysis of these clones has identified an open reading frame of 1959 bases coding for a protein of 72.5 kDa. Northern analysis has shown the mRNA for the gene to be approximately 2.5 kb, and comparison of the cDNA and the genomic clones shows that the coding region of the gene lacks introns. The 5' end of the transcript has been mapped by primer extension, and the position of the gene in the genome mapped using in situ analysis. Computer analysis has been carried out on both coding and non coding regions of the gene. The protein sequence shows some homology to the analogous subunit in the S. cerevisiae DNA polymerase alpha, however a search of the data banks failed to reveal other homologies, or provide any clues as to the function of the protein. Analysis of the non-coding regions indicates some potential control regions for the gene. The 73 kDa protein has been overproduced, but a preliminary analysis failed to reveal any enzymatic activities. Images PMID:1508723

  11. A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver.

    PubMed Central

    Serizawa, H; Conaway, R C; Conaway, J W

    1992-01-01

    We previously purified RNA polymerase II transcription factor delta from rat liver and found that it has an associated DNA-dependent ATPase (dATPase) activity. In this report, we show that delta is also closely associated with a protein kinase activity that catalyzes phosphorylation of the largest subunit of RNA polymerase II. Kinase activity copurifies with transcription and DNA-dependent ATPase (dATPase) activities when delta is analyzed by anion- and cation-exchange HPLC as well as by sucrose gradient sedimentation, arguing that delta possesses all three activities. Phosphorylation of the largest subunits of both rat and yeast RNA polymerase II is stimulated by DNA, whereas phosphorylation of a synthetic peptide containing multiple copies of the carboxyl-terminal heptapeptide repeat is not. Although both ATP and GTP appear to function as phosphate donors, GTP is utilized less than 10% as well as ATP. These findings suggest that delta may exert its action in transcription at least in part through a mechanism involving phosphorylation of the largest subunit of RNA polymerase II. Images PMID:1386928

  12. A Perspective on the Enhancer Dependent Bacterial RNA Polymerase

    PubMed Central

    Zhang, Nan; Buck, Martin

    2015-01-01

    Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed. PMID:26010401

  13. Mammalian Cells with Altered Forms of RNA Polymerase II

    PubMed Central

    Chan, V. L.; Whitmore, G. F.; Siminovitch, Louis

    1972-01-01

    Mutants of Chinese hamster ovary cells that are resistant to α-amanitin can be isolated. At least some of these mutants contain an altered form of DNA-dependent RNA polymerase II, as indicated by its resistance to α-amanitin. These results indicate that mutation to α-amanitin resistance involves a change of a structural gene. PMID:4508306

  14. A perspective on the enhancer dependent bacterial RNA polymerase.

    PubMed

    Zhang, Nan; Buck, Martin

    2015-01-01

    Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed. PMID:26010401

  15. RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

    PubMed Central

    Altmann, C R; Solow-Cordero, D E; Chamberlin, M J

    1994-01-01

    In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein. Images PMID:7513426

  16. Ancient Origin and Recent Innovations of RNA Polymerase IV and V

    PubMed Central

    Huang, Yi; Kendall, Timmy; Forsythe, Evan S.; Dorantes-Acosta, Ana; Li, Shaofang; Caballero-Pérez, Juan; Chen, Xuemei; Arteaga-Vázquez, Mario; Beilstein, Mark A.; Mosher, Rebecca A.

    2015-01-01

    Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence. PMID:25767205

  17. The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi.

    PubMed Central

    Hinkle, G; Leipe, D D; Nerad, T A; Sogin, M L

    1994-01-01

    The small subunit ribosomal RNA (rRNA) of the anaerobic amoeba Phreatamoeba balamuthi is the longest 16S-like rRNA sequenced to date. Secondary structure analysis suggests that the additional sequence is incorporated in canonical eukaryotic expansion regions and is not due to the presence of introns. Reverse transcriptase sequencing of total RNA extracts confirmed that two uncommonly long expansion regions are present in native P. balamuthi 16S-like rRNA. Primary sequence comparison and similar secondary structure indicate a 61 base stem and loop repeat within an expansion region; a mechanism whereby the repeat may have been incorporated is presented. P. balamuthi provides further evidence that 16S-like rRNA length does not correlate with phylogenetic position. PMID:8127686

  18. PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases.

    PubMed

    Croci, Romina; Tarantino, Delia; Milani, Mario; Pezzullo, Margherita; Rohayem, Jacques; Bolognesi, Martino; Mastrangelo, Eloise

    2014-05-01

    Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex.

  19. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    PubMed Central

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  20. Highly selective affinity labelling of RNA polymerase B (II) from wheat germ.

    PubMed

    Grachev, M A; Hartmann, G R; Maximova, T G; Mustaev, A A; Schäffner, A R; Sieber, H; Zaychikov, E F

    1986-05-12

    DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(beta-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH4. Reaction of the modified enzyme with [alpha-32P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with Mr 140 000 by covalently linked ApU. Labelling was inhibited by 1 microgram/ml alpha-amanitin.

  1. Single molecule studies of RNA polymerase II transcription in vitro.

    PubMed

    Horn, Abigail E; Goodrich, James A; Kugel, Jennifer F

    2014-01-01

    Eukaryotic mRNA transcription by RNA polymerase II (RNAP II) is the first step in gene expression and a key determinant of cellular regulation. Elucidating the mechanism by which RNAP II synthesizes RNA is therefore vital to determining how genes are controlled under diverse biological conditions. Significant advances in understanding RNAP II transcription have been achieved using classical biochemical and structural techniques; however, aspects of the transcription mechanism cannot be assessed using these approaches. The application of single-molecule techniques to study RNAP II transcription has provided new insight only obtainable by studying molecules in this complex system one at a time.

  2. Mitochondrial DNA polymerase from embryos of Drosophila melanogaster: purification, subunit structure, and partial characterization

    SciTech Connect

    Wernette, C.M.; Kaguni, L.S.

    1986-11-05

    The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase ..gamma.. is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phiX174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase ..gamma.. as partially purified from several vertebrates.

  3. Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle.

    PubMed

    Pfannschmidt, Thomas; Blanvillain, Robert; Merendino, Livia; Courtois, Florence; Chevalier, Fabien; Liebers, Monique; Grübler, Björn; Hommel, Elisabeth; Lerbs-Mache, Silva

    2015-12-01

    Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations. PMID:26355147

  4. Yeast telomerase RNA: a flexible scaffold for protein subunits.

    PubMed

    Zappulla, David C; Cech, Thomas R

    2004-07-01

    In the yeast Saccharomyces cerevisiae, distinct regions of the 1.2-kb telomerase RNA (TLC1) bind to the catalytic subunit Est2p and to accessory proteins. In particular, a bulged stem structure binds the essential regulatory subunit Est1p. We now show that the Est1p-binding domain of the RNA can be moved to three distant locations with retention of telomerase function in vivo. We present the Est1p relocation experiment in the context of a working model for the secondary structure of the entire TLC1 RNA, based on thermodynamic considerations and comparative analysis of sequences from four species. The model for TLC1 has three long quasihelical arms that bind the Ku, Est1p, and Sm proteins. These arms emanate from a central catalytic core that contains the template and Est2p-binding region. Deletion mutagenesis provides evidence that the Sm arm exists in vivo and can be shortened by 42 predicted base pairs with retention of function; therefore, precise positioning of Sm proteins, like Est1p, is not required within telomerase. In the best-studied ribonucleoprotein enzyme, the ribosome, the RNAs have specific three-dimensional structures that orient the functional elements. In the case of yeast telomerase, we propose that the RNA serves a very different function, providing a flexible tether for the protein subunits. PMID:15226497

  5. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

    PubMed

    Viktorovskaya, Olga V; Engel, Krysta L; French, Sarah L; Cui, Ping; Vandeventer, Paul J; Pavlovic, Emily M; Beyer, Ann L; Kaplan, Craig D; Schneider, David A

    2013-09-12

    Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

  6. Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae.

    PubMed

    Engel, Christoph

    2016-01-01

    Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography. PMID:27576712

  7. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II

    PubMed Central

    Viktorovskaya, Olga V.; Engel, Krysta L.; French, Sarah L.; Cui, Ping; Vandeventer, Paul J.; Pavlovic, Emily M.; Beyer, Ann L.; Kaplan, Craig D.; Schneider, David A.

    2013-01-01

    SUMMARY Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss-of-function in Pol I [Pol II: rpb1- E1103G; Pol I: rpa190-E1224G]. Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase function, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps. PMID:23994471

  8. The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II

    PubMed Central

    Bharati, Akhilendra Pratap; Singh, Neha; Kumar, Vikash; Kashif, Md.; Singh, Amit Kumar; Singh, Priyanka; Singh, Sudhir Kumar; Siddiqi, Mohammad Imran; Tripathi, Timir; Akhtar, Md. Sohail

    2016-01-01

    RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription. PMID:27503426

  9. A multiplicity of factors contributes to selective RNA polymerase III occupancy of a subset of RNA polymerase III genes in mouse liver.

    PubMed

    Canella, Donatella; Bernasconi, David; Gilardi, Federica; LeMartelot, Gwendal; Migliavacca, Eugenia; Praz, Viviane; Cousin, Pascal; Delorenzi, Mauro; Hernandez, Nouria

    2012-04-01

    The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that only ∼40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.

  10. Separation of lymphocyte chromatin into template-active fractions with specificity for eukaryotic RNA polymerase II or prokaryotic RNA polymerase.

    PubMed Central

    Magee, B B; Paoletti, J; Magee, P T

    1975-01-01

    When chromatin prepared from WI-L2 lymphocytes by low salt extraction and shearing is centrifuged on a glycerol gradient, one area of the gradient yields chromatin enriched in template activity for Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) as compared to Saccharomyces cerevisiae RNA polymerase II (or B). Another area yields chromatin preferred by the eukaryotic enzyme. Kinetic studies indicate that the differences in activity cannot be explained by differences in affinity of the enzymes for the various templates. The DNA isolated from either fraction has a molecular weight of 8.5 X 106. The "yeast active" fraction seems enriched in proteins. Mixing experiments indicate that the yeast enzyme does not alter the template in such a way as to improve it for the bacterial enzyme. PMID:1108005

  11. Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly

    NASA Astrophysics Data System (ADS)

    Male, Gary; von Appen, Alexander; Glatt, Sebastian; Taylor, Nicholas M. I.; Cristovao, Michele; Groetsch, Helga; Beck, Martin; Müller, Christoph W.

    2015-06-01

    In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

  12. Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly

    PubMed Central

    Male, Gary; von Appen, Alexander; Glatt, Sebastian; Taylor, Nicholas M. I.; Cristovao, Michele; Groetsch, Helga; Beck, Martin; Müller, Christoph W.

    2015-01-01

    In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131–τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation. PMID:26060179

  13. Foraminifera and Cercozoa share a common origin according to RNA polymerase II phylogenies.

    PubMed

    Longet, David; Archibald, John M; Keeling, Patrick J; Pawlowski, Jan

    2003-11-01

    Phylogenetic analysis of small and large subunits of rDNA genes suggested that Foraminifera originated early in the evolution of eukaryotes, preceding the origin of other rhizopodial protists. This view was recently challenged by the analysis of actin and ubiquitin protein sequences, which revealed a close relationship between Foraminifera and Cercozoa, an assemblage of various filose amoebae and amoeboflagellates that branch in the so-called crown of the SSU rDNA tree of eukaryotes. To further test this hypothesis, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1) from five foraminiferans, two cercozoans and the testate filosean Gromia oviformis. Analysis of our data confirms a close relationship between Foraminifera and Cercozoa and points to Gromia as the closest relative of Foraminifera.

  14. The SWI/SNF chromatin remodeling complex influences transcription by RNA polymerase I in Saccharomyces cerevisiae.

    PubMed

    Zhang, Yinfeng; Anderson, Susan J; French, Sarah L; Sikes, Martha L; Viktorovskaya, Olga V; Huband, Jacalyn; Holcomb, Katherine; Hartman, John L; Beyer, Ann L; Schneider, David A

    2013-01-01

    SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5' end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5' end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.

  15. Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

    PubMed Central

    Campagnola, Grace; McDonald, Seth; Beaucourt, Stéphanie; Vignuzzi, Marco

    2014-01-01

    ABSTRACT Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCE Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating

  16. A Broad Anti-influenza Hybrid Small Molecule That Potently Disrupts the Interaction of Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Subunits.

    PubMed

    Massari, Serena; Nannetti, Giulio; Desantis, Jenny; Muratore, Giulia; Sabatini, Stefano; Manfroni, Giuseppe; Mercorelli, Beatrice; Cecchetti, Violetta; Palù, Giorgio; Cruciani, Gabriele; Loregian, Arianna; Goracci, Laura; Tabarrini, Oriana

    2015-05-14

    In continuing our efforts to identify small molecules able to disrupt the interaction of the polymerase acidic protein-basic protein 1 (PA-PB1) subunits of influenza virus (Flu) RNA-dependent RNA polymerase, this paper is devoted to the optimization of a dihydrotriazolopyrimidine derivative, previously identified through structure-based drug discovery. The structure modifications performed around the bicyclic core led to the identification of compounds endowed with both the ability to disrupt PA-PB1 subunits interaction and anti-Flu activity with no cytotoxicity. Very interesting results were obtained with the hybrid molecules 36 and 37, designed by merging some peculiar structural features known to impart PA-PB1 interaction inhibition, with compound 36 that emerged as the most potent PA-PB1 interaction inhibitor (IC50 = 1.1 μM) among all the small molecules reported so far. Calculations showed a very favored H-bonding between the 2-amidic carbonyl of 36 and Q408, which seems to justify its potent ability to interfere with the interaction of the polymerase subunits.

  17. Interaction of amatoxins with plant cells and RNA polymerases II: selection of amanitin-resistant cell lines and synthesis of amanitin-based affinity ligands

    SciTech Connect

    Little, M.C.

    1984-01-01

    A series of experiments directed toward deriving basic information regarding plant RNA polymerase II is presented. The experiments described relate to the potential of isolating RNA polymerase II mutants in plants, using carrot cell cultures as models. Additionally, the synthesis of amanitin-based affinity ligands to immobilize isolated plant RNA polymerase II and associated transcriptional complexes is described. RNA polymerase II activities have been isolated from suspension cultures of carrot and compared to other plant RNA polymerases II with respect to subunit analysis and inhibition with ..cap alpha..-amanitin. RNA polymerase II purified by polymin P absorption, DE52, phosphocellulose, and RNA-agarose chromatography is shown to copurify with proteins of 175 (and 200), 135, 70, 43, 28, 22, and 17 kdaltons apparent molecular weights. Conditions for accurate determination of amanitin inhibition of the enzyme are established using /sup 3/H-amanitin and are presented for the first time for plant RNA polymerase II; RNA polymerase II from these cultures is shown to be inhibited by 50% at 3-5 nM by ..cap alpha..-amanitin, a value 10-50 times lower than previously reported.

  18. Physicochemical properties and interactions of Escherichia coli ribonucleic acid polymerase holoenzyme, core enzyme, subunits, and subassembly alpha 2 beta.

    PubMed

    Levine, B J; Orphanos, P D; Fischmann, B S; Beychok, S

    1980-10-14

    We have investigated several physicochemical properties of Escherichia coli DNA-dependent RNa polymerase, its constituent subunits alpha, beta, beta', and sigma, and the subassembly alpha 2 beta. These included ultraviolet (UV) absorption, isoelectric points, sulfhydryl content, extinction coefficients, and circular dichroism (CD). Among the most notable results is the observation, based on CD measurements, that the sigma subunit, free and combined in holoenzyme, is a highly structured protein with approximately 75% of its residues folded in alpha-helical conformation and little or no detectable beta sheet. No secondary structure changes in either sigma or core accompany formation of holoenzyme. In contrast to the conformational independence of the subunits in assembly of holoenzyme, the protein and its components exhibit conformational flexibility as glycerol concentration is varied and in their interaction with DNA. The effect of glycerol on the conformation of sigma, core, and holoenzyme was monitored by circular dichroism measurements. In the far-ultraviolet, the residue ellipticity at 220 nm ([theta]220) increased approximately 15% from 0 to 10% glycerol for both core and holoenzyme. In the near-ultraviolet, the residue ellipticity at a peak near 280 nm also varied with glycerol concentration, decreasing in intensity by about 50% with holoenzyme, when glycerol was raised from 5 to 10%, then increasing at still higher glycerol contents. Electrophoretic and molecular sieve anaysis showed core and sigma to have greater affinity for each other in 50% glycerol than in 10% glycerol. The presence of 10% glycerol in the assay buffer increased the activity of the enzyme. The effect of various DNA templates on the conformations of core, holoenzyme, alpha 2 beta subassembly, and beta' subunit was also monitored by far-ultraviolet circular dichroism. All the protein samples showed between 10 and 20% decrease in secondary structure upon the addition of the DNA. PMID

  19. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  20. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  1. A heteromeric transcription factor required for mammalian RNA polymerase II.

    PubMed Central

    Kitajima, S; Tanaka, Y; Kawaguchi, T; Nagaoka, T; Weissman, S M; Yasukochi, Y

    1990-01-01

    A general transcription factor, FC, essential for specific initiation of in vitro transcription by mammalian RNA polymerase II was identified and a procedure developed to purify it to near homogeneity from HeLa cell nuclei. Purified FC is composed of two polypeptides of apparent molecular masses 80 kDa and 30 kDa, on SDS-PAGE, and has a native size of 280 kDa estimated by gel filtration column. Both polypeptides were shown to be essential for reconstituting in vitro transcription activity. Biochemical analysis showed that the 80 kDa and 30 kDa components were present in a 1:1 molar ratio. FC was also demonstrated to interact directly or indirectly with purified RNA polymerase II. Similarities between FC and transcription factors reported by others from human, rat or Drosophila cells are discussed. Images PMID:2395645

  2. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  3. Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

    PubMed Central

    Rocha, Antônio Augusto; Moretti, Nilmar Silvio

    2014-01-01

    The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing. PMID:24813189

  4. Structure, subunit composition, and molecular weight of RD-114 RNA.

    PubMed Central

    Kung, H J; Bailey, J M; Davidson, N; Nicolson, M O; McAllister, R M

    1975-01-01

    The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic

  5. RNA Polymerase Backtracking in Gene Regulation and Genome Instability

    PubMed Central

    Nudler, Evgeny

    2013-01-01

    RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. The amount of backtracking (reversible sliding of the enzyme along DNA and RNA) varies from one to many nucleotides. Since its first description 15 years ago, backtracking has been implicated in a plethora of critical processes in bacteria and eukaryotic cells. Here we review the most fundamental roles of this phenomenon in controlling transcription elongation, pausing, termination, fidelity, and genome instability. We also discuss recent progress in understanding the structural and mechanistic properties of the backtracking process. PMID:22726433

  6. Mechanism of histone survival during transcription by RNA polymerase II.

    PubMed

    Kulaeva, Olga I; Studitsky, Vasily M

    2010-01-01

    This work is related to and stems from our recent NSMB paper, "Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II" (December 2009). Synopsis. Recent genomic studies from many laboratories have suggested that nucleosomes are not displaced from moderately transcribed genes. Furthermore, histones H3/H4 carrying the primary epigenetic marks are not displaced or exchanged (in contrast to H2A/H2B histones) during moderate transcription by RNA polymerase II (Pol II) in vivo. These exciting observations suggest that the large molecule of Pol II passes through chromatin structure without even transient displacement of H3/H4 histones. The most recent analysis of the RNA polymerase II (Pol II)-type mechanism of chromatin remodeling in vitro (described in our NSMB 2009 paper) suggests that nucleosome survival is tightly coupled with formation of a novel intermediate: a very small intranucleosomal DNA loop (Ø-loop) containing transcribing Pol II. In the submitted manuscript we critically evaluate one of the key predictions of this model: the lack of even transient displacement of histones H3/H4 during Pol II transcription in vitro. The data suggest that, indeed, histones H3/H4 are not displaced during Pol II transcription in vitro. These studies are directly connected with the observation in vivo on the lack of exchange of histones H3/H4 during Pol II transcription.

  7. Recent advances in understanding transcription termination by RNA polymerase II

    PubMed Central

    Loya, Travis J.; Reines, Daniel

    2016-01-01

    Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery. A greater appreciation for the coupling of termination to RNA processing and metabolism has been recognized. In addition to serving as an essential step at the end of the transcription cycle, termination is involved in the regulation of a broad range of cellular processes. More recently, a role for termination in pervasive transcription, non-coding RNA regulation, genetic stability, chromatin remodeling, the immune response, and disease has come to the fore. Interesting mechanistic questions remain, but the last several years have resulted in significant insights into termination and an increasing recognition of its biological importance. PMID:27408690

  8. Favipiravir (T-705), a novel viral RNA polymerase inhibitor.

    PubMed

    Furuta, Yousuke; Gowen, Brian B; Takahashi, Kazumi; Shiraki, Kimiyasu; Smee, Donald F; Barnard, Dale L

    2013-11-01

    Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus. It is phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5'-triphosphate (RTP). Its antiviral effect is attenuated by the addition of purine nucleic acids, indicating the viral RNA polymerase mistakenly recognizes favipiravir-RTP as a purine nucleotide. Favipiravir is active against a broad range of influenza viruses, including A(H1N1)pdm09, A(H5N1) and the recently emerged A(H7N9) avian virus. It also inhibits influenza strains resistant to current antiviral drugs, and shows a synergistic effect in combination with oseltamivir, thereby expanding influenza treatment options. A Phase III clinical evaluation of favipiravir for influenza therapy has been completed in Japan and two Phase II studies have been completed in the United States. In addition to its anti-influenza activity, favipiravir blocks the replication of many other RNA viruses, including arenaviruses (Junin, Machupo and Pichinde); phleboviruses (Rift Valley fever, sandfly fever and Punta Toro); hantaviruses (Maporal, Dobrava, and Prospect Hill); flaviviruses (yellow fever and West Nile); enteroviruses (polio- and rhinoviruses); an alphavirus, Western equine encephalitis virus; a paramyxovirus, respiratory syncytial virus; and noroviruses. With its unique mechanism of action and broad range of antiviral activity, favipiravir is a promising drug candidate for influenza and many other RNA viral diseases for which there are no approved therapies.

  9. Favipiravir (T-705), a novel viral RNA polymerase inhibitor

    PubMed Central

    Furuta, Yousuke; Gowen, Brian B.; Takahashi, Kazumi; Shiraki, Kimiyasu; Smee, Donald F.; Barnard, Dale L.

    2013-01-01

    Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus. It is phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5′-triphosphate (RTP). Its antiviral effect is attenuated by the addition of purine nucleic acids, indicating the viral RNA polymerase mistakenly recognizes favipiravir-RTP as a purine nucleotide. Favipiravir is active against a broad range of influenza viruses, including A(H1N1)pdm09, A(H5N1) and the recently emerged A(H7N9) avian virus. It also inhibits influenza strains resistant to current antiviral drugs, and shows a synergistic effect in combination with oseltamivir, thereby expanding influenza treatment options. A Phase III clinical evaluation of favipiravir for influenza therapy has been completed in Japan and two Phase II studies have been completed in the United States. In addition to its anti-influenza activity, favipiravir blocks the replication of many other RNA viruses, including arenaviruses (Junin, Machupo and Pichinde); phleboviruses (Rift Valley fever, sandfly fever and Punta Toro); hantaviruses (Maporal, Dobrava, and Prospect Hill); flaviviruses (yellow fever and West Nile); enteroviruses (polio- and rhinoviruses); an alphavirus, Western equine encephalitis virus; a paramyxovirus, respiratory syncytial virus; and noroviruses. With its unique mechanism of action and broad range of antiviral activity, favipiravir is a promising drug candidate for influenza and many other RNA viral diseases for which there are no approved therapies. PMID:24084488

  10. Distinguishing Core and Holoenzyme Mechanisms of Transcription Termination by RNA Polymerase III

    PubMed Central

    Arimbasseri, Aneeshkumar G.

    2013-01-01

    Transcription termination by RNA polymerase (Pol) III serves multiple purposes; it delimits interference with downstream genes, forms 3′ oligo(U) binding sites for the posttranscriptional processing factor, La protein, and resets the polymerase complex for reinitiation. Although an interplay of several Pol III subunits is known to collectively control these activities, how they affect molecular function of the active center during termination is incompletely understood. We have approached this using immobilized Pol III-nucleic acid scaffolds to examine the two major components of termination, transcription pausing and RNA release. This allowed us to distinguish two mechanisms of termination by isolated Saccharomyces cerevisiae Pol III. A core mechanism can operate in the absence of C53/37 and C11 subunits but requires synthesis of 8 or more 3′ U nucleotides, apparently reflecting inherent sensitivity to an oligo(rU·dA) hybrid that is the termination signal proper. The holoenzyme mechanism requires fewer U nucleotides but uses C53/37 and C11 to slow elongation and prevent terminator arrest. N-terminal truncation of C53 or point mutations that disable the cleavage activity of C11 impair their antiarrest activities. The data are consistent with a model in which C53, C37, and C11 activities are functionally integrated with the active center of Pol III during termination. PMID:23401852

  11. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

    PubMed Central

    Thiffault, Isabelle; Wolf, Nicole I.; Forget, Diane; Guerrero, Kether; Tran, Luan T.; Choquet, Karine; Lavallée-Adam, Mathieu; Poitras, Christian; Brais, Bernard; Yoon, Grace; Sztriha, Laszlo; Webster, Richard I.; Timmann, Dagmar; van de Warrenburg, Bart P.; Seeger, Jürgen; Zimmermann, Alíz; Máté, Adrienn; Goizet, Cyril; Fung, Eva; van der Knaap, Marjo S.; Fribourg, Sébastien; Vanderver, Adeline; Simons, Cas; Taft, Ryan J.; Yates III, John R.; Coulombe, Benoit; Bernard, Geneviève

    2015-01-01

    A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. PMID:26151409

  12. SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination.

    PubMed

    Zhao, Dorothy Yanling; Gish, Gerald; Braunschweig, Ulrich; Li, Yue; Ni, Zuyao; Schmitges, Frank W; Zhong, Guoqing; Liu, Ke; Li, Weiguo; Moffat, Jason; Vedadi, Masoud; Min, Jinrong; Pawson, Tony J; Blencowe, Benjamin J; Greenblatt, Jack F

    2016-01-01

    The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. Here we show that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires protein arginine methyltransferase 5 (PRMT5) and recruits the Tudor domain of the survival of motor neuron (SMN, also known as GEMIN1) protein, which is mutated in spinal muscular atrophy. SMN interacts with senataxin, which is sometimes mutated in ataxia oculomotor apraxia type 2 and amyotrophic lateral sclerosis. Because POLR2A R1810me2s and SMN, like senataxin, are required for resolving RNA-DNA hybrids created by RNA polymerase II that form R-loops in transcription termination regions, we propose that R1810me2s, SMN, and senataxin are components of an R-loop resolution pathway. Defects in this pathway can influence transcription termination and may contribute to neurodegenerative disorders. PMID:26700805

  13. Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II

    PubMed Central

    Jendrisak, Jerome J.; Becker, Wayne M.

    1974-01-01

    A procedure is described for the purification of the α-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH4)2SO4, chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material. ImagesPLATE 1PLATE 2 PMID:4853970

  14. Nucleolin provides a link between RNA polymerase I transcription and pre-ribosome assembly.

    PubMed

    Roger, Benoit; Moisand, André; Amalric, François; Bouvet, Philippe

    2003-03-01

    Despite the identification of numerous factors involved in ribosomal RNA synthesis and maturation, the molecular mechanisms of ribosome biogenesis, and in particular the relationship between the different steps, are still largely unknown. We have investigated the consequences of an increased amount of a major nucleolar non-ribosomal protein, nucleolin, in Xenopus laevisstage VI oocytes on the production of ribosomal subunits. We show that a threefold increase in nucleolin leads to the complete absence of pre-rRNA maturation in addition to significant repression of RNA polymerase I transcription. Observation of "Christmas trees" by electron microscopy and analysis of the sedimentation properties of 40S pre-ribosomal particles suggest that an increased amount of nucleolin leads to incorrect packaging of the 40S particle. Interestingly, nucleolin affects the maturation of the 40S particle only when it is present at the time of transcription. These results indicate that nucleolin participates in the co-transcriptional packaging of the pre-rRNA, and that the quality of this packaging will determine whether the 40S precursor undergoes maturation or is degraded. The interaction of nucleolin with nascent pre-rRNA could help the co-transcriptional assembly on pre-rRNA of factors necessary for the subsequent maturation of the pre-ribosomal particle containing the 40S pre-rRNA.

  15. Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation.

    PubMed

    Arimbasseri, Aneeshkumar G; Rijal, Keshab; Maraia, Richard J

    2014-01-01

    In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.

  16. RNA polymerase III in Cajal bodies and lampbrush chromosomes of the Xenopus oocyte nucleus.

    PubMed

    Murphy, Christine; Wang, Zhengxin; Roeder, Robert G; Gall, Joseph G

    2002-10-01

    We used immunofluorescence to study the distribution and targeting of RNA polymerase (pol) III subunits and pol III transcription factors in the Xenopus laevis oocyte nucleus. Antibodies against several of these proteins stained Cajal bodies and approximately 90 specific sites on the lampbrush chromosomes. Some of the chromosomal sites had been identified previously by in situ hybridization as the genes for 5S rRNA. The remaining sites presumably encode tRNAs and other pol III transcripts. Pol III sites were often resolvable as loops similar to the much more abundant pol II loops, but without a matrix detectable by phase contrast or differential interference contrast. This morphology is consistent with the transcription of short repeated sequences. Hemagglutinin-tagged transcripts encoding core subunits and transcription factors were injected into the oocyte cytoplasm, and the distribution of newly translated proteins inside the nucleus was monitored by immunostaining. Cajal bodies were preferentially targeted by these proteins, and in some cases the chromosomal sites were also weakly stained. The existence of pol III subunits and pol III transcription factors in Cajal bodies and their targeting to these organelles are consistent with a model of Cajal bodies as sites for preassembly of the nuclear transcription machinery. PMID:12388750

  17. Analyses of in vivo interactions between transcription factors and the archaeal RNA polymerase.

    PubMed

    Walker, Julie E; Santangelo, Thomas J

    2015-09-15

    Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation.

  18. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  19. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  20. De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase.

    PubMed

    Maida, Yoshiko; Yasukawa, Mami; Masutomi, Kenkichi

    2016-04-01

    RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such as Caenorhabditis elegans and Neurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAs de novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However, de novo synthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5'-triphosphorylated ends, which indicates de novo synthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity. PMID:26830230

  1. The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme.

    PubMed

    Tomer, G; Reuven, N B; Livneh, Z

    1998-11-24

    The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

  2. Characterization of the RNA polymerase II and III complexes in Leishmania major.

    PubMed

    Martínez-Calvillo, Santiago; Saxena, Alka; Green, Amanda; Leland, Aaron; Myler, Peter J

    2007-04-01

    Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function. PMID:17275824

  3. Synergistic action of RNA polymerases in overcoming the nucleosomal barrier.

    PubMed

    Jin, Jing; Bai, Lu; Johnson, Daniel S; Fulbright, Robert M; Kireeva, Maria L; Kashlev, Mikhail; Wang, Michelle D

    2010-06-01

    During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10-base pair periodicity and backtracked by approximately 10-15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAP's elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

  4. Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core.

    PubMed

    Conte, Emanuele; Vincelli, Gabriele; Schaaper, Roel M; Bressanin, Daniela; Stefan, Alessandra; Dal Piaz, Fabrizio; Hochkoeppler, Alejandro

    2012-07-15

    Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.

  5. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.

    PubMed Central

    van der Werf, S; Bradley, J; Wimmer, E; Studier, F W; Dunn, J J

    1986-01-01

    Plasmids containing the entire cDNA sequence of poliovirus type 1 (Mahoney strain) under control of a promoter for T7 RNA polymerase have been constructed. Purified T7 RNA polymerase efficiently transcribes the entire poliovirus cDNA in either direction to produce full-length poliovirus RNA [(+)RNA] or its complement [(-)RNA]. The (+)RNA produced initially had 60 nucleotides on the 5' side of the poliovirus RNA sequence, including a string of 18 consecutive guanine residues generated in the original cloning and an additional 626 nucleotides of pBR322 sequence beyond the poly(A) tract at the 3' end. Such RNA, while much more infectious than the plasmid DNA, is only about 0.1% as infectious as RNA isolated from the virus. Subsequently, a T7 promoter was placed only 2 base pairs ahead of the poliovirus sequence, so that T7 RNA polymerase synthesizes poliovirus RNA with only 2 additional guanine residues at the 5' end and no more than seven nucleotides past the poly(A) tract at the 3' end. Such RNA has much higher specific infectivity, about 5% that of RNA isolated from the virus. The ability to make infectious poliovirus RNA efficiently from cloned DNA makes it possible to apply techniques of in vitro mutagenesis to the analysis of poliovirus functions and the construction of novel and perhaps useful derivatives of poliovirus. A source of variant RNAs should also allow detailed study of the synthesis and processing of poliovirus proteins in vitro. Images PMID:3010307

  6. Ccr4-Not Regulates RNA Polymerase I Transcription and Couples Nutrient Signaling to the Control of Ribosomal RNA Biogenesis

    PubMed Central

    Laribee, R. Nicholas; Hosni-Ahmed, Amira; Workman, Jason J.; Chen, Hongfeng

    2015-01-01

    Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis. PMID:25815716

  7. Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).

    PubMed Central

    Laquel, P.; Litvak, S.; Castroviejo, M.

    1994-01-01

    DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants. PMID:12232187

  8. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  9. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  10. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  11. A small post-translocation energy bias aids nucleotide selection in T7 RNA polymerase transcription.

    PubMed

    Yu, Jin; Oster, George

    2012-02-01

    The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr(639) that moves its side chain into the active site, pushing aside the 3'-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr(639) closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.

  12. Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate.

    PubMed

    García, Alicia; Collin, Alejandro; Calvo, Olga

    2012-11-01

    The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.

  13. Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate

    PubMed Central

    García, Alicia; Collin, Alejandro; Calvo, Olga

    2012-01-01

    The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3′-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5–Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5–Rpb1 complex levels and consequently transcription elongation rate. PMID:22973055

  14. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

    PubMed

    Chung, Kwan-Ho; Hart, Christopher C; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D; Vojtek, Anne B; Turner, David L

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

  15. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    PubMed Central

    Chung, Kwan-Ho; Hart, Christopher C.; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D.; Vojtek, Anne B.; Turner, David L.

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications. PMID:16614444

  16. Double strand breaks: hurdles for RNA polymerase II transcription?

    PubMed

    Pankotai, Tibor; Soutoglou, Evi

    2013-01-01

    DNA lesions pose a physical obstacle to DNA-dependent cellular transactions such as replication and transcription. A great deal is known regarding RNA polymerase II (RNAP II) transcription stalling in the presence of lesions induced by UV, but recent studies have uncovered previously uncharacterized behavior of the RNAP II machinery in the presence of double strand breaks (DSBs). These new data, although contradictory, contribute to our understanding of a vital cellular mechanism that defends against the production of aberrant transcripts and protects cell viability.

  17. Chromatin modification by the RNA Polymerase II elongation complex

    PubMed Central

    Tanny, Jason C.

    2014-01-01

    Transcription elongation by RNA polymerase II (RNAP II) involves the coordinated action of numerous regulatory factors. Among these are chromatin-modifying enzymes, which generate a stereotypic and conserved pattern of histone modifications along transcribed genes. This pattern implies a precise coordination between regulators of histone modification and the RNAP II elongation complex. Here I review the pathways and molecular events that regulate co-transcriptional histone modifications. Insight into these events will illuminate the assembly of functional RNAP II elongation complexes and how the chromatin landscape influences their composition and function. PMID:25494544

  18. Paused RNA polymerase II as a developmental checkpoint.

    PubMed

    Levine, Michael

    2011-05-13

    The textbook view of gene activation is that the rate-limiting step is the interaction of RNA polymerase II (Pol II) with the gene's promoter. However, studies in a variety of systems, including human embryonic stem cells and the early Drosophila embryo, have begun to challenge this view. There is increasing evidence that differential gene expression often depends on the regulation of transcription elongation via the release of Pol II from the proximal promoter. I review the implications of this mechanism of gene activation with respect to the orderly unfolding of complex gene networks governing animal development. PMID:21565610

  19. Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II.

    PubMed

    Stiller, J W; Duffield, E C; Hall, B D

    1998-09-29

    Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.

  20. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  1. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  2. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  3. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  4. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1990-01-01

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  5. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  6. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  7. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  8. Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers.

    PubMed Central

    Kao, C C; Sun, J H

    1996-01-01

    Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp. PMID:8794323

  9. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    PubMed

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T(1) protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

  10. DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

    PubMed Central

    Parshin, Andrey; Shiver, Anthony L.; Lee, Jookyung; Ozerova, Maria; Schneidman-Duhovny, Dina; Gross, Carol A.; Borukhov, Sergei

    2015-01-01

    Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo–cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription. PMID:26604313

  11. Evidence for Moonlighting Functions of the θ Subunit of Escherichia coli DNA Polymerase III

    PubMed Central

    Dietrich, M.; Pedró, L.; García, J.; Pons, M.; Hüttener, M.; Paytubi, S.; Madrid, C.

    2014-01-01

    The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no detectable homology to other ORFs in a database). It encodes the θ subunit of the DNA polymerase III core complex. The precise function of the θ subunit within this complex is not well established, and loss of holE does not result in a noticeable phenotype. Paralogs of holE are also present on many conjugative plasmids and on phage P1 (hot gene). In this study, we provide evidence indicating that θ (HolE) exhibits structural and functional similarities to a family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins that are also encoded by enterobacterial ORFan genes. Microarray studies comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT mutants revealed highly similar expression patterns for strains harboring holE and ydgT alleles. Among the genes differentially regulated in both mutants were genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE and YdgT downregulate expression of the tna operon by possibly increasing the level of Rho-dependent transcription termination at the tna operon's leader region. Thus, for the first time, a regulatory function can be attributed to HolE, in addition to its role as structural component of the DNA polymerase III complex. PMID:24375106

  12. In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus.

    PubMed Central

    Rao, L; Karls, R K; Betley, M J

    1995-01-01

    The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70. PMID:7751267

  13. The Ess1 prolyl isomerase: Traffic cop of the RNA polymerase II transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5–Pro6 bonds, and to a lesser extent pSer2–Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs. PMID:24530645

  14. 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.

  15. 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

    2016-07-15

    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.

  16. 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

  17. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    SciTech Connect

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud

    2007-06-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.

  18. The X-ray Crystal Structure of RNA Polymerase from Archaea

    SciTech Connect

    Hirata,A.; Klein, B.; Murakami, K.

    2008-01-01

    The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits3, 4. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 Angstroms resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 Angstroms ) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

  19. Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex

    PubMed Central

    Sourimant, Julien; Plemper, Richard K.

    2016-01-01

    The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pave the path towards the structure-based design and optimization of next-generation RdRp inhibitors. PMID:27626440

  20. Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex.

    PubMed

    Sourimant, Julien; Plemper, Richard K

    2016-01-01

    The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pave the path towards the structure-based design and optimization of next-generation RdRp inhibitors. PMID:27626440

  1. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

    PubMed Central

    Himmelfarb, H J; Simpson, E M; Friesen, J D

    1987-01-01

    Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain. PMID:3299061

  2. Intragenic and Extragenic Suppressors of Mutations in the Heptapeptide Repeat Domain of Saccharomyces Cerevisiae RNA Polymerase II

    PubMed Central

    Nonet, M. L.; Young, R. A.

    1989-01-01

    The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2Δ10) was constructed. The srb2Δ10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II. PMID:2693207

  3. Mammalian. cap alpha. -polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

    SciTech Connect

    SenGupta, D.N.; Kumar, P.; Zmudzka, B.Z.; Coughlin, S.; Vishwanatha, J.K.; Robey, F.A.; Parrott, C.; Wilson, S.H.

    1987-02-10

    A new polyclonal antibody against the ..cap alpha..-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell..cap alpha..-polymerase. The antibody neutralized ..cap alpha..-polymerase activity and was strong and specific for the ..cap alpha..-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambdagt11. A positive phage was identified and plaque purified. This phage, designated lambdapol..cap alpha..1.2, also was found to be positive with an antibody against Drosophila ..cap alpha..-polymerase. The insert in lambdapol..cap alpha..1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified ..cap alpha..-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating ..cap alpha..-polymerase. This indicated the lambdapol..cap alpha..1.2 insert encoded an ..cap alpha..-polymerase epitope and suggested that the cDNA corresponded to an ..cap alpha..-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding ..cap alpha..-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approx.5.4 kilobases.

  4. Mutations in the RNA Polymerase II Transcription Machinery Suppress the Hyperrecombination Mutant Hpr1Δ of Saccharomyces Cerevisiae

    PubMed Central

    Fan, H. Y.; Cheng, K. K.; Klein, H. L.

    1996-01-01

    The soh1, soh2 and soh4 mutants were isolated as suppressors of the temperature-dependent growth of the hyperrecombination mutant hpr1 of Saccharomyces cerevisiae. Cloning and sequence analysis of these suppressor genes has unexpectedly shown them to code for components of the RNA polymerase II transcription complex. SOH2 is identical to RPB2, which encodes the second largest subunit of RNA polymerase II, and SOH4 is the same as SUA7, encoding the yeast transcription initiation factor TFIIB. SOH1 encodes a novel 14-kD protein with limited sequence similarity to RNA polymerases. Interestingly, SOH1 not only interacts with factors involved in DNA repair, but transcription as well. Thus, the Soh1 protein may serve to couple these two processes. The Soh1 protein interacts with a DNA repair protein, Rad5p, in a two-hybrid system assay. Soh1p may functionally interact with components of the RNA polymerase II complex as suggested from the synthetic lethality observed in soh1 rpb1Δ104, soh1 soh2-1 (rpb2), and soh1 soh4 (sua7) double mutants. Because mutations in SOH1, RPB2 and SUA7 suppress the hyperrecombination phenotype of hpr1 mutants, this suggests a link between recombination in direct repeats and transcription. PMID:8849885

  5. Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

    SciTech Connect

    Gleghorn, M.; Davydova, E; Rothman-Denes, L; Murakami, K

    2008-01-01

    Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

  6. RNA polymerase II mutants defective in transcription of a subset of genes.

    PubMed Central

    Scafe, C; Nonet, M; Young, R A

    1990-01-01

    Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species. Images PMID:2406558

  7. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.

    2016-01-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

  8. Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

    PubMed Central

    Treich, I; Carles, C; Sentenac, A; Riva, M

    1992-01-01

    In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site. Images PMID:1408783

  9. Inhibition of acetyl phosphate-dependent transcription by an acetylatable lysine on RNA polymerase.

    PubMed

    Lima, Bruno P; Thanh Huyen, Tran Thi; Bäsell, Katrin; Becher, Dörte; Antelmann, Haike; Wolfe, Alan J

    2012-09-14

    The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment. PMID:22829598

  10. Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase*

    PubMed Central

    Lima, Bruno P.; Thanh Huyen, Tran Thi; Bäsell, Katrin; Becher, Dörte; Antelmann, Haike; Wolfe, Alan J.

    2012-01-01

    The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment. PMID:22829598

  11. Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation.

    PubMed

    Chlamydas, Sarantis; Holz, Herbert; Samata, Maria; Chelmicki, Tomasz; Georgiev, Plamen; Pelechano, Vicent; Dündar, Friederike; Dasmeh, Pouria; Mittler, Gerhard; Cadete, Filipe Tavares; Ramírez, Fidel; Conrad, Thomas; Wei, Wu; Raja, Sunil; Manke, Thomas; Luscombe, Nicholas M; Steinmetz, Lars M; Akhtar, Asifa

    2016-06-01

    Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies. PMID:27183194

  12. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain.

    PubMed

    Jeronimo, Célia; Collin, Pierre; Robert, François

    2016-06-19

    The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this "Perspective," we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed.

  13. DNA-Dependent RNA Polymerase Detects Hidden Giant Viruses in Published Databanks

    PubMed Central

    Sharma, Vikas; Colson, Philippe; Giorgi, Roch; Pontarotti, Pierre; Raoult, Didier

    2014-01-01

    Environmental metagenomic studies show that there is a “dark matter,” composed of sequences not linked to any known organism, as determined mainly using ribosomal DNA (rDNA) sequences, which therefore ignore giant viruses. DNA-dependent RNA polymerase (RNAP) genes are universal in microbes and conserved in giant viruses and may replace rDNA for identifying microbes. We found while reconstructing RNAP subunit 2 (RNAP2) phylogeny that a giant virus sequenced together with the genome of a large eukaryote, Hydra magnipapillata, has been overlooked. To explore the dark matter, we used viral RNAP2 and reconstructed putative ancestral RNAP2, which were significantly superior in detecting distant clades than current sequences, and we revealed two additional unknown mimiviruses, misclassified as an euryarchaeote and an oomycete plant pathogen, and detected unknown putative viral clades. We suggest using RNAP systematically to decipher the black matter and identify giant viruses. PMID:24929085

  14. DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

    PubMed Central

    Coulombe, Benoit; Burton, Zachary F.

    1999-01-01

    A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. PMID:10357858

  15. Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye.

    PubMed Central

    Chen, X; Li, Q; Fischer, J A

    2000-01-01

    The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, we identified mutations in DNAprim. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles were determined and these define essential protein domains. We show that while flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs. Mechanisms by which DNA primase levels might influence the faf-dependent cell communication pathway are discussed. PMID:11102374

  16. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    DOE PAGES

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A.

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

  17. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    SciTech Connect

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A.

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

  18. Direct observation of base-pair stepping by RNA polymerase

    NASA Astrophysics Data System (ADS)

    Abbondanzieri, Elio A.; Greenleaf, William J.; Shaevitz, Joshua W.; Landick, Robert; Block, Steven M.

    2005-11-01

    During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6Å, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

  19. Direct observation of base-pair stepping by RNA polymerase.

    PubMed

    Abbondanzieri, Elio A; Greenleaf, William J; Shaevitz, Joshua W; Landick, Robert; Block, Steven M

    2005-11-24

    During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

  20. Retrotransposon profiling of RNA polymerase III initiation sites

    PubMed Central

    Qi, Xiaojie; Daily, Kenneth; Nguyen, Kim; Wang, Haoyi; Mayhew, David; Rigor, Paul; Forouzan, Sholeh; Johnston, Mark; Mitra, Robi David; Baldi, Pierre; Sandmeyer, Suzanne

    2012-01-01

    Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III–transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III–transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes. PMID:22287102

  1. Retrotransposon profiling of RNA polymerase III initiation sites.

    PubMed

    Qi, Xiaojie; Daily, Kenneth; Nguyen, Kim; Wang, Haoyi; Mayhew, David; Rigor, Paul; Forouzan, Sholeh; Johnston, Mark; Mitra, Robi David; Baldi, Pierre; Sandmeyer, Suzanne

    2012-04-01

    Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes. PMID:22287102

  2. Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae

    PubMed Central

    Barillà, Daniela; Lee, Barbara A.; Proudfoot, Nick J.

    2001-01-01

    The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript. PMID:11149954

  3. Intergenic transcriptional interference is blocked by RNA polymerase III transcription factor TFIIIB in Saccharomyces cerevisiae.

    PubMed

    Korde, Asawari; Rosselot, Jessica M; Donze, David

    2014-02-01

    The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such "extratranscriptional" activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467-ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins.

  4. Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit

    SciTech Connect

    Guu, Tom S.Y.; Dong Liping; Wittung-Stafshede, Pernilla; Tao, Yizhi J.

    2008-09-15

    The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed {alpha}/{beta} structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.

  5. Defining the status of RNA polymerase at promoters.

    PubMed

    Core, Leighton J; Waterfall, Joshua J; Gilchrist, Daniel A; Fargo, David C; Kwak, Hojoong; Adelman, Karen; Lis, John T

    2012-10-25

    Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila. PMID:23062713

  6. Defining the status of RNA polymerase at promoters.

    PubMed

    Core, Leighton J; Waterfall, Joshua J; Gilchrist, Daniel A; Fargo, David C; Kwak, Hojoong; Adelman, Karen; Lis, John T

    2012-10-25

    Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.

  7. 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

  8. Is actin a transcription initiation factor for RNA polymerase B?

    PubMed Central

    Egly, J M; Miyamoto, N G; Moncollin, V; Chambon, P

    1984-01-01

    We have previously reported that two fractions derived from HeLa cell S100 extracts, the heparin flow-through and the heparin 0.6 M KCl eluate are required in vitro for efficient and accurate transcription by RNA polymerase class B (II). We have further purified a factor present in the heparin flow-through fraction, which markedly stimulates specific transcription catalyzed by the heparin 0.6 M KCl eluate. We report here that some of the properties of the stimulatory factor present in our most purified fractions are strikingly similar to those of actin. We demonstrate also that this factor acts at the pre-initiation level of the transcription reaction. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. Fig. 8. Fig. 9. Fig. 10. Fig. 11. Fig. 12. Fig. 13. PMID:6499833

  9. ELL facilitates RNA polymerase II pause site entry and release.

    PubMed

    Byun, Jung S; Fufa, Temesgen D; Wakano, Clay; Fernandez, Alfonso; Haggerty, Cynthia M; Sung, Myong-Hee; Gardner, Kevin

    2012-01-01

    Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex, containing the eleven-nineteen lysine-rich leukaemia (ELL) protein, as a key regulator of transcriptional elongation. Here we show that ELL has a diverse and kinetically distinct role before its assembly into the super elongation complex by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the pre-initiation complexes and results in disruption of early elongation and promoter proximal chromatin structure before recruitment of AFF4 and other super elongation complex components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL has an early and essential role during rapid high-amplitude gene expression that is required for both Pol II pause site entry and release. PMID:22252557

  10. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.

    1984-03-30

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties.

  11. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

    PubMed

    Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P

    2016-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7. PMID:26979928

  12. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

    PubMed

    Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P

    2016-03-16

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

  13. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

    PubMed Central

    Hunter, Lydia J. R.; Brockington, Samuel F.; Murphy, Alex M.; Pate, Adrienne E.; Gruden, Kristina; MacFarlane, Stuart A.; Palukaitis, Peter; Carr, John P.

    2016-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7. PMID:26979928

  14. Microarray data analyses of yeast RNA Pol I subunit RPA12 deletion strain.

    PubMed

    Yadav, Kamlesh Kumar; Rajasekharan, Ram

    2016-06-01

    The ribosomal RNA (rRNA) biosynthesis is the most energy consuming process in all living cells and the majority of total transcription activity is dedicated for synthesizing rRNA. The cells may adjust the synthesis of rRNA with the availability of resources. rRNA is mainly synthesized by RNA polymerase I that is composed of 14 subunits. Deletion of RPA12, 14, 39 and 49 are viable. RPA12 is a very small protein (13.6 kDa), and the amount of protein in the cells is very high (12,000 molecules per cell), but the role of this protein is unknown in other cellular metabolic processes (Kulak et al., 2014 [1]). RPA12 consists of two zinc-binding domains and it is required for the termination of rRNA synthesis (Mullem et al., 2002 [2]). Deletions of RPA12 in Saccharomyces cerevisiae and Schizosaccharomyces pombe cause a conditional growth defect (Nogi et al., 1993 [3]). In S. pombe, C-terminal deletion behaves like wild-type (Imazawa et al., 2001 [4]). This prompted us to investigate in detail the physiological role of RPA12 in S. cerevisiae, we performed the microarray of rpa12 ∆ strain and deposited into Gene Expression Omnibus under GSE68731. The analysis of microarray data revealed that the expression of major cellular metabolism genes is high. The amino acid biosynthesis, nonpolar lipid biosynthesis and glucose metabolic genes are highly expressed. The analyses also revealed that the rpa12 ∆ cells have an uncontrolled synthesis of cell metabolites, so RPA12 could be a master regulator for whole cellular metabolism. PMID:27222810

  15. The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

    PubMed Central

    Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

    2014-01-01

    ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated

  16. Transcription by single molecules of RNA polymerase observed by light microscopy.

    PubMed

    Schafer, D A; Gelles, J; Sheetz, M P; Landick, R

    1991-08-01

    The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.

  17. Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

    PubMed Central

    Canella, Donatella; Praz, Viviane; Reina, Jaime H.; Cousin, Pascal; Hernandez, Nouria

    2010-01-01

    Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units. PMID:20413673

  18. Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

    PubMed Central

    Lin, Xiaoyan; Thorne, Lucy; Jin, Zhinan; Hammad, Loubna A.; Li, Serena; Deval, Jerome; Goodfellow, Ian G.; Kao, C. Cheng

    2015-01-01

    The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp. PMID:25520198

  19. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus

    PubMed Central

    Guo, Yusong R.; Toh, Yukimatsu; Poranen, Minna M.; Tao, Yizhi J.

    2016-01-01

    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly. PMID:27078841

  20. Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases*

    PubMed Central

    Qian, Xinlei; Hamid, Fursham M.; El Sahili, Abbas; Darwis, Dina Amallia; Wong, Yee Hwa; Bhushan, Shashi; Makeyev, Eugene V.; Lescar, Julien

    2016-01-01

    Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro. However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1Ncr) and related fungi, Thielavia terrestris (QDE-1Tte) and Myceliophthora thermophila (QDE-1Mth), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1Ncr counterpart favoring processive RNA synthesis, QDE-1Tte and QDE-1Mth produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1Tte reveals a quasisymmetric dimer similar to QDE-1Ncr. Further electron microscopy analyses confirm that QDE-1Tte occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure. PMID:26907693

  1. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  2. Bacterial RNA polymerase can retain σ70 throughout transcription

    PubMed Central

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

    2016-01-01

    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, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–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 σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 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 σ70 during transcription. PMID:26733675

  3. RNA polymerase II ternary transcription complexes generated in vitro.

    PubMed Central

    Ackerman, S; Bunick, D; Zandomeni, R; Weinmann, R

    1983-01-01

    Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation. Images PMID:6193489

  4. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus.

    PubMed

    Hengrung, Narin; El Omari, Kamel; Serna Martin, Itziar; Vreede, Frank T; Cusack, Stephen; Rambo, Robert P; Vonrhein, Clemens; Bricogne, Gérard; Stuart, David I; Grimes, Jonathan M; Fodor, Ervin

    2015-11-01

    Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

  5. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus

    PubMed Central

    Hengrung, Narin; El Omari, Kamel; Martin, Itziar Serna; Vreede, Frank T.; Cusack, Stephen; Rambo, Robert P.; Vonrhein, Clemens; Bricogne, Gérard; Stuart, David I.; Grimes, Jonathan M.; Fodor, Ervin

    2016-01-01

    Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome1. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching2. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5′ and 3′ termini of viral genome segments), showing FluPol in transcription pre-initiation states3,4. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new ‘closed’ conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols3,4. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication. PMID:26503046

  6. A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

    PubMed Central

    Bourquin, J P; Stagljar, I; Meier, P; Moosmann, P; Silke, J; Baechi, T; Georgiev, O; Schaffner, W

    1997-01-01

    The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription. PMID:9153302

  7. Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44

    SciTech Connect

    Silva, Laurie A.; Strang, Blair L.; Lin, Eric W.; Kamil, Jeremy P.; Coen, Donald M.

    2011-09-01

    The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis.

  8. Structure of the gene for the catalytic subunit of human DNA polymerase {delta} (POLD1)

    SciTech Connect

    Chang, Lon-Sheng; Zhao, Lingyun; Zhu, Longyun

    1995-08-10

    We have isolated genomic DNA clones covering the gene for human DNA polymerase {delta} catalytic subunit (POLD1) and its 5{prime} flanking sequence. This gene is divided into 27 exons and is distributed over at least 32 kb of DNA. The exons and most of the introns are relatively small. The sizes of the exons range from 55 to 201 bp. Seven introns are smaller than 100 bp. Intron 1 is the largest intron, with a size of greater than 10 kb. All of the intron-exon junctions match well with the reported consensus sequence and the variable number of tandem repeats were found in several introns. Transcription of POLD1 appears to initiate at multiple sites. The major start site was 53 nucleotides upstream of the ATG start codon. The sequence of the promoter and upstream DNA is G+C rich and does not contain a TATA sequence. Several potential transcription factor-binding sites, including the AP2-, CTF-, Ets1-, GCF, MBF-1, NF-E1, and Sp1-binding sites, were found in this region. A 1.8-kb pol {delta} promoter DNA directed the expression of a luciferase reporter gene when transfected into HeLa cells. 69 refs., 4 figs., 3 tabs.

  9. Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

    PubMed Central

    Dias, João D; Rito, Tiago; Torlai Triglia, Elena; Kukalev, Alexander; Ferrai, Carmelo; Chotalia, Mita; Brookes, Emily; Kimura, Hiroshi; Pombo, Ana

    2015-01-01

    Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels. DOI: http://dx.doi.org/10.7554/eLife.11215.001 PMID:26687004

  10. Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation.

    PubMed

    Shu, Bo; Gong, Peng

    2016-07-12

    Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template-product duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases. PMID:27339134

  11. Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B

    PubMed Central

    Powdrill, Megan H.; Bernatchez, Jean A.; Götte, Matthias

    2010-01-01

    More than 20 years after the identification of the hepatitis C virus (HCV) as a novel human pathogen, the only approved treatment remains a combination of pegylated interferon-α and ribavirin. This rather non-specific therapy is associated with severe side effects and by far not everyone benefits from treatment. Recently, progress has been made in the development of specifically targeted antiviral therapy for HCV (STAT-C). A major target for such direct acting antivirals (DAAs) is the HCV RNA-dependent RNA polymerase or non-structural protein 5B (NS5B), which is essential for viral replication. This review will examine the current state of development of inhibitors targeting the polymerase and issues such as the emergence of antiviral resistance during treatment, as well as strategies to address this problem. PMID:21994615

  12. Specific threonine-4 phosphorylation and function of RNA polymerase II CTD during M phase progression

    PubMed Central

    Hintermair, Corinna; Voß, Kirsten; Forné, Ignasi; Heidemann, Martin; Flatley, Andrew; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2016-01-01

    Dynamic phosphorylation of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 heptad-repeats in the C-terminal domain (CTD) of the large subunit coordinates progression of RNA polymerase (Pol) II through the transcription cycle. Here, we describe an M phase-specific form of Pol II phosphorylated at Thr4, but not at Tyr1, Ser2, Ser5, and Ser7 residues. Thr4 phosphorylated Pol II binds to centrosomes and midbody and interacts with the Thr4-specific Polo-like kinase 1. Binding of Pol II to centrosomes does not require the CTD but may involve subunits of the non-canonical R2TP-Prefoldin-like complex, which bind to and co-localize with Pol II at centrosomes. CTD Thr4 mutants, but not Ser2 and Ser5 mutants, display severe mitosis and cytokinesis defects characterized by multipolar spindles and polyploid cells. We conclude that proper M phase progression of cells requires binding of Pol II to centrosomes to facilitate regulation of mitosis and cytokinesis in a CTD Thr4-P dependent manner. PMID:27264542

  13. X-ray crystal structure of Escherichia coli RNA polymerase σ70 holoenzyme.

    PubMed

    Murakami, Katsuhiko S

    2013-03-29

    Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ(70) holoenzyme, which shows σ region 1.1 (σ1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.

  14. Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase

    PubMed Central

    James, Tamara D.; Cardozo, Timothy; Abell, Lauren E.; Hsieh, Meng-Lun; Jenkins, Lisa M. Miller; Jha, Saheli S.; Hinton, Deborah M.

    2016-01-01

    The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ70 subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ70 Region 4, the N-terminal domain of MotA [MotANTD], and the C-terminal domain of MotA [MotACTD]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the β subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation. PMID:27458207

  15. Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase.

    PubMed

    James, Tamara D; Cardozo, Timothy; Abell, Lauren E; Hsieh, Meng-Lun; Jenkins, Lisa M Miller; Jha, Saheli S; Hinton, Deborah M

    2016-09-19

    The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ(70) subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ(70) Region 4, the N-terminal domain of MotA [MotA(NTD)], and the C-terminal domain of MotA [MotA(CTD)]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the β subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation. PMID:27458207

  16. Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene.

    PubMed

    Longet, David; Pawlowski, Jan

    2007-08-01

    Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.

  17. The genome of AR9, a giant transducing Bacillus phage encoding two multisubunit RNA polymerases.

    PubMed

    Lavysh, Daria; Sokolova, Maria; Minakhin, Leonid; Yakunina, Maria; Artamonova, Tatjana; Kozyavkin, Sergei; Makarova, Kira S; Koonin, Eugene V; Severinov, Konstantin

    2016-08-01

    Bacteriophage AR9 and its close relative PBS1 have been extensively used to construct early Bacillus subtilis genetic maps. Here, we present the 251,042bp AR9 genome, a linear, terminally redundant double-stranded DNA containing deoxyuridine instead of thymine. Multiple AR9 genes are interrupted by non-coding sequences or sequences encoding putative endonucleases. We show that these sequences are group I and group II self-splicing introns. Eight AR9 proteins are homologous to fragments of bacterial RNA polymerase (RNAP) subunits β/β'. These proteins comprise two sets of paralogs of RNAP largest subunits, with each paralog encoded by two disjoint phage genes. Thus, AR9 is a phiKZ-related giant phage that relies on two multisubunit viral RNAPs to transcribe its genome independently of host transcription apparatus. Purification of one of PBS1/AR9 RNAPs has been reported previously, which makes AR9 a promising object for further studies of RNAP evolution, assembly and mechanism. PMID:27236306

  18. Initiation of (-)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides.

    PubMed

    Sun, J H; Adkins, S; Faurote, G; Kao, C C

    1996-12-01

    RNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (-)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (-)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (-)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (-)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRp in vitro. Addition of heparin or Mn2+ differentially affected production of the 8-mer and full-length (-)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (-)-strand RNA synthesis.

  19. RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms

    PubMed Central

    Ferrer-Orta, Cristina; Ferrero, Diego; Verdaguer, Núria

    2015-01-01

    RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies. PMID:26258787

  20. RNA Polymerase II cluster dynamics predict mRNA output in living cells

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

    2016-01-01

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

  1. Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

    PubMed Central

    Rodriguez, P L; Carrasco, L

    1992-01-01

    The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme. Images PMID:1372367

  2. Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

    PubMed

    Markov, Dmitriy A; Wojtas, Ireneusz D; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T

    2014-07-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  3. Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

    PubMed

    Palmer, Adam C; Egan, J Barry; Shearwin, Keith E

    2011-01-01

    Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

  4. Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

    PubMed Central

    Crerar, M M; Leather, R; David, E; Pearson, M L

    1983-01-01

    To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation. Images PMID:6865946

  5. MYC Regulation of Cell Growth through Control of Transcription by RNA Polymerases I and III

    PubMed Central

    Campbell, Kirsteen J.; White, Robert J.

    2014-01-01

    MYC’s tumorigenic potential involves increased ribosome biogenesis and translational capacity, which supply the cell with protein required for enhanced cell growth and subsequent cell division. In addition to activation of protein-encoding genes transcribed by RNA polymerase II, MYC must stimulate transcription by RNA polymerase I and RNA polymerase III to meet this synthetic demand. In the past decade our knowledge of the mechanisms and importance of MYC regulation of RNA polymerases I and III has flourished. Here we discuss MYC’s influence on transcription by these “odd” RNA polymerases and the physiological impact of this regulation is evaluated with relevance to cancer development and treatment. PMID:24789877

  6. Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

    PubMed

    Jishage, M; Ishihama, A

    1999-06-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

  7. Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation.

    PubMed

    Lefèvre, Stéphane; Dumay-Odelot, Hélène; El-Ayoubi, Leyla; Budd, Aidan; Legrand, Pierre; Pinaud, Noël; Teichmann, Martin; Fribourg, Sébastien

    2011-03-01

    The 17-subunit human RNA polymerase III (hPol III) transcribes small, untranslated RNA genes that are involved in the regulation of transcription, splicing and translation. hPol III subunits hRPC62, hRPC39 and hRPC32 form a stable ternary subcomplex required for promoter-specific transcription initiation by hPol III. Here, we report the crystal structure of hRPC62. This subunit folds as a four-tandem extended winged helix (eWH) protein that is structurally related to the transcription factor TFIIEα N terminus. Through biochemical analyses, we mapped the protein-protein interactions of hRPC62, hRPC32 and hRPC39. In addition, we demonstrated that hRPC62 and hRPC39 bind single-stranded and duplex DNA, respectively, in a sequence-independent manner. Overall, we shed light on structural similarities between the hPol III-specific subunit hRPC62 and TFIIEα and propose specific functions for hRPC39 and hRPC62 in transcription initiation by hPol III. PMID:21358628

  8. Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

    PubMed Central

    Brown, David D.; Rima, Bertus K.; Allen, Ingrid V.; Baron, Michael D.; Banyard, Ashley C.; Barrett, Thomas; Duprex, W. Paul

    2005-01-01

    Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPVKO) can be rescued. A recombinant virus, rRPVKOL-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPVKO in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 104 TCID50 of the rRPVKO parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPVKOL-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo. PMID:16254367

  9. Rational attenuation of a morbillivirus by modulating the activity of the RNA-dependent RNA polymerase.

    PubMed

    Brown, David D; Rima, Bertus K; Allen, Ingrid V; Baron, Michael D; Banyard, Ashley C; Barrett, Thomas; Duprex, W Paul

    2005-11-01

    Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

  10. Molecular identification of nanoplanktonic protists based on small subunit ribosomal RNA gene sequences for ecological studies.

    PubMed

    Lim, E L

    1996-01-01

    Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.

  11. Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

    PubMed Central

    Archambault, J; Lacroute, F; Ruet, A; Friesen, J D

    1992-01-01

    Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII. Images PMID:1508210

  12. Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

    PubMed

    Patton, J T; Jones, M T; Kalbach, A N; He, Y W; Xiaobo, J

    1997-12-01

    Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

  13. RNA polymerase supply and flux through the lac operon in Escherichia coli.

    PubMed

    Sendy, Bandar; Lee, David J; Busby, Stephen J W; Bryant, Jack A

    2016-11-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'. PMID:27672157

  14. RNA polymerase supply and flux through the lac operon in Escherichia coli

    PubMed Central

    Sendy, Bandar; Lee, David J.; Bryant, Jack A.

    2016-01-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli. By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase. This article is part of the themed issue ‘The new bacteriology’. PMID:27672157

  15. RNA polymerase supply and flux through the lac operon in Escherichia coli.

    PubMed

    Sendy, Bandar; Lee, David J; Busby, Stephen J W; Bryant, Jack A

    2016-11-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'.

  16. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

    PubMed

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription. PMID:24406346

  17. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

    PubMed Central

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription. PMID:25764111

  18. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

    PubMed

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription. PMID:25764111

  19. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

    PubMed

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

  20. Genome-Wide Mapping of Yeast RNA Polymerase II Termination

    PubMed Central

    Schaughency, Paul; Merran, Jonathan; Corden, Jeffry L.

    2014-01-01

    Yeast RNA polymerase II (Pol II) terminates transcription of coding transcripts through the polyadenylation (pA) pathway and non-coding transcripts through the non-polyadenylation (non-pA) pathway. We have used PAR-CLIP to map the position of Pol II genome-wide in living yeast cells after depletion of components of either the pA or non-pA termination complexes. We show here that Ysh1, responsible for cleavage at the pA site, is required for efficient removal of Pol II from the template. Depletion of Ysh1 from the nucleus does not, however, lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators, but in contrast to Nrd1, this readthrough is less processive, or more susceptible to pausing. The data presented here provide delineation of in vivo Pol II termination regions and highlight differences in the sequences that signal termination of different classes of non-pA transcripts. PMID:25299594

  1. Bacterial control of host gene expression through RNA polymerase II

    PubMed Central

    Lutay, Nataliya; Ambite, Ines; Hernandez, Jenny Grönberg; Rydström, Gustav; Ragnarsdóttir, Bryndís; Puthia, Manoj; Nadeem, Aftab; Zhang, Jingyao; Storm, Petter; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2013-01-01

    The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II–dependent (Pol II–dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II–dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur. PMID:23728172

  2. VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase.

    PubMed

    von Einem, Ursula I; Gorbalenya, Alexander E; Schirrmeier, Horst; Behrens, Sven-Erik; Letzel, Tobias; Mundt, Egbert

    2004-08-01

    Segment B of the bisegmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) encodes the viral protein VP1. This has been presumed to represent the RNA-dependent RNA polymerase (RdRp) as it contains motifs that are typical for the RdRp of plus-strand RNA viruses. Here it is demonstrated that baculovirus-expressed wild-type but not motif A mutated VP1 acts as an RdRp on IBDV-specific RNA templates. Thus, on a plus-strand IBDV segment A cRNA template, minus-strand synthesis occurred in such a way that a covalently linked double-stranded RNA product was generated (by a 'copy-back' mechanism). Importantly, enzyme activity was observed only with templates that comprised the 3' non-coding region of plus-strand RNAs transcribed from IBDV segments A and B, indicating template specificity. RdRp activity was shown to have a temperature optimum of 37 degrees C and required magnesium ions for enzyme activity. Thus, it has been demonstrated unequivocally that VP1 represents the RdRp of IBDV.

  3. Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II.

    PubMed

    Carrillo Oesterreich, Fernando; Herzel, Lydia; Straube, Korinna; Hujer, Katja; Howard, Jonathon; Neugebauer, Karla M

    2016-04-01

    Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) and introns are removed from pre-mRNA by the spliceosome. Understanding the time lag between Pol II progression and splicing could provide mechanistic insights into the regulation of gene expression. Here, we present two single-molecule nascent RNA sequencing methods that directly determine the progress of splicing catalysis as a function of Pol II position. Endogenous genes were analyzed on a global scale in budding yeast. We show that splicing is 50% complete when Pol II is only 45 nt downstream of introns, with the first spliced products observed as introns emerge from Pol II. Perturbations that slow the rate of spliceosome assembly or speed up the rate of transcription caused splicing delays, showing that regulation of both processes determines in vivo splicing profiles. We propose that matched rates streamline the gene expression pathway, while allowing regulation through kinetic competition.

  4. Nuclear Distribution of RNA Polymerase II and mRNA Processing Machinery in Early Mammalian Embryos

    PubMed Central

    Bogolyubova, Irina O.; Bogolyubov, Dmitry S.

    2014-01-01

    Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of gene expression. While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughly traced in mammalian somatic cells, similar data for the preimplantation embryos are scanty and fragmental. However, the period of cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation. In this minireview, we try to summarize the results of studies concerning distribution of major factors involved in RNA polymerase II-dependent transcription, pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals. PMID:24868542

  5. RNA polymerase pausing regulates translation initiation by providing additional time for TRAP-RNA interaction.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2006-11-17

    RNA polymerase (RNAP) pause sites have been identified in several prokaryotic genes. Although the presumed biological function of RNAP pausing is to allow synchronization of RNAP position with regulatory factor binding and/or RNA folding, a direct causal link between pausing and changes in gene expression has been difficult to establish. RNAP pauses at two sites in the Bacillus subtilis trpEDCFBA operon leader. Pausing at U107 and U144 participates in transcription attenuation and trpE translation control mechanisms, respectively. Substitution of U144 caused a substantial pausing defect in vitro and in vivo. These mutations led to increased trp operon expression that was suppressed by overproduction of TRAP, indicating that pausing at U144 provides additional time for TRAP to bind to the nascent transcript and promote formation of an RNA structure that blocks translation of trpE. These results establish that pausing is capable of playing a role in regulating translation in bacteria. PMID:17114058

  6. Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit.

    PubMed

    McCauley, Micah J; Shokri, Leila; Sefcikova, Jana; Venclovas, Ceslovas; Beuning, Penny J; Williams, Mark C

    2008-09-19

    The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding. PMID:18652472

  7. The Structure of Fcp1, an Essential RNA Polymerase II CTD Phosphatase

    SciTech Connect

    Ghosh, A.; Shuman, S.; Lima, C.D.

    2009-03-27

    Kinases and phosphatases regulate mRNA synthesis and processing by phosphorylating and dephosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Fcp1 is an essential CTD phosphatase that preferentially hydrolyzes Ser2-PO{sub 4} of the tandem YSPTSPS CTD heptad array. Fcp1 crystal structures were captured at two stages of the reaction pathway: a Mg-BeF{sub 3} complex that mimics the aspartylphosphate intermediate and a Mg-AlF{sub 4}{sup -} complex that mimics the transition state of the hydrolysis step. Fcp1 is a Y-shaped protein composed of an acylphosphatase domain located at the base of a deep canyon formed by flanking modules that are missing from the small CTD phosphatase (SCP) clade: an Fcp1-specific helical domain and a C-terminal BRCA1 C-terminal (BRCT) domain. The structure and mutational analysis reveals that Fcp1 and Scp1 (a Ser5-selective phosphatase) adopt different CTD-binding modes; we surmise the CTD threads through the Fcp1 canyon to access the active site.

  8. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

    PubMed

    Jácome, Rodrigo; Becerra, Arturo; Ponce de León, Samuel; Lazcano, Antonio

    2015-01-01

    The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  9. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications

    PubMed Central

    Jácome, Rodrigo; Becerra, Arturo; Ponce de León, Samuel; Lazcano, Antonio

    2015-01-01

    The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, “fingertips” that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection. PMID:26397100

  10. A Caenorhabditis Elegans RNA Polymerase II Gene, Ama-1 Iv, and Nearby Essential Genes

    PubMed Central

    Rogalski, T. M.; Riddle, D. L.

    1988-01-01

    The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20° but are arrested as larvae at 25°, and two others are fertile at 20° and sterile at 25°. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25° is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four γ-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development. PMID:8608933

  11. Identification of host factors that regulate the influenza virus RNA polymerase activity.

    PubMed

    Momose, F; Handa, H; Nagata, K

    1996-01-01

    Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.

  12. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    PubMed

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.

  13. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    PubMed

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups. PMID:26485449

  14. RNA-directed DNA polymerase from particles released by normal goose cells.

    PubMed Central

    Bauer, G; Temin, H M

    1979-01-01

    Cells from a goose embryo were shown to release particle-associated RNA-directed DNA polymerase and RNase H activities that required the presence of Nonidet P-40 for detection. The particles were not infectious and did not have endogenous DNA synthesis. The goose particle DNA polymerase was related to the DNA polymerase of spleen necrosis virus with respect to size and was inhibited by immunoglobulin G to spleen necrosis virus DNA polymerase. However, goose cells producing DNA polymerase-containing particles did not contain reticuloendotheliosis virus-related nucleotide sequences in their DNA. PMID:87517

  15. Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification.

    PubMed

    Banerjee, Rajdeep; Rudra, Paulami; Prajapati, Ranjit Kumar; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2014-07-01

    Mycobacterium tuberculosis, the human pathogen that causes tuberculosis, warrants enormous attention due to the emergence of multi drug resistant and extremely drug resistant strains. RNA polymerase (RNAP), the key enzyme in gene regulation, is an attractive target for anti-TB drugs. Understanding the structure-function relationship of M. tuberculosis RNAP and the mechanism of gene regulation by RNAP in conjunction with different σ factors and transcriptional regulators would provide significant information for anti-tuberculosis drug development targeting RNAP. Studies with M. tuberculosis RNAP remain tedious because of the extremely slow-growing nature of the bacteria and requirement of special laboratory facility. Here, we have developed and optimized recombinant methods to prepare M. tuberculosis RNAP core and RNAP holo enzymes assembled in vivo in Escherichia coli. These methods yield high amounts of transcriptionally active enzymes, free of E. coli RNAP contamination. The recombinant M. tuberculosis RNAP is used to develop a highly sensitive fluorescence based in vitro transcription assay that could be easily adopted in a high-throughput format to screen RNAP inhibitors. These recombinant methods would be useful to set a platform for M. tuberculosis RNAP targeted anti TB drug development, to analyse the structure/function of M. tuberculosis RNAP and to analyse the interactions among promoter DNA, RNAP, σ factors, and transcription regulators of M. tuberculosis in vitro, avoiding the hazard of handling of pathogenic bacteria.

  16. Structure-based inhibition of Norovirus RNA-dependent RNA polymerases.

    PubMed

    Mastrangelo, Eloise; Pezzullo, Margherita; Tarantino, Delia; Petazzi, Roberto; Germani, Francesca; Kramer, Dorothea; Robel, Ivonne; Rohayem, Jacques; Bolognesi, Martino; Milani, Mario

    2012-06-01

    Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

  17. Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor λQ

    PubMed Central

    Vorobiev, Sergey M.; Gensler, Yocheved; Vahedian-Movahed, Hanif; Seetharaman, Jayaraman; Su, Min; Huang, Janet Y.; Xiao, Rong; Kornhaber, Gregory; Montelione, Gaetano T.; Tong, Liang; Ebright, Richard H.; Nickels, Bryce E.

    2014-01-01

    SUMMARY The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ’s interaction with the paused early elongation complex involves interactions between λQ and two regions of RNA polymerase: region 4 of the σ70 subunit and the flap domain of the β subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ70, and interaction with the β flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex. PMID:24440517

  18. Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA.

    PubMed

    Gvozdeva, Olga V; Prassolov, Vladimir S; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-08-01

    Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2'-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.

  19. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  20. Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays

    PubMed Central

    Shakhmin, Anton; Jones, John-Paul; Bychinskaya, Inessa; Zibinsky, Mikhail; Oertell, Keriann; Goodman, Myron F.; Prakash, G.K. Surya

    2015-01-01

    Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA polymerase β. PMID:26279588

  1. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    PubMed

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

  2. RNA initiation with dinucleoside monophosphates during transcription of bacteriophage T4 DNA with RNA polymerase of Escherichia coli.

    PubMed

    Hoffman, D J; Niyogi, S K

    1973-02-01

    The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5'-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 "early" type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of (3)H-labeled stimulatory dinucleoside monophosphates support the above conclusions.

  3. The RNA polymerase II of an alpha-amanitin-resistant Chinese hamster ovary cell line.

    PubMed

    Lobban, P E; Siminovitch, L; Ingles, C J

    1976-05-01

    Amal, an alpha-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to alpha-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to alpha-amanitin and more thermolabile than the wild-type enzyme. PMID:954093

  4. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  5. RNA as an RNA Polymerase: Net Elongation of an RNA Primer Catalyzed by the Tetrahymena Ribozyme

    NASA Astrophysics Data System (ADS)

    Been, Michael D.; Cech, Thomas R.

    1988-03-01

    A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3', 5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.

  6. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    PubMed Central

    Galiano, Vicente; Garcia-Valtanen, Pablo; Micol, Vicente; Encinar, José Antonio

    2016-01-01

    The dengue virus (DENV) nonstructural protein 5 (NS5) contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds) to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <−10.5 kcal/mol) were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. PMID:27784988

  7. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases.

    PubMed

    Bruenn, J A

    1991-01-25

    The sequences of 50 RNA-dependent RNA polymerases (RDRPs) from 43 positive strand and 7 double strand RNA (dsRNA) viruses have been compared. The alignment permitted calculation of distances among the 50 viruses and a resultant dendrogram based on every amino acid, rather than just those amino acids in the conserved motifs. Remarkably, a large subgroup of these viruses, including vertebrate, plant, and insect viruses, forms a single cluster whose only common characteristic is exploitation of insect hosts or vectors. This similarity may be due to molecular constraints associated with a present and/or past ability to infect insects and/or to common descent from insect viruses. If common descent is important, as it appears to be, all the positive strand RNA viruses of eucaryotes except for the picornaviruses may have evolved from an ancestral dsRNA virus. Viral RDRPs appear to be inherited as modules rather than as portions of single RNA segments, implying that RNA recombination has played an important role in their dissemination.

  8. Identification of distinct biological functions for four 3'-5' RNA polymerases.

    PubMed

    Long, Yicheng; Abad, Maria G; Olson, Erik D; Carrillo, Elisabeth Y; Jackman, Jane E

    2016-09-30

    The superfamily of 3'-5' polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNA(His) guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNA(His) maturation reaction, which is distinct from the tRNA(His) maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5'-editing in vivo and in vitro, establishing template-dependent 3'-5' polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3'-5' polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3'-5' polymerases in eukaryotes.

  9. Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes

    PubMed Central

    Ernens, Isabelle; Goodfellow, Sarah J.; Innes, Fiona; Kenneth, Niall S.; Derblay, Louise E.; White, Robert J.; Scott, Pamela H.

    2006-01-01

    RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes. PMID:16407335

  10. Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress.

    PubMed

    Peltonen, Karita; Colis, Laureen; Liu, Hester; Jäämaa, Sari; Zhang, Zhewei; Af Hällström, Taija; Moore, Henna M; Sirajuddin, Paul; Laiho, Marikki

    2014-11-01

    Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9- and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting.

  11. Small Molecule BMH-compounds that Inhibit RNA Polymerase I and Cause Nucleolar Stress

    PubMed Central

    Peltonen, Karita; Colis, Laureen; Liu, Hester; Jäämaa, Sari; Zhang, Zhewei; Hällström, Taija af; Moore, Henna M.; Sirajuddin, Paul; Laiho, Marikki

    2014-01-01

    Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53 activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194 and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22 and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9 and BMH-22 –mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogs showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22 and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally we show that the Pol I transcription stress by BMH-9, BMH-22 and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and potentially, cancer targeting. PMID:25277384

  12. Genomewide Recruitment Analysis of Rpb4, a Subunit of Polymerase II in Saccharomyces cerevisiae, Reveals Its Involvement in Transcription Elongation▿ †

    PubMed Central

    Verma-Gaur, Jiyoti; Rao, Sudha Narayana; Taya, Toshiki; Sadhale, Parag

    2008-01-01

    The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in Saccharomyces cerevisiae is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in S. cerevisiae and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation (“ChIP on-chip”) technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the rpb4Δ mutant. Unlike most phenotypes of rpb4Δ, the 6AU sensitivity of the rpb4Δ strain was not rescued by overexpression of RPB7. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7. PMID:18441121

  13. Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World

    PubMed Central

    Siegel, Robert W.; Bellon, Laurent; Beigelman, Leonid; Kao, C. Cheng

    1999-01-01

    All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes. PMID:10400735

  14. Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Joyce, Gerald F.

    2001-01-01

    A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the non-template and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and P-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that employ T7 RNA polymerase for in vitro transcription.

  15. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.

    PubMed Central

    Wuarin, J; Schibler, U

    1994-01-01

    In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates. Images PMID:7523861

  16. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    PubMed

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  17. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    PubMed

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

  18. Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

    PubMed Central

    Sesmero, Ester; Thorpe, Ian F.

    2015-01-01

    Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process. PMID:26193306

  19. RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus.

    PubMed Central

    Dion, A S; Williams, C J; Moore, D H

    1977-01-01

    The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses. Images PMID:67221

  20. UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

    PubMed Central

    Bregman, D B; Halaban, R; van Gool, A J; Henning, K A; Friedberg, E C; Warren, S L

    1996-01-01

    Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876179

  1. UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

    PubMed

    Bregman, D B; Halaban, R; van Gool, A J; Henning, K A; Friedberg, E C; Warren, S L

    1996-10-15

    Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription. PMID:8876179

  2. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain.

    PubMed

    Jeronimo, Célia; Collin, Pierre; Robert, François

    2016-06-19

    The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this "Perspective," we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed. PMID:26876604

  3. Interaction of CarD with RNA polymerase mediates Mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis.

    PubMed

    Weiss, Leslie A; Harrison, Phillip G; Nickels, Bryce E; Glickman, Michael S; Campbell, Elizabeth A; Darst, Seth A; Stallings, Christina L

    2012-10-01

    Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

  4. Mutant cohesin affects RNA polymerase II regulation in Cornelia de Lange syndrome

    PubMed Central

    Mannini, Linda; C. Lamaze, Fabien; Cucco, Francesco; Amato, Clelia; Quarantotti, Valentina; Rizzo, Ilaria M; Krantz, Ian D; Bilodeau, Steve; Musio, Antonio

    2015-01-01

    In addition to its role in sister chromatid cohesion, genome stability and integrity, the cohesin complex is involved in gene transcription. Mutations in core cohesin subunits SMC1A, SMC3 and RAD21, or their regulators NIPBL and HDAC8, cause Cornelia de Lange syndrome (CdLS). Recent evidence reveals that gene expression dysregulation could be the underlying mechanism for CdLS. These findings raise intriguing questions regarding the potential role of cohesin-mediated transcriptional control and pathogenesis. Here, we identified numerous dysregulated genes occupied by cohesin by combining the transcriptome of CdLS cell lines carrying mutations in SMC1A gene and ChIP-Seq data. Genome-wide analyses show that genes changing in expression are enriched for cohesin-binding. In addition, our results indicate that mutant cohesin impairs both RNA polymerase II (Pol II) transcription initiation at promoters and elongation in the gene body. These findings highlight the pivotal role of cohesin in transcriptional regulation and provide an explanation for the typical gene dysregulation observed in CdLS patients. PMID:26581180

  5. Bases in 16S rRNA important for subunit association, tRNA binding, and translocation.

    PubMed

    Shi, Xinying; Chiu, Katie; Ghosh, Srikanta; Joseph, Simpson

    2009-07-28

    Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated so their role in translation could be studied. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding, and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association, and tRNA binding. In contrast, the A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far as 100 A from the tRNA binding sites can be important for translation.

  6. Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

    SciTech Connect

    Opalka, N.; Brown, J; Lane, W; Twist, K; Landick, R; Asturias, F; Darst, S

    2010-01-01

    The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program. Transcription, or the synthesis of RNA from DNA, is one of the most important processes in the cell. The central enzyme of transcription is the DNA-dependent RNA polymerase (RNAP), a large, macromolecular assembly consisting of at least five subunits. Historically, much of our fundamental information on the process of transcription has come from genetic and biochemical studies of RNAP from the model bacterium Escherichia coli. More recently, major breakthroughs in our understanding of the mechanism of action of RNAP have come from high resolution crystal structures of various bacterial, archaebacterial, and eukaryotic enzymes. However, all of our high-resolution bacterial RNAP structures are of enzymes from the thermophiles Thermus aquaticus or T. thermophilus, organisms with poorly characterized transcription systems. It has thus far proven impossible to obtain a high-resolution structure of E. coli RNAP, which has made it difficult to relate the large collection

  7. Role of DNA-Dependent RNA Polymerase III in the Transcription of the tRNA and 5S RNA Genes

    PubMed Central

    Weinmann, Roberto; Roeder, Robert G.

    1974-01-01

    Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated IIIA and IIIB). The enzymes are unaffected by low α-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for IIIA and IIIB) at higher toxin concentrations. RNA polymerase I was unaffected at all α-amanitin concentrations tested. Myeloma RNA polymerases II, IIIA, and IIIB appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic α-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases. Isolated myeloma nuclei and nucleoli contińue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by α-amanitin, but only at high concentrations; and the α-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases IIIA and IIIB. In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by α-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high α-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems. These results unequivocally demonstrate the role of RNA

  8. DIS3 shapes the RNA polymerase II transcriptome in humans by degrading a variety of unwanted transcripts.

    PubMed

    Szczepińska, Teresa; Kalisiak, Katarzyna; Tomecki, Rafal; Labno, Anna; Borowski, Lukasz S; Kulinski, Tomasz M; Adamska, Dorota; Kosinska, Joanna; Dziembowski, Andrzej

    2015-11-01

    Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome.

  9. DIS3 shapes the RNA polymerase II transcriptome in humans by degrading a variety of unwanted transcripts

    PubMed Central

    Szczepińska, Teresa; Kalisiak, Katarzyna; Tomecki, Rafal; Labno, Anna; Borowski, Lukasz S.; Kulinski, Tomasz M.; Adamska, Dorota; Kosinska, Joanna; Dziembowski, Andrzej

    2015-01-01

    Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome. PMID:26294688

  10. Picornavirus RNA polyadenylation by 3Dpol, the viral RNA-dependent RNA polymerase

    PubMed Central

    Kempf, Brian J; Barton, David J.

    2016-01-01

    Poly(A) tails are functionally important features of all picornavirus RNA genomes. Some viruses have genomes with relatively short poly(A) tails (encephalomyocarditis virus) whereas others have genomes with longer poly(A) tails (polioviruses and rhinoviruses). Here we review the polyadenylation of picornavirus RNA as it relates to the structure and function of 3Dpol. Poliovirus 3Dpol uses template-dependent reiterative transcription mechanisms as it replicates the poly(A) tails of viral RNA (Steil et al., 2010). These mechanisms are analogous to those involved in the polyadenylation of vesicular stomatitis virus and influenza virus mRNAs. 3Dpol residues intimately associated with viral RNA templates and products regulate the size of poly(A) tails in viral RNA (Kempf et al., 2013). Consistent with their ancient evolutionary origins, picornavirus 3Dpol and telomerase reverse transcriptase (TERT) share structural and functional features. Structurally, both 3Dpol and TERT assume a “right-hand” conformation with thumb, palm and fingers domains encircling templates and products. Functionally, both 3Dpol and TERT use template-dependent reiterative transcription mechanisms to synthesize repetitive sequences: poly(A) tails in the case of picornavirus RNA genomes and DNA telomeres in the case of eukaryotic chromosomes. Thus, picornaviruses and their eukaryotic hosts (humans & animals) maintain the 3’ ends of their respective genomes via evolutionarily related mechanisms. PMID:25559071

  11. The Whole Set of Constitutive Promoters Recognized by RNA Polymerase RpoD Holoenzyme of Escherichia coli

    PubMed Central

    Shimada, Tomohiro; Yamazaki, Yukiko; Tanaka, Kan; Ishihama, Akira

    2014-01-01

    The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature. PMID:24603758

  12. Genetic Transformation of Citrus Paradisi with Antisense and untranslatable RNA-dependent RNA Polymerase Genes of Citrus Tristeza Closterovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although 56 kDa CTV-RdRp is thought to be expressed by a +1 translational frameshift at the carboxyl te...

  13. Effect of DNA-interacting drugs on phage T7 RNA polymerase.

    PubMed

    Piestrzeniewicz, M; Studzian, K; Wilmańska, D; Płucienniczak, G; Gniazdowski, M

    1998-01-01

    9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands. PMID:9701505

  14. Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.

    PubMed

    Dmitrieva, Tatiana M; Alexeevski, Andrei V; Shatskaya, Galina S; Tolskaya, Elena A; Gmyl, Anatoly P; Khitrina, Elena V; Agol, Vadim I

    2007-08-15

    Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed. PMID:17467026

  15. Disruption of the 5' stem-loop of yeast U6 RNA induces trimethylguanosine capping of this RNA polymerase III transcript in vivo.

    PubMed Central

    Kwan, S; Gerlach, V L; Brow, D A

    2000-01-01

    Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Pol III, are modified by addition of a 7-methylguanosine (m7G) residue to the triphosphate 5' end shortly after it emerges from the polymerase. The m7G "caps" of small nuclear and small nucleolar RNAs, but not messenger RNAs, are subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) residue. U6 RNA, the only small nuclear RNA synthesized by Pol III in most eukaryotes, does not receive a methylguanosine cap. However, human U6 RNA is O-methylated on the 5'-terminal (gamma) phosphate by an enzyme that recognizes the 5' stem-loop of U6. Here we show that variant yeast U6 RNAs truncated or substituted within the 5' stem-loop are TMG capped in vivo. Accumulation of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming that it is indeed synthesized by Pol III. Thus, methylguanosine capping and cap hypermethylation are not exclusive to Pol II transcripts in yeast. We propose that TMG capping of variant U6 RNAs occurs posttranscriptionally due to exposure of the 5' triphosphate by disruption of protein binding and/or gamma-methyl phosphate capping. 5' truncation and TMG capping of U6 RNA does not appear to affect its normal function in splicing, suggesting that assembly and action of the spliceosome is not very sensitive to the 5' end structure of U6 RNA. PMID:11142384

  16. Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin

    PubMed Central

    Böhmdorfer, Gudrun; Sethuraman, Shriya; Rowley, M Jordan; Krzyszton, Michal; Rothi, M Hafiz; Bouzit, Lilia; Wierzbicki, Andrzej T

    2016-01-01

    RNA-mediated transcriptional gene silencing is a conserved process where small RNAs target transposons and other sequences for repression by establishing chromatin modifications. A central element of this process are long non-coding RNAs (lncRNA), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. Here we show that non-coding transcription by Pol V is controlled by preexisting chromatin modifications located within the transcribed regions. Most Pol V transcripts are associated with AGO4 but are not sliced by AGO4. Pol V-dependent DNA methylation is established on both strands of DNA and is tightly restricted to Pol V-transcribed regions. This indicates that chromatin modifications are established in close proximity to Pol V. Finally, Pol V transcription is preferentially enriched on edges of silenced transposable elements, where Pol V transcribes into TEs. We propose that Pol V may play an important role in the determination of heterochromatin boundaries. DOI: http://dx.doi.org/10.7554/eLife.19092.001 PMID:27779094

  17. Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds.

    PubMed

    Croci, Romina; Pezzullo, Margherita; Tarantino, Delia; Milani, Mario; Tsay, Shwu-Chen; Sureshbabu, Radhakrishnan; Tsai, Yi-Jin; Mastrangelo, Eloise; Rohayem, Jacques; Bolognesi, Martino; Hwu, Jih Ru

    2014-01-01

    Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3-7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

  18. Structural Bases of Norovirus RNA Dependent RNA Polymerase Inhibition by Novel Suramin-Related Compounds

    PubMed Central

    Tarantino, Delia; Milani, Mario; Tsay, Shwu-Chen; Sureshbabu, Radhakrishnan; Tsai, Yi-Jin; Mastrangelo, Eloise; Rohayem, Jacques; Bolognesi, Martino; Hwu, Jih Ru

    2014-01-01

    Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3–7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability. PMID:24622391

  19. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    PubMed

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  20. The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases.

    PubMed

    Ferrero, Diego S; Buxaderas, Mònica; Rodríguez, José F; Verdaguer, Núria

    2015-12-01

    Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5'-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups. PMID:26625123

  1. Nonnucleoside Inhibitor of Measles Virus RNA-Dependent RNA Polymerase Complex Activity▿ †

    PubMed Central

    White, Laura K.; Yoon, Jeong-Joong; Lee, Jin K.; Sun, Aiming; Du, Yuhong; Fu, Haian; Snyder, James P.; Plemper, Richard K.

    2007-01-01

    Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex. PMID:17470652

  2. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

    PubMed Central

    Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

    2014-01-01

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

  3. Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase.

    PubMed

    Lee, M T Michael; Bishop, Konrad; Medcalf, Liz; Elton, Debra; Digard, Paul; Tiley, Laurence

    2002-01-15

    The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

  4. Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases*

    PubMed Central

    Selvakumar, Tharakeswari; Gjidoda, Alison; Hovde, Stacy L.; Henry, R. William

    2012-01-01

    The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks. PMID:22219193

  5. Structural Characterization of the Catalytic Subunit of a Novel RNA Splicing Endonuclease

    SciTech Connect

    Calvin, Kate; Hall, Michelle D.; Xu, Fangmin; Xue, Song; Li, Hong

    2010-07-13

    The RNA splicing endonuclease is responsible for recognition and excision of nuclear tRNA and all archaeal introns. Despite the conserved RNA cleavage chemistry and a similar enzyme assembly, currently known splicing endonuclease families have limited RNA specificity. Different from previously characterized splicing endonucleases in Archaea, the splicing endonuclease from archaeum Sulfolobus solfataricus was found to contain two different subunits and accept a broader range of substrates. Here, we report a crystal structure of the catalytic subunit of the S. solfataricus endonuclease at 3.1 {angstrom} resolution. The structure, together with analytical ultracentrifugation analysis, identifies the catalytic subunit as an inactive but stable homodimer, thus suggesting the possibility of two modes of functional assembly for the active enzyme.

  6. Role of DNA-Dependent RNA Polymerases II and III in Transcription of the Adenovirus Genome Late in Productive Infection

    PubMed Central

    Weinmann, Roberto; Raskas, Heschel J.; Roeder, Robert G.

    1974-01-01

    DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of α-amanitin (50% inhibition at 0.02 μg/ml), RNA polymerase III was completely sensitive to high concentrations of α-amanitin (50% inhibition at 20 μg/ml) and RNA polymerase I was totally resistant to concentrations of α-amanitin less than or equal to 200 μg/ml. RNA synthesis by the endogenous RNA polymerase activities in nuclei isolated from infected cells was completely sensitive to α-amanitin, thus suggesting that RNA polymerase I is not involved in viral DNA transcription even though it is present in these cells. The α-amanitin inhibition curve was biphasic and showed inflection points at about 0.02 and 20 μg/ml, suggesting the participation of both RNA polymerases II and III in the synthesis of RNA in these nuclei. Furthermore, at least a large fraction of the synthesis of the nuclear precursors to viral mRNA, monitored by hybridization to viral DNA, showed the same sensitivity to α-amanitin as did RNA polymerase II; and the synthesis of both viral 5.5S RNA and (presumably cellular) 5S RNA in the isolated nuclei exhibited the same sensitivity to α-amanitin as did purified RNA polymerase III. Thus, these data provide strong supporting evidence for previous studies which suggested the involvement of an RNA polymerase II in transcription of the adenovirus genome and demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA. PMID:4530313

  7. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

    PubMed

    Arimbasseri, Aneeshkumar G; Blewett, Nathan H; Iben, James R; Lamichhane, Tek N; Cherkasova, Vera; Hafner, Markus; Maraia, Richard J

    2015-12-01

    Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells. PMID:26720005

  8. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification

    PubMed Central

    Arimbasseri, Aneeshkumar G.; Blewett, Nathan H.; Iben, James R.; Lamichhane, Tek N.; Cherkasova, Vera; Hafner, Markus; Maraia, Richard J.

    2015-01-01

    Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m2 2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m2 2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m2 2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m2 2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m2 2G26 modification and that this response is conserved among highly divergent yeasts and human cells. PMID:26720005

  9. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

    SciTech Connect

    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.

    2009-12-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

  10. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    PubMed

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

  11. Identification of distinct biological functions for four 3′-5′ RNA polymerases

    PubMed Central

    Long, Yicheng; Abad, Maria G.; Olson, Erik D.; Carrillo, Elisabeth Y.; Jackman, Jane E.

    2016-01-01

    The superfamily of 3′-5′ polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNAHis guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNAHis maturation reaction, which is distinct from the tRNAHis maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5′-editing in vivo and in vitro, establishing template-dependent 3′-5′ polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3′-5′ polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3′-5′ polymerases in eukaryotes. PMID:27484477

  12. Primer-Dependent and Primer-Independent Initiation of Double Stranded RNA Synthesis by Purified Arabidopsis RNA-Dependent RNA Polymerases RDR2 and RDR6

    PubMed Central

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  13. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.

    PubMed

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  14. Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

    PubMed

    Byrn, Randal A; Jones, Steven M; Bennett, Hamilton B; Bral, Chris; Clark, Michael P; Jacobs, Marc D; Kwong, Ann D; Ledeboer, Mark W; Leeman, Joshua R; McNeil, Colleen F; Murcko, Mark A; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W; Zhou, Yi; Charifson, Paul S

    2015-03-01

    VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

  15. In Vitro Synthesis of Rous Sarcoma Virus-Specific RNA is Catalyzed by a DNA-Dependent RNA Polymerase

    PubMed Central

    Rymo, L.; Parsons, J. T.; Coffin, J. M.; Weissmann, C.

    1974-01-01

    Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [α-32P]triphosphates, Mn++, Mg++ and (NH4)2SO4. Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37°. About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA</