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Sample records for rna polymerase subunit

  1. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  2. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits.

    PubMed

    Carey, Lucas B

    2015-12-10

    Errors during transcription may play an important role in determining cellular phenotypes: the RNA polymerase error rate is >4 orders of magnitude higher than that of DNA polymerase and errors are amplified >1000-fold due to translation. However, current methods to measure RNA polymerase fidelity are low-throughout, technically challenging, and organism specific. Here I show that changes in RNA polymerase fidelity can be measured using standard RNA sequencing protocols. I find that RNA polymerase is error-prone, and these errors can result in splicing defects. Furthermore, I find that differential expression of RNA polymerase subunits causes changes in RNA polymerase fidelity, and that coding sequences may have evolved to minimize the effect of these errors. These results suggest that errors caused by RNA polymerase may be a major source of stochastic variability at the level of single cells.

  3. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits

    PubMed Central

    Carey, Lucas B

    2015-01-01

    Errors during transcription may play an important role in determining cellular phenotypes: the RNA polymerase error rate is >4 orders of magnitude higher than that of DNA polymerase and errors are amplified >1000-fold due to translation. However, current methods to measure RNA polymerase fidelity are low-throughout, technically challenging, and organism specific. Here I show that changes in RNA polymerase fidelity can be measured using standard RNA sequencing protocols. I find that RNA polymerase is error-prone, and these errors can result in splicing defects. Furthermore, I find that differential expression of RNA polymerase subunits causes changes in RNA polymerase fidelity, and that coding sequences may have evolved to minimize the effect of these errors. These results suggest that errors caused by RNA polymerase may be a major source of stochastic variability at the level of single cells. DOI: http://dx.doi.org/10.7554/eLife.09945.001 PMID:26652005

  4. 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

  5. On the evolution of the single-subunit RNA polymerases.

    PubMed

    Cermakian, N; Ikeda, T M; Miramontes, P; Lang, B F; Gray, M W; Cedergren, R

    1997-12-01

    Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

  6. A genetic analysis of Plasmodium falciparum RNA polymerase II subunits in yeast.

    PubMed

    Hazoume, Adonis; Naderi, Kambiz; Candolfi, Ermanno; Kedinger, Claude; Chatton, Bruno; Vigneron, Marc

    2011-04-01

    RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors.

  7. 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.

  8. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

    PubMed Central

    Jones, E V; Puckett, C; Moss, B

    1987-01-01

    Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A. Images PMID:3033308

  9. The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases.

    PubMed Central

    Patel, D D; Pickup, D J

    1989-01-01

    We have characterized the poxvirus gene encoding the second-largest subunit of the viral DNA-dependent RNA polymerase. This gene, designated rpo132, is located in the HindIII A fragment of the DNA of the Brighton Red strain of cowpox virus. A similar gene is located in the corresponding position in the HindIII A fragment of the DNA of the Western Reserve strain of vaccinia virus. The rpo132 gene is transcribed throughout the viral multiplication cycle. It has two transcriptional start sites; one is operative at late times only, and the other (80 base pairs downstream) is operative both at early times and at late times. Neither early nor late transcripts originating from the latter RNA start site contain long 5'-terminal poly(A) sequences. The rpo132 gene has the capacity to encode primary gene products of two types. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 133-kilodalton (kDa) protein. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 132-kDa protein. Transcripts of the latter type are more abundant, suggesting that the 132-kDa protein is the major primary product of this gene. The predicted amino acid sequences of both gene products share extensive similarities with the amino acid sequences of the second-largest subunits of the following enzymes: the RNA polymerase of Escherichia coli, the RNA polymerase II of Saccharomyces cerevisiae, and the RNA polymerase II of Drosophila melanogaster. This result provides further evidence of relatedness between multisubunit DNA-dependent RNA polymerases. Images PMID:2915377

  10. Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ 1

    PubMed Central

    Jendrisak, Jerry

    1981-01-01

    A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH4)2SO4 precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000. Images PMID:16661690

  11. Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

    PubMed Central

    Cozens, A L; Walker, J E

    1986-01-01

    The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

  12. 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

  13. 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

  14. Relatedness of archaebacterial RNA polymerase core subunits to their eubacterial and eukaryotic equivalents.

    PubMed Central

    Berghöfer, B; Kröckel, L; Körtner, C; Truss, M; Schallenberg, J; Klein, A

    1988-01-01

    The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme. PMID:2843811

  15. Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

    PubMed Central

    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; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-01-01

    Summary Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, 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 twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses 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 non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/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. PMID:25284785

  16. Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

    DOE PAGES

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; ...

    2014-10-02

    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 analyses 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, yieldsmore » 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.« less

  17. 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

  18. 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.

  19. 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

  20. Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

    PubMed Central

    Liljelund, P; Mariotte, S; Buhler, J M; Sentenac, A

    1992-01-01

    The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant. Images PMID:1409638

  1. 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

  2. Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

    PubMed Central

    Klinger, C; Huet, J; Song, D; Petersen, G; Riva, M; Bautz, E K; Sentenac, A; Oudet, P; Schultz, P

    1996-01-01

    Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. Images PMID:8887555

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

    PubMed

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

    2014-03-06

    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.

  4. 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.

  5. 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.

  6. The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus.

    PubMed

    Weiss, Andy; Moore, Brittney D; Tremblay, Miguel H J; Chaput, Dale; Kremer, Astrid; Shaw, Lindsey N

    2017-01-15

    Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the β' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α2ββ') a fifth, smaller subunit, ω, is present in all forms of life. Although this

  7. Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

    PubMed

    Sloan, Amy E; Sidorenko, Lyudmila; McGinnis, Karen M

    2014-11-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. Copyright © 2014 by the Genetics Society of America.

  8. Diverse Gene-Silencing Mechanisms with Distinct Requirements for RNA Polymerase Subunits in Zea mays

    PubMed Central

    Sloan, Amy E.; Sidorenko, Lyudmila; McGinnis, Karen M.

    2014-01-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes—mop1-1, Mop2-1, and mop3-1—suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. PMID:25164883

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

    USDA-ARS?s Scientific Manuscript database

    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. Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae.

    PubMed

    Chhetri, Gaurav; Ghosh, Arabinda; Chinta, Ramesh; Akhtar, Sohail; Tripathi, Timir

    2015-01-01

    We report the molecular cloning, expression, and single-step homogeneous purification of RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae. RPB5 is a 210 amino acid nuclear protein that functions as the fifth largest subunit of polymerase II and plays a central role in transcription. The gene that codes for RPB5 was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pET28a(+) under the transcriptional control of the bacteriophage T7 promoter and lac operator. BL21(DE3) Escherichia coli strain transformed with the rpb5 expression vector pET28a(+)-rpb5 accumulates large amounts of a soluble protein of about 30 kDa (25 kDa plus 5 kDa double His6-Tag at N and C-terminal). The protein was purified to homogeneity using immobilized metal affinity chromatography. RPB5 recombinant protein was further confirmed by immunoblotting with anti-His antibody. In this study, the expression and purification procedures have provided a simple and efficient method to obtain pure RPB5 in large quantities. This will provide an opportunity to study the role of S. cerevisiae RPB5 in gene expression and transcription regulation. Furthermore, it can provide additional knowledge of the interaction partners of RPB5 during various steps of transcription and gene expression.

  11. Positioning of Two Alpha Subunit Carboxy-Terminal Domains of RNA Polymerase at Promoters by Two Transcription Factors

    NASA Astrophysics Data System (ADS)

    Murakami, Katsuhiko; Owens, Jeffrey T.; Belyaeva, Tamara A.; Meares, Claude F.; Busby, Stephen J. W.; Ishihama, Akira

    1997-10-01

    Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase α subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to α . Each CRP dimer was found to direct the positioning of one of the two α subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two α subunits and two different species of transcription factors.

  12. 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

  13. Recognition of overlapping nucleotides by AraC and the sigma subunit of RNA polymerase.

    PubMed

    Dhiman, A; Schleif, R

    2000-09-01

    The Escherichia coli promoter p(BAD), under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p(BAD) overlaps the RNA polymerase -35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the -35 region or whether both AraC and sigma make important contacts with the DNA in the -35 region. If sigma does not contact DNA near the -35 region, p(BAD) activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p(BAD) promoter and in a second promoter where the AraC binding site overlaps the -35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the -35 region. Since sigma and AraC are thus closely positioned at p(BAD), it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.

  14. Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit*

    PubMed Central

    Sarkar, Paramita; Sardesai, Abhijit A.; Murakami, Katsuhiko S.; Chatterji, Dipankar

    2013-01-01

    The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β′ subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β′2112, a variant β′ bearing Y457S substitution, immediately adjacent to the β′ catalytic center. Our results suggest that the enhanced ω6-β′ interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state. PMID:23843456

  15. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

    PubMed

    Tabib-Salazar, Aline; Liu, Bing; Doughty, Philip; Lewis, Richard A; Ghosh, Somadri; Parsy, Marie-Laure; Simpson, Peter J; O'Dwyer, Kathleen; Matthews, Steve J; Paget, Mark S

    2013-06-01

    RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

  16. Promoter Structure of the RNA Polymerase II Large Subunit Gene in Caenorhabditis elegans and C. briggsae.

    PubMed

    Bird, D M; Kaloshian, I; Molinari, S

    1997-06-01

    The 5'-end of the Caenorhabditis elegans ama-1 gene transcript, which encodes the largest subunit of RNA polymerase II, was cloned. Sequencing revealed that the message is trans-spliced. To characterize the Ce-ama-1 promoter, DNA sequence spanning 3 kb upstream from the initiation codon was determined. Typical elements, such as TATA and Spl sites, were absent. The homologue of ama-1 in C. briggsae, Cb-ama-1, was isolated and its 5' flanking sequence compared with that of Ce-ama-1, revealing only limited similarity, although both sequences included a potential initiator-class transcriptional regulator and phased repeats of an ATC motif. The latter elements are postulated to facilitate DNA bending and may play a role in transcription regulation.

  17. The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid.

    PubMed

    Naryshkina, Tatyana; Kuznedelov, Konstantin; Severinov, Konstantin

    2006-08-25

    Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the "lid", is formed by an evolutionarily conserved segment of the RNAP largest subunit (beta' in bacterial RNAP). The beta' lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated from the DNA template-strand and double-stranded upstream DNA is formed. To test the beta' lid functions, we generated bacterial RNAP lacking the lid and studied the mutant enzyme's properties in vitro. Our results demonstrate that removal of the lid has minimal consequences on transcription elongation from double-stranded DNA. On single-stranded DNA, the mutant RNAP generates full-sized transcripts that remain annealed to the DNA throughout their length. In contrast, the wild-type enzyme produces short, 18-22 nucleotide transcripts that remain part of the transcription complex but cannot be further elongated. The cessation of transcription is apparently triggered by a clash between the lid and the nascent RNA 5' end. The results show that the lid's function is redundant in the presence of the non-template DNA strand, which alone can control the proper geometry of nucleic acids at the upstream edge of the transcription complex. Structural considerations suggest that in the absence of the non-template strand and the lid, a new channel opens within the RNAP molecule that allows continuous DNA-RNA hybrid to exit RNAP.

  18. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos 1

    PubMed Central

    Jendrisak, Jerry; Skuzeski, Jim

    1983-01-01

    DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights × 10−3 of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:16663122

  20. Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

    PubMed

    Leffers, H; Gropp, F; Lottspeich, F; Zillig, W; Garrett, R A

    1989-03-05

    The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

  1. Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

    PubMed Central

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

    2014-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life. PMID:24452800

  2. The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

    PubMed

    Gabig, M; Obuchowski, M; Ciesielska, A; Latała, B; Wegrzyn, A; Thomas, M S; Wegrzyn, G

    1998-01-01

    Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

  3. Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7.

    PubMed

    Qiu, Zilong; Jiang, Rongrong

    2017-01-01

    Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.

  4. 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

  5. RNA polymerase II termination involves CTD tyrosine dephosphorylation by CPF subunit Glc7

    PubMed Central

    Etzold, Stefanie; Wiederhold, Katrin; Lidschreiber, Michael; Cramer, Patrick; Passmore, Lori A.

    2014-01-01

    At the 3′ end of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine (Tyr1) residues of its C-terminal domain (CTD). In addition, the associated cleavage and polyadenylation (pA) factor (CPF) cleaves the transcript and adds a polyA tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the pA site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II Tyr1 dephosphorylation. PMID:24413056

  6. Orthologs of the small RPB8 subunit of the eukaryotic RNA polymerases are conserved in hyperthermophilic Crenarchaeota and "Korarchaeota".

    PubMed

    Koonin, Eugene V; Makarova, Kira S; Elkins, James G

    2007-12-14

    Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea.

  7. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea.

    PubMed

    Blombach, Fabian; Makarova, Kira S; Marrero, Jeannette; Siebers, Bettina; Koonin, Eugene V; van der Oost, John

    2009-10-14

    One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA polymerase II with respect to the subunit composition. Here we identify archaeal orthologs of the eukaryotic RNA polymerase III subunit RPC34. Genome context analysis supports a function of this archaeal protein in the transcription of non-coding RNAs. These findings suggest that functional separation of RNA polymerases for protein-coding genes and non-coding RNAs might predate the origin of the Eukaryotes.

  8. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea

    PubMed Central

    Blombach, Fabian; Makarova, Kira S; Marrero, Jeannette; Siebers, Bettina; Koonin, Eugene V; Oost, John van der

    2009-01-01

    One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA polymerase II with respect to the subunit composition. Here we identify archaeal orthologs of the eukaryotic RNA polymerase III subunit RPC34. Genome context analysis supports a function of this archaeal protein in the transcription of non-coding RNAs. These findings suggest that functional separation of RNA polymerases for protein-coding genes and non-coding RNAs might predate the origin of the Eukaryotes. Reviewers: This article was reviewed by Andrei Osterman and Patrick Forterre (nominated by Purificación López-García) PMID:19828044

  9. 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

  10. 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

    2016-04-18

    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.

  11. 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.

  12. Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

    PubMed Central

    Xue, Xiaoli; Sztajer, Helena; Buddruhs, Nora; Petersen, Jörn; Rohde, Manfred; Talay, Susanne R.; Wagner-Döbler, Irene

    2011-01-01

    The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased. PMID:21625504

  13. Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology.

    PubMed

    Khazak, V; Sadhale, P P; Woychik, N A; Brent, R; Golemis, E A

    1995-07-01

    Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.

  14. Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology.

    PubMed Central

    Khazak, V; Sadhale, P P; Woychik, N A; Brent, R; Golemis, E A

    1995-01-01

    Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS. Images PMID:7579693

  15. Crystal Structure in the Vivo-Assembled Bacillus subtilis Spx/RNA Polymerase alpha Subunit C-Terminal Domain Complex

    SciTech Connect

    Lamour, V.; Westblade, L; Campbell, E; Darst, S

    2009-01-01

    The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase {alpha} subunit ({alpha}CTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/{alpha}CTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

  16. Similarity and divergence between the RNA polymerase alpha subunits from hyperthermophilic Thermotoga maritima and mesophilic Escherichia coli bacteria.

    PubMed

    Braun, Frederique; Marhuenda, Fanny B; Morin, Amelie; Guevel, Laetitia; Fleury, Fabrice; Takahashi, Masayuki; Sakanyan, Vehary

    2006-10-01

    The alpha subunit (alphaTm) of Thermotoga maritima RNA polymerase has been characterized to investigate its role in transcriptional regulation in one of the few known anaerobic hyperthermophilic bacteria. The highly thermostable alphaTm shares 54% similarity with its Escherichia coli analogue (alphaEc). The T. maritima rpoA gene coding the alpha subunit does not complement the thermosensitive rpoA112 mutation of E. coli. However, alphaTm and alphaEc show similar folding patterns as determined by circular dichroism. Purified alphaTm binds to the T. maritima PargGo promoter region (probably to a UP-element) and Arg282 appears to be crucial for DNA binding. The thermostable protein is also able to interact with transcription regulatory proteins, like ArgR from T. neapolitana or CRP from E. coli. These data indicate that the RNA polymerase alpha subunit might play a crucial role in the modulation of gene expression in hyperthermophiles.

  17. Quantitative Proteomics Demonstrates That the RNA Polymerase II Subunits Rpb4 and Rpb7 Dissociate during Transcriptional Elongation*

    PubMed Central

    Mosley, Amber L.; Hunter, Gerald O.; Sardiu, Mihaela E.; Smolle, Michaela; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

    2013-01-01

    Eukaryotic RNA polymerase II (RNAPII) is a 12-subunit enzyme that is responsible for the transcription of messenger RNA. Two of the subunits of RNA polymerase II, Rpb4 and Rpb7, have been shown to dissociate from the enzyme under a number of specific laboratory conditions. However, a biological context for the dissociation of Rpb4 and Rpb7 has not been identified. We have found that Rpb4/7 dissociate from RNAPII upon interaction with specific transcriptional elongation-associated proteins that are recruited to the hyperphosphorylated form of the C-terminal domain. However, the dissociation of Rpb4/7 is likely short lived because a significant level of free Rpb4/7 was not detected by quantitative proteomic analyses. In addition, we have found that RNAPII that is isolated through Rpb7 is depleted in serine 2 C-terminal domain phosphorylation. In contrast to previous reports, these data indicate that Rpb4/7 are dispensable during specific stages of transcriptional elongation in Saccharomyces cerevisiae. PMID:23418395

  18. Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II.

    PubMed

    Petermann, R; Mossier, B M; Aryee, D N; Khazak, V; Golemis, E A; Kovar, H

    1998-08-06

    As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.

  19. 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

  20. 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

  1. The primary structure of E. coli RNA polymerase, Nucleotide sequence of the rpoC gene and amino acid sequence of the beta'-subunit.

    PubMed

    Ovchinnikov YuA; Monastyrskaya, G S; Gubanov, V V; Guryev, S O; Salomatina, I S; Shuvaeva, T M; Lipkin, V M; Sverdlov, E D

    1982-07-10

    The primary structure of the E. coli rpoC gene (5321 base pairs) coding the beta'-subunit of RNA polymerase as well as its adjacent segment have been determined. The structure analysis of the peptides obtained by cleavage of the protein with cyanogen bromide and trypsin has confirmed the amino acid sequence of the beta'-subunit deduced from the nucleotide sequence analysis. The beta'-subunit of E. coli RNA polymerase contains 1407 amino acid residues. Its translation is initiated by codon GUG and terminated by codon TAA. It has been detected that the sequence following the terminating codon is strikingly homologous to known sequences of rho-independent terminators.

  2. Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and Phage T7 single-subunit RNA polymerases.

    PubMed

    Sultana, Shemaila; Solotchi, Mihai; Ramachandran, Aparna; Patel, Smita S

    2017-09-07

    Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rate of T7 RNAP (2×10-6), yeast mitochondrial Rpo41 (6×10-6), and human mitochondrial POLRMT (2×10-5) indicates high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A to G errors in RNA with rates of ~10-4. The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine to purine mismatches were relatively frequent (10-5 to10-6), whereas C→G, U→A, G→C, and C→A errors from purine to purine and pyrimidine to pyrimidine mismatches were rare (10-7 to10-10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (~10-4). Strikingly, POLRMT shows high mutagenic bypass rate, which is exacerbated by TEFM. The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM that allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in case of T7 RNAP, to assess the quality of in vitro transcribed RNAs. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  3. A hypothetical hierarchical mechanism of the self-assembly of the Escherichia coli RNA polymerase σ(70) subunit.

    PubMed

    Koroleva, O N; Dubrovin, E V; Tolstova, A P; Kuzmina, N V; Laptinskaya, T V; Yaminsky, I V; Drutsa, V L

    2016-02-21

    Diverse morphology of aggregates of amyloidogenic proteins has been attracting much attention in the last few years, and there is still no complete understanding of the relationships between various types of aggregates. In this work, we propose the model, which universally explains the formation of morphologically different (wormlike and rodlike) aggregates on the example of a σ(70) subunit of RNA polymerase, which has been recently shown to form amyloid fibrils. Aggregates were studied using AFM in solution and depolarized dynamic light scattering. The obtained results demonstrate comparably low Young's moduli of the wormlike structures (7.8-12.3 MPa) indicating less structured aggregation of monomeric proteins than that typical for β-sheet formation. To shed light on the molecular interaction of the protein during the aggregation, early stages of fibrillization of the σ(70) subunit were modeled using all-atom molecular dynamics. Simulations have shown that the σ(70) subunit is able to form quasi-symmetric extended dimers, which may further interact with each other and grow linearly. The proposed general model explains different pathways of σ(70) subunit aggregation and may be valid for other amyloid proteins.

  4. Transcription-Independent Functions of an RNA Polymerase II Subunit, Rpb2, During Genome Rearrangement in the Ciliate, Oxytricha trifallax

    PubMed Central

    Khurana, Jaspreet S.; Wang, Xing; Chen, Xiao; Perlman, David H.; Landweber, Laura F.

    2014-01-01

    The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10–12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus. The gene encoding the second-largest subunit of Pol-II, Rpb2, has undergone gene duplication, and the two paralogs, Rpb2-a and -b, display different expression patterns. Immunoprecipitation of double-stranded RNAs identified an association with Rpb2-a. Through immunoprecipitation and mass spectrometry, we show that Rpb2-a in early zygotes appears surprisingly unassociated with other Pol II subunits. A partial loss of function of Rpb2-a leads to an increase in expression of transposons and other germline-limited satellite repeats. We propose that evolutionary divergence of the Rpb2 paralogs has led to acquisition of transcription-independent functions during sexual reproduction that may contribute to the negative regulation of germline gene expression. PMID:24793090

  5. Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

    PubMed

    Wooddell, C I; Burgess, R R

    2000-11-07

    The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

  6. A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly

    PubMed Central

    Markov, Dmitriy; Naryshkina, Tatyana; Mustaev, Arkady; Severinov, Konstantin

    1999-01-01

    All multisubunit DNA-dependent RNA polymerases (RNAP) are zinc metalloenzymes, and at least two zinc atoms are present per enzyme molecule. RNAP residues involved in zinc binding and the functional role of zinc ions in the transcription mechanism or RNAP structure are unknown. Here, we locate four cysteine residues in the Escherichia coli RNAP largest subunit, β′, that coordinate one of the two zinc ions tightly associated with the enzyme. In the absence of zinc, or when zinc binding is prevented by mutation, the in vitro-assembled RNAP retains the proper subunit stoichiometry but is not functional. We demonstrate that zinc acts as a molecular chaperone, converting denatured β′ into a compact conformation that productively associates with other RNAP subunits. The β′ residues coordinating zinc are conserved throughout eubacteria and chloroplasts, but are absent from homologs from eukaryotes and archaea. Thus, the involvement of zinc in the RNAP assembly may be a unique feature of eubacterial-type enzymes. PMID:10500100

  7. Identification of a novel compound with antiviral activity against influenza A virus depending on PA subunit of viral RNA polymerase.

    PubMed

    Yamada, Kazunori; Koyama, Hiroko; Hagiwara, Kyoji; Ueda, Atsushi; Sasaki, Yutaka; Kanesashi, Shin-Nosuke; Ueno, Ryuki; Nakamura, Hironori K; Kuwata, Kazuo; Shimizu, Kazufumi; Suzuki, Masaaki; Aida, Yoko

    2012-08-01

    Influenza viruses have developed resistance to current drugs, creating a need for new antiviral targets and new drugs to treat influenza virus infections. In this study, computational and experimental screening of an extensive compound library identified THC19, which was able to suppress influenza virus replication. This compound had no cytotoxic effects and did not disrupt cell cycle progression or induce apoptosis in MDCK cells as confirmed by WST-1 assays, flow cytometry analysis, and caspase-3 assays. Time-of-addition experiments showed that THC19 acts at a relatively early stage of the viral lifecycle. Subsequent mini-genome assays revealed that THC19 inhibited viral genome replication and/or transcription, suggesting that it interferes with one or more of the viral components that form the ribonucleoprotein complexes, namely polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP) and viral RNA. Finally, mini-genome assays where PB2, PB1, PA or NP from A/WSN/33 (H1N1) virus were replaced with those from A/Udorn/307/1972 (H3N2) virus effectively demonstrated that THC19 inhibited viral multiplication in a manner dependent upon the PA subunit. Taken together, these results suggest that influenza virus PA protein is a potential target for, and may aid the development of, novel compounds that inhibit influenza A virus replication. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  8. Ligand-free RAR can interact with the RNA polymerase II subunit hsRPB7 and repress transcription.

    PubMed

    Shen, X Q; Bubulya, A; Zhou, X F; Khazak, V; Golemis, E A; Shemshedini, L

    1999-06-01

    Upon binding retinoic acid (RA), the retinoic acid receptors (RARs) are able to positively and negatively regulate transcription. It has been shown that the DNA-binding domain and carboxy terminus of RARs are necessary for the ligand-dependent ability of the receptor to repress AP-1 transcriptional activity. A fusion of these two regions, shown to constitutively inhibit AP-1 activity, was used in a yeast two-hybrid screen to identify a novel hRARalpha-interacting protein. This protein, hsRPB7, a subunit of RNA polymerase II, interacts with hRARalpha in the absence of RA and addition of RA disrupts the interaction. Truncation analysis indicates that hsRPB7 specifically interacts with the hRARalpha DNA-binding domain. This interaction appears to compromise transcription, since overexpressed hRARalpha, in the absence of RA, is able to repress the activity of several RNA polymerase II-dependent activators, including AP-1 and the glucocorticoid receptor. This repression is relieved by transfected hsRPB7, strongly suggesting that ligand-free hRARalpha can block AP-1 activity by sequestering hsRPB7. The repression is dependent on the integrity of the hRARalpha DBD, since a mutation within the DBD blocks both the hRARalpha-hsRPB7 interaction and ligand-free hRARalpha repression of AP-1. These results provide evidence that non-liganded hRARalpha can regulate transcription by directly interacting with RNA polymerase II, and thus suggest a novel pathway by which hRARalpha can cross-talk with AP-1 and perhaps other families of transcriptional activators.

  9. 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. © 2016. Published by The Company of Biologists Ltd.

  10. 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

  11. RPAP1, a Novel Human RNA Polymerase II-Associated Protein Affinity Purified with Recombinant Wild-Type and Mutated Polymerase Subunits

    PubMed Central

    Jeronimo, Célia; Langelier, Marie-France; Zeghouf, Mahel; Cojocaru, Marilena; Bergeron, Dominique; Baali, Dania; Forget, Diane; Mnaimneh, Sanie; Davierwala, Armaity P.; Pootoolal, Jeff; Chandy, Mark; Canadien, Veronica; Beattie, Bryan K.; Richards, Dawn P.; Workman, Jerry L.; Hughes, Timothy R.; Greenblatt, Jack; Coulombe, Benoit

    2004-01-01

    We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction. PMID:15282305

  12. 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

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

    PubMed

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

    1990-04-11

    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.

  14. Underproduction of the largest subunit of RNA polymerase II causes temperature sensitivity, slow growth, and inositol auxotropy in Saccharomyces cerevisiae

    SciTech Connect

    Archambault, J.; Jansma, D.B.; Friesen, J.D.

    1996-03-01

    In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery. 70 refs., 7 figs., 1 tab.

  15. The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator of Streptococcus mutans Environmental Adaptation▿ †

    PubMed Central

    Xue, Xiaoli; Tomasch, Jürgen; Sztajer, Helena; Wagner-Döbler, Irene

    2010-01-01

    The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation. PMID:20675470

  16. 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

  17. 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.

  18. 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.

  19. The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase

    PubMed Central

    Shimada, Tomohiro; Tanaka, Kan

    2017-01-01

    The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the “constitutive promoters” that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of “inducible promoters” in the presence of additional supporting factors. PMID:28666008

  20. Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function.

    PubMed

    Bowman, Elizabeth Anne; Riddle, Donald L; Kelly, William

    2011-11-01

    Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism.

  1. Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function

    PubMed Central

    Bowman, Elizabeth Anne; Riddle, Donald L.; Kelly, William

    2011-01-01

    Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism. PMID:22384351

  2. Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II.

    PubMed

    Sakurai, H; Ishihama, A

    2001-01-01

    The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III. At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits. To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay. The intracellular concentrations of the 12 Rpb subunits in growing S. pombe cells are different, within a range of 15-fold difference between the least abundant Rpb3 and the most abundant Rpb12. The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites. The promoters of the first group of genes contain the TATA box sequence between -26 and -62, while the second group of genes carry TATA-less promoters. Several common sequence segments, tentatively designated 'Rpb motifs', were identified in the promoter regions of the rpb genes. Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40-fold higher than the Rpb3 mRNA level. The levels of rpb mRNAs do not correlate with those of Rpb proteins. The protein-to-mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits beta', beta, alpha and alpha, respectively, of the prokaryotic RNA polymerase core enzyme.

  3. Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans.

    PubMed

    Randall, Eva; Young, Vanessa; Sierotzki, Helge; Scalliet, Gabriel; Birch, Paul R J; Cooke, David E L; Csukai, Michael; Whisson, Stephen C

    2014-09-01

    Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.

  4. The primary structure of E. coli RNA polymerase, Nucleotide sequence of the rpoC gene and amino acid sequence of the beta'-subunit.

    PubMed Central

    Ovchinnikov YuA; Monastyrskaya, G S; Gubanov, V V; Guryev, S O; Salomatina, I S; Shuvaeva, T M; Lipkin, V M; Sverdlov, E D

    1982-01-01

    The primary structure of the E. coli rpoC gene (5321 base pairs) coding the beta'-subunit of RNA polymerase as well as its adjacent segment have been determined. The structure analysis of the peptides obtained by cleavage of the protein with cyanogen bromide and trypsin has confirmed the amino acid sequence of the beta'-subunit deduced from the nucleotide sequence analysis. The beta'-subunit of E. coli RNA polymerase contains 1407 amino acid residues. Its translation is initiated by codon GUG and terminated by codon TAA. It has been detected that the sequence following the terminating codon is strikingly homologous to known sequences of rho-independent terminators. PMID:6287430

  5. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. 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.

  7. The sequence of the largest subunit of RNA polymerase II is a useful marker for inferring seed plant phylogeny.

    PubMed

    Nickerson, Jennifer; Drouin, Guy

    2004-05-01

    We used RT-PCR to sequence approximately 3 kb of the gene coding for the largest subunit of RNA polymerase II (rpb1) from nine land plants. Our results show that plant rpb1 genes all have a similar GC-content and that their amino acid sequences evolve at a similar rate in most species we examined, except for the Arabidopsis thaliana and rice sequences which evolve faster. This gene also exists as a single copy in most species and contains enough phylogenetically informative sites to resolve the evolutionary relationships among seed plants. Protein maximum parsimony, as well as neighbor-joining and maximum likelihood analyses of DNA and protein sequences, all generated identical tree topologies with similar strong support values at each node. The angiosperms are a clade comprising Amborella as a sister group to all other angiosperms, followed by Nymphaea, Magnolia, Arabidopsis, and a monocot clade containing maize and rice. The gymnosperms also form a monophyletic clade with Welwitschia and pine grouped together and sister to a Cycas and Zamia clade. These findings concur with recent studies that refute the Anthophyte Hypothesis and place Amborella at the base of the angiosperm tree. These rpb1 sequences also give a more consistent picture of seed plant relationships than similar analyses performed on data sets made of 18S rDNA, atpB, and rbcL sequences from the same species. These sequences therefore show great promise to help further resolve the phylogenetic relationships of seed plants.

  8. X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase.

    PubMed

    Demo, Gabriel; Papoušková, Veronika; Komárek, Jan; Kadeřávek, Pavel; Otrusinová, Olga; Srb, Pavel; Rabatinová, Alžbeta; Krásný, Libor; Zídek, Lukáš; Sklenář, Vladimír; Wimmerová, Michaela

    2014-08-01

    The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni(2+) ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

    PubMed Central

    Greiner, D P; Hughes, K A; Gunasekera, A H; Meares, C F

    1996-01-01

    We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8552678

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

    PubMed

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

    2014-04-01

    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.

  11. Basic amino acids in the N-terminal half of the PB2 subunit of influenza virus RNA polymerase are involved in both transcription and replication.

    PubMed

    Hara, Koyu; Kashiwagi, Takahito; Hamada, Nobuyuki; Watanabe, Hiroshi

    2017-05-01

    The PB2 subunit of influenza virus RNA polymerase is known to be involved in the initiation of transcription of the virus genome via cap binding. However, other specific roles of PB2 for viral RNA synthesis are not well understood. Here, we demonstrate that basic residues, 124R, 142R, 143R, 268R and 331K/332R, in the N-terminal half of PB2 are important for the polymerase activity. Notably, R124A mutation remarkably reduced the synthesis of mRNA, cRNA and vRNA in vivo, which was in good agreement with the data obtained in vitro. Cross-linking studies suggested that a reduction of the polymerase activity in the R124A mutant was due to a significant decrease in binding to the viral RNA promoter. In the three-dimensional structure of the polymerase, 124R is visible through the NTP tunnel and is located close to the polymerase active site. We propose that 124R plays a key role in promoter binding during RNA synthesis.

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

    PubMed

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

    2016-08-22

    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.

  13. 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

  14. 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.

  15. 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.

  16. 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; ...

    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

  17. Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters.

    PubMed

    Landini, P; Bown, J A; Volkert, M R; Busby, S J

    1998-05-22

    The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613). Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma70 interaction.

  18. Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

    PubMed

    Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2015-04-24

    The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component.

  19. Structure and expression of the gene coding for the alpha-subunit of DNA-dependent RNA polymerase from the chloroplast genome of Zea mays.

    PubMed Central

    Ruf, M; Kössel, H

    1988-01-01

    The rpoA gene coding for the alpha-subunit of DNA-dependent RNA polymerase located on the DNA of Zea mays chloroplasts has been characterized with respect to its position on the chloroplast genome and its nucleotide sequence. The amino acid sequence derived for a 39 Kd polypeptide shows strong homology with sequences derived from the rpoA genes of other chloroplast species and with the amino acid sequence of the alpha-subunit from E. coli RNA polymerase. Transcripts of the rpoA gene were identified by Northern hybridization and characterized by S1 mapping using total RNA isolated from maize chloroplasts. Antibodies raised against a synthetic C-terminal heptapeptide show cross reactivity with a 39 Kd polypeptide contained in the stroma fraction of maize chloroplasts. It is concluded that the rpoA gene is a functional gene and that therefore, at least the alpha-subunit of plastidic RNA polymerase, is expressed in chloroplasts. Images PMID:3399379

  20. Signal Transduction and Regulatory Mechanisms Involved in Control of the σS (RpoS) Subunit of RNA Polymerase

    PubMed Central

    Hengge-Aronis, Regine

    2002-01-01

    The σS (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little σS, exposure to many different stress conditions results in rapid and strong σS induction. Consequently, transcription of numerous σS-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular σS level is achieved by rpoS transcriptional and translational control as well as by regulated σS proteolysis, with various stress conditions differentially affecting these levels of σS control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of σS, which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of σS regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For σS proteolysis, the response regulator RssB is essential. RssB is a specific direct σS recognition factor, whose affinity for σS is modulated by phosphorylation of its receiver domain. RssB delivers σS to the ClpXP protease, where σS is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system. PMID:12208995

  1. Trichomonas vaginalis initiator binding protein (IBP39) and RNA polymerase II large subunit carboxy terminal domain interaction.

    PubMed

    Lau, Audrey O T; Smith, Alias J; Brown, Mark T; Johnson, Patricia J

    2006-11-01

    The core promoter that directs RNA polymerase to the start of transcription in the protist Trichomonas vaginalis is an initiator (Inr) element recognized by the Inr Binding Protein, IBP39. This nuclear protein is composed of two domains: a 14.5 kDa amino (N-terminal) and a 25 kDa carboxy terminal domain (C-domain). Here we describe the identification of an IBP39-interacting protein by screening a T. vaginalis expression library using a two-hybrid system with the IBP39 C-domain as bait. The CTD of the large subunit of RNAP II was found to specifically interact with the C-domain. The specificity and nature of the interaction between the CTD of RNAP II and the C-domain of IBP39 was validated by three independent biochemical methods: co-immunoprecipitation with epitope-tagged proteins, affinity chromatography and enzyme linked ligand sorbent (ELLSA) assays. Binding was shown to involve hydrophobic bonds and to have a disassociation constant (K(d)) of 690 nM (+/-55). These results confirm and extend our previous binding studies using a peptide composed of the last nine amino acids of RNAP II CTD [Schumacher MA, Lau AOT, Johnson PJ. Structural basis of core promoter recognition in a primitive eukaryote. Cell 2003;115:413-24] that predicted an interaction between the CTD and IBP39. These data further demonstrate that IBP39 minimally possesses two functional domains: a N-terminal DNA binding domain (that recognizes the Inr) [Liston DR, Johnson PJ. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 1999;19:2380-8] and a C-terminal protein binding domain that recognizes the RNAP II CTD, an interaction that may be critical for recruiting RNAP II for initiation of transcription.

  2. Association Between Germline Mutations in BRF1, a subunit of the RNA Polymerase III Transcription Complex, and Hereditary Colorectal Cancer.

    PubMed

    Bellido, Fernando; Sowada, Nadine; Mur, Pilar; Lázaro, Conxi; Pons, Tirso; Valdés-Mas, Rafael; Pineda, Marta; Aiza, Gemma; Iglesias, Silvia; Soto, José Luís; Urioste, Miguel; Caldés, Trinidad; Balbín, Milagros; Blay, Pilar; Rueda, Daniel; Durán, Mercedes; Valencia, Alfonso; Moreno, Victor; Brunet, Joan; Blanco, Ignacio; Navarro, Matilde; Calin, George A; Borck, Guntram; Puente, Xose S; Capellá, Gabriel; Valle, Laura

    2017-09-11

    Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. We collected blood samples from 3 relatives with CRC in Spain (65, 62 and 40 years old at diagnosis) and perfomed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next-generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases. We carried out protein-dependent yeast-growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. A total of 42 unique or rare (population minor allele frequency below 1%) non-synonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11/503 individuals in families with a history of CRC or early-onset CRC,- a significantly greater proportion than in control population (34/4,300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability or expression and/or function. In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that

  3. The model of amyloid aggregation of Escherichia coli RNA polymerase σ70 subunit based on AFM data and in vitro assays.

    PubMed

    Koroleva, Olga N; Dubrovin, Evgeniy V; Khodak, Yu A; Kuzmina, Natalia V; Yaminsky, Igor V; Drutsa, Valeriy L

    2013-07-01

    To propose a model for recently described amyloid aggregation of E.coli RNA polymerase σ(70) subunit, we have investigated the role of its N-terminal region. For this purpose, three mutant variants of protein with deletions Δ1-73, Δ1-100 and Δ74-100 were constructed and studied in a series of in vitro assays and using atomic force microscopy (AFM). Specifically, all RNA polymerase holoenzymes, reconstituted with the use of mutant σ subunits, have shown reduced affinity for promoter-containing DNA and reduced activity in run-off transcription experiments (compared to that of WT species), thus substantiating the modern concept on the modulatory role of N-terminus in formation of open complex and transcription initiation. The ability of mutant proteins to form amyloid-like structures has been investigated using AFM, which revealed the increased propensity of mutant proteins to form rodlike aggregates with the effect being more pronounced for the mutant with the deletion Δ1-73 (10 fold increase). σ(70) subunit aggregation ability has shown complex dependence on the ionic surrounding, which we explain by Debye screening effect and the change of the internal state of the protein. Basing on the obtained data, we propose the model of amyloid fibril formation by σ(70) subunit, implying the involvement of N-terminal region according to the domain swapping mechanism.

  4. Archaeal RNA polymerase

    PubMed Central

    Hirata, Akira; Murakami, Katsuhiko S.

    2010-01-01

    Summary The recently solved X-ray crystal structures of archaeal RNA polymerase allows a structural comparison of the transcription machinery among all three domains of life. Archaeal transcription is very simple and all components, including the structures of general transcription factors and RNA polymerase, are highly conserved in eukaryotes. Therefore, it could be a new model for dissection of the eukaryotic transcription apparatus. The archaeal RNA polymerase structure also provides a framework for addressing the functional role that Fe–S clusters play within the transcription machinery of archaea and eukaryotes. A comparison between bacterial and archaeal open complex models reveals likely key motifs of archaeal RNA polymerase for DNA unwinding during the open complex formation. PMID:19880312

  5. A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

    PubMed

    Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi

    2014-09-05

    The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The Old and New Testaments of gene regulation. Evolution of multi-subunit RNA polymerases and co-evolution of eukaryote complexity with the RNAP II CTD.

    PubMed

    Burton, Zachary F

    2014-01-01

    I relate a story of genesis told from the point of view of multi-subunit RNA polymerases (RNAPs) including an Old Testament (core RNAP motifs in all cellular life) and a New Testament (the RNAP II heptad repeat carboxy terminal domain (CTD) and CTD interactome in eukarya). The Old Testament: at their active site, one class of eukaryotic interfering RNAP and ubiquitous multi-subunit RNAPs each have two-double psi β barrel (DPBB) motifs (a distinct pattern for compact 6-β sheet barrels). Between β sheets 2 and 3 of the β subunit type DPBB of all multi-subunit RNAPs is a sandwich barrel hybrid motif (SBHM) that interacts with conserved initiation and elongation factors required to utilize a DNA template. Analysis of RNAP core protein motifs, therefore, indicates that RNAP evolution can be traced from the RNA-protein world to LUCA (the last universal common ancestor) branching to LECA (the last eukaryotic common ancestor) and to the present day, spanning about 4 billion years. The New Testament: in the eukaryotic lineage, I posit that splitting RNAP functions into RNAPs I, II and III and innovations developed around the CTD heptad repeat of RNAP II and the extensive CTD interactome helps to describe how greater structural, cell cycle, epigenetic and signaling complexity co-evolved in eukaryotes relative to eubacteria and archaea.

  7. A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

    PubMed Central

    Cahyani, Inswasti; Cridge, Andrew G.; Engelke, David R.; Ganley, Austen R. D.

    2014-01-01

    The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

  8. TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

    PubMed Central

    McBryant, S J; Meier, E; Leresche, A; Sharp, S J; Wolf, V J; Gottesfeld, J M

    1996-01-01

    The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides. PMID:8756620

  9. Transcription coupled nucleotide excision repair in Escherichia coli can be affected by changing the arginine at position 529 of the β subunit of RNA polymerase

    PubMed Central

    Ganesan, Ann K.; Smith, Abigail J.; Savery, Nigel J.; Zamos, Portia; Hanawalt, Philip C.

    2008-01-01

    The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the β subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of Escherichia coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the β subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR. PMID:17532270

  10. Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation*

    PubMed Central

    Miropolskaya, Nataliya; Ignatov, Artem; Bass, Irina; Zhilina, Ekaterina; Pupov, Danil; Kulbachinskiy, Andrey

    2012-01-01

    RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ70 and T. aquaticus σA subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ70 subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the −10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation. PMID:22605342

  11. The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

    PubMed Central

    Pezet-Valdez, Marisol; Fernández-Retana, Jorge; Ospina-Villa, Juan David; Ramírez-Moreno, María Esther; Orozco, Esther; Charcas-López, Socorro; Soto-Sánchez, Jacqueline; Mendoza-Hernández, Guillermo; López-Casamicha, Mavil; López-Camarillo, César; Marchat, Laurence A.

    2013-01-01

    In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite. PMID:23840799

  12. Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

    PubMed Central

    Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

    1994-01-01

    Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

  13. RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

    PubMed Central

    Mann, C; Micouin, J Y; Chiannilkulchai, N; Treich, I; Buhler, J M; Sentenac, A

    1992-01-01

    RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants. Images PMID:1406624

  14. 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

  15. 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.

  16. [Involvement of sigma S and sigma 70 subunits of RNA polymerase and the CRP protein in the regulation of microcin C51 operon expression].

    PubMed

    Veselovskiĭ, A M; Bass, I A; Zolotukhina, M A; Mironov, A S; Metlitskaia, A Z; Khmel', I A

    2004-11-01

    Expression of the microcin C51 operon in Escherichia coli cells is activated during cell entry into the stationary growth phase and depends on the sigmaS subunit of RNA polymerase (RpoS). The null rpoS mutations retained the residual expression level of the transcriptional P(mcc)-lac fusion, which indicates that other sigma subunit can participate in the regulation of transcription of the microcin C51 operon. Data presented in this work show that the overproduction of sigma70 in rpoS- cells diminished the level of P(mcc)-lac expression, as in wild-type cells, which seems to be the consequence of competition between sigma factors for a limited number of core RNA polymerase molecules. In the presence of the rpoD800 mutation that renders sigma70 temperature-sensitive, expression of P(mcc)-lac was not induced in the phase of delayed culture growth at nonpermissive temperature, which indicates that sigma70 is indispensable for microcin operon expression. Point substitutions in the -10 P(mcc) region, leading to the formation of 5'-TGaTATAAT-3' site, enhanced promoter activity but did not affect the relationship between P(mcc)-lac transcription and growth phase, sigmaS, and the activator protein CRP. The activator protein CRP was shown to bind a DNA fragment containing the TGTGA(AATGAA)TCTAT site in the -59.5 bp position relative to the start site of transcription. Mutation in the ssrI gene encoding 6S RNA did not disturb P9mcc)-lac expression; these results indicate that 6S RNA does not participate in the regulation of microcin C51 operon expression.

  17. 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

  18. Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II.

    PubMed

    Khazak, V; Estojak, J; Cho, H; Majors, J; Sonoda, G; Testa, J R; Golemis, E A

    1998-04-01

    Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.

  19. Analysis of the Interaction of the Novel RNA Polymerase II (pol II) Subunit hsRPB4 with Its Partner hsRPB7 and with pol II

    PubMed Central

    Khazak, Vladimir; Estojak, Joanne; Cho, Helen; Majors, Jenifer; Sonoda, Gonosuke; Testa, Joseph R.; Golemis, Erica A.

    1998-01-01

    Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4− phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme. PMID:9528765

  20. RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts.

    PubMed

    Aung-Aud-Chariya, Amornrat; Bangrak, Phuwadol; Lumyong, Saisamorn; Phupong, Worrapong; Aggangan, Nelly Siababa; Kamlangdee, Niyom

    2015-03-01

    Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further.

  1. The β subunit gate loop is required for RNA polymerase modification by RfaH and NusG

    PubMed Central

    Sevostyanova, Anastasia; Belogurov, Georgiy A.; Mooney, Rachel A.; Landick, Robert; Artsimovitch, Irina

    2011-01-01

    SUMMARY In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β′ clamp helices. We hypothesize that, upon binding to the clamp helices and the gate loop, RfaH bridges the gap across the DNA channel, stabilizing RNAP/nucleic acid contacts and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity. PMID:21777814

  2. Molecular basis of the interaction for an essential subunit PA-PB1 in influenza virus RNA polymerase: insights from molecular dynamics simulation and free energy calculation.

    PubMed

    Liu, Huanxiang; Yao, Xiaojun

    2010-02-01

    The emergence of the extremely aggressive influenza recently has highlighted the urgent need for new effective treatments. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer including PA, PB1 and PB2 has crucial roles in viral RNA replication and transcription. The highly conserved PB1 binding site on PA can be considered as a novel potential drug target site. The interaction between PB1 binding site and PA is crucial to many functions of the virus. In this study, to understand the detailed interaction profile and to characterize the binding hot spots in the interactions of the PA-PB1 complex, an 8 ns molecular dynamics simulation of the subunit PA-PB1 combined with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area), MM-GBSA (molecular mechanics generalized Born surface area) computations and virtual alanine scanning were performed. The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process. Through the pair interaction analysis and virtual alanine scanning, we identified the binding hot spots of PA and the basic binding motif of PB1. This information can provide some insights for the structure-based RNA-dependent RNA polymerase inhibitors design. The identified binding motif can be used as the starting point for the rational design of small molecules or peptide mimics. This study will also lead to new opportunities toward the development of new generation therapeutic agents exhibiting specificity and low resistance to influenza virus.

  3. Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit.

    PubMed

    Poznański, Jarosław; Bolewska, Krystyna; Zhukov, Igor; Wierzchowski, Kazimierz L

    2003-11-25

    Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1

  4. Pivotal Role for a Tail Subunit of the RNA Polymerase II Mediator Complex CgMed2 in Azole Tolerance and Adherence in Candida glabrata

    PubMed Central

    Borah, Sapan; Shivarathri, Raju; Srivastava, Vivek Kumar; Ferrari, Sélène; Sanglard, Dominique

    2014-01-01

    Antifungal therapy failure can be associated with increased resistance to the employed antifungal agents. Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of all Candida bloodstream infections. Here, we show that C. glabrata MED2 (CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs in C. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of the Cgmed2Δ mutant toward azole antifungals. We also report for the first time that the Cgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of the Cgmed2Δ mutant was attributed to the elevated expression of the EPA1 and EPA7 genes. Further, our data demonstrate that CgMED2 is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals. PMID:25070095

  5. Evidence that sigma factors are components of chloroplast RNA polymerase.

    PubMed Central

    Troxler, R F; Zhang, F; Hu, J; Bogorad, L

    1994-01-01

    Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae. PMID:8159791

  6. The fission yeast protein Ker1p is an ortholog of RNA polymerase I subunit A14 in Saccharomyces cerevisiae and is required for stable association of Rrn3p and RPA21 in RNA polymerase I.

    PubMed

    Imazawa, Yukiko; Hisatake, Koji; Mitsuzawa, Hiroshi; Matsumoto, Masahito; Tsukui, Tohru; Nakagawa, Kaori; Nakadai, Tomoyoshi; Shimada, Miho; Ishihama, Akira; Nogi, Yasuhisa

    2005-03-25

    A heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+ using a yeast two-hybrid system, including rpa21+, which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker1+ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1delta was suppressed by overexpression of either rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-log-phase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription.

  7. Evaluation of the DNA-dependent RNA polymerase ß-subunit gene (rpoB) for phytoplasma classification and phylogeny

    USDA-ARS?s Scientific Manuscript database

    Phytoplasmas are classified into 16Sr groups, subgroups, and ‘Candidatus Phytoplasma’ species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related ‘Candidatus Phytoplasma’ species and strains based on 16S rRNA gene sequences alone has limitation...

  8. Activation of Antibiotic Biosynthesis by Specified Mutations in the rpoB Gene (Encoding the RNA Polymerase β Subunit) of Streptomyces lividans

    PubMed Central

    Hu, Haifeng; Zhang, Qin; Ochi, Kozo

    2002-01-01

    We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the rpoB gene that confer resistance to rifampin to Streptomyces lividans 66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the rpoB gene was shown to be dependent on the mutation's position and the amino acid species substituted in the β-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the rpoB gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of actII-ORF4 and redD, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the rpoB gene of S. lividans, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes, actII-ORF4 and redD. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in Streptomyces. PMID:12081971

  9. A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes

    PubMed Central

    Sidorenko, Lyudmila; Dorweiler, Jane E.; Cigan, A. Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L.

    2009-01-01

    Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2

  10. Chloroplast RNA polymerases: Role in chloroplast biogenesis.

    PubMed

    Börner, Thomas; Aleynikova, Anastasia Yu; Zubo, Yan O; Kusnetsov, Victor V

    2015-09-01

    Plastid genes are transcribed by two types of RNA polymerase in angiosperms: the bacterial type plastid-encoded RNA polymerase (PEP) and one (RPOTp in monocots) or two (RPOTp and RPOTmp in dicots) nuclear-encoded RNA polymerase(s) (NEP). PEP is a bacterial-type multisubunit enzyme composed of core subunits (coded for by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes) and additional protein factors (sigma factors and polymerase associated protein, PAPs) encoded in the nuclear genome. Sigma factors are required by PEP for promoter recognition. Six different sigma factors are used by PEP in Arabidopsis plastids. NEP activity is represented by phage-type RNA polymerases. Only one NEP subunit has been identified, which bears the catalytic activity. NEP and PEP use different promoters. Many plastid genes have both PEP and NEP promoters. PEP dominates in the transcription of photosynthesis genes. Intriguingly, rpoB belongs to the few genes transcribed exclusively by NEP. Both NEP and PEP are active in non-green plastids and in chloroplasts at all stages of development. The transcriptional activity of NEP and PEP is affected by endogenous and exogenous factors. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. 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

    Akhrymuk, Ivan; Kulemzin, Sergey V; Frolova, Elena I

    2012-07-01

    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.

  12. 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

  13. Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

    PubMed Central

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

    2014-01-01

    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. PMID:24786462

  14. Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

    PubMed

    Mühlbacher, Wolfgang; Mayer, Andreas; Sun, Mai; Remmert, Michael; Cheung, Alan C M; Niesser, Jürgen; Soeding, Johannes; Cramer, Patrick

    2015-10-01

    CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

  15. Amebic colitis in an antigenically and serologically negative patient: usefulness of a small-subunit ribosomal RNA gene-based polymerase chain reaction in diagnosis.

    PubMed

    Solaymani-Mohammadi, Shahram; Coyle, Christina M; Factor, Stephen M; Petri, William A

    2008-11-01

    Specific identification of Entamoeba histolytica in clinical specimens is an essential confirmatory diagnostic step in the management of amebiasis. Here, we report an unusual case of amebic colitis in a 20-year-old female immigrant from South China. The patient had experienced diarrhea, crampy abdominal pain, and fever for approximately 3 weeks prior to admission to hospital and had treated herself at home with metronidazole. On admission, stool microscopy and serology for E. histolytica were negative. Because the clinical findings raised the suspicion of Clostridium difficile fulminant colitis, she underwent a subtotal colectomy. Histopathology revealed flask-shaped ulcers characteristic of amebic colitis. Consequently, E. histolytica DNA was detected by a sensitive small-subunit rRNA polymerase chain reaction (PCR) from feces, and the patient was successfully treated for amebiasis with metronidazole. This case exemplifies the relative insensitivity of serologic tests for the diagnosis of intestinal amebiasis and the difficulties encountered in detecting the parasite antigen in a patient partially treated with metronidazole. We conclude that when the possibility of invasive intestinal amebiasis is suspected, detecting the parasite DNA directly in the stool sample by PCR using E. histolytica-specific primers may be an alternative, noninvasive, and reliable tool for the specific diagnosis of the disease.

  16. 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.

  17. RNA Polymerase in Mumps Virion

    PubMed Central

    Bernard, Jacqueline P.; Northrop, Robert L.

    1974-01-01

    Mumps virions of the Enders' strain were examined for polymerase activity in vitro. An RNA-dependent RNA polymerase was found to be associated with the virion. The general properties of the reaction appear to be similar to those described for other paramyxoviruses. PMID:4836602

  18. The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

    PubMed Central

    Kojima, Ikuo; Kasuga, Kano; Kobayashi, Masayuki; Fukasawa, Akira; Mizuno, Satoshi; Arisawa, Akira; Akagawa, Hisayoshi

    2002-01-01

    The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (ω), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete ω of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy. PMID:12426327

  19. An allele of the yeast RPB7 gene, encoding an essential subunit of RNA polymerase II, reduces cellular resistance to the antitumor drug bleomycin.

    PubMed

    He, C H; Ramotar, D

    1999-01-01

    Bleomycin is an antitumor drug that kills cells by introducing lesions in DNA. Thus, normal cells exposed to bleomycin must rely on efficient DNA repair mechanisms to survive. In the yeast Saccharomyces cerevisiae, the transcriptional activator Imp2 is required to fend off the toxic effects of bleomycin. However, it remains unclear whether Imp2 controls the expression of a protein that either repairs bleomycin-induced DNA lesions, or detoxifies the drug, and or both. To gain further insight into the mechanisms by which yeast cells mount a response towards bleomycin, we began to sequentially characterize the genetic defect in a collection of bleomycin-sensitive mutants that were previously isolated by mini-Tn3 transposon mutagenesis. A rescue plasmid designed to integrate at the site of the mini-Tn3 insertion was used to identify the defective gene in one of the mutant strains, HCY53, which was not allelic to IMP2. We showed that in strain HCY53, the mini-Tn3 was inserted at the distal end of an essential gene RPB7, which encodes one of the two subunits, Rpb4-Rbp7, that forms a subcomplex with RNA polymerase II. Since rpb7 null mutants are nonviable, it would appear that the rpb7::mini-Tn3 allele produces a protein that retains partial biological function thus permitting cell viability, but which is unable to provide bleomycin resistance to strain HCY53. The defective phenotype of strain HCY53 could be corrected by a plasmid bearing the entire RPB7 gene. Two dimensional gel analysis revealed that the expression of several proteins were diminished or absent in the rpb7::mini-Tn3 mutant when challenged with bleomycin. These results are in accord with our previous report that bleomycin resistance in yeast is controlled at the transcriptional level.

  20. Basic mechanism of transcription by RNA polymerase II

    PubMed Central

    Svetlov, Vladimir; Nudler, Evgeny

    2012-01-01

    RNA polymerase II-like enzymes carry out transcription of genomes in Eukaryota, Archaea, and some viruses. They also exhibit fundamental similarity to RNA polymerases from bacteria, chloroplasts, and mitochondria. In this review we take an inventory of recent studiesilluminating different steps of basic transcription mechanism, likely common for most multi-subunit RNA polymerases. Through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible reaction pathway for the two-metal nucleotidyl transfer mechanism, and to evaluate the way catalysis can be linked to translocation in the mechano-chemical cycle catalyzed by RNA polymerase II. PMID:22982365

  1. 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.

  2. Multisubunit RNA Polymerase Cleavage Factors Modulate the Kinetics and Energetics of Nucleotide Incorporation: An RNA Polymerase I Case Study.

    PubMed

    Appling, Francis D; Schneider, David A; Lucius, Aaron L

    2017-10-11

    All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called "cleavage factors" appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase's cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase-cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.

  3. 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.

  4. Simultaneous Gain and Loss of Functions Caused by a Single Amino Acid Substitution in the β Subunit of Escherichia Coli RNA Polymerase: Suppression of Nusa and Rho Mutations and Conditional Lethality

    PubMed Central

    Sparkowski, J.; Das, A.

    1992-01-01

    Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage λ antitermination protein, N. The E. coli nusA1 mutant fails to support λ development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant (rif(R)) variants of the nusA1 host support λ growth. We report here the isolation and pleiotropic properties of one such rif(R) mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase β subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42°. Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature. Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities. ts8 is an allele-specific suppressor of nusA1. It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and λ growth. A mutation in the N gene, punA1, that restores λ growth in the nusA1 mutant host but not in the nusAsal host, compensates for the nusAsal allele in the ts8 mutant. This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function. ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein. Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain. By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition

  5. Activation of Bacteriophage Mu mom Transcription by C Protein Does Not Require Specific Interaction with the Carboxyl-Terminal Region of the α or ς70 Subunit of Escherichia coli RNA Polymerase

    PubMed Central

    Sun, Weiyong; Hattman, Stanley; Fujita, Noboyuki; Ishihama, Akira

    1998-01-01

    Late in its growth cycle, transcription of the phage Mu mom promoter (Pmom) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the α or ς70 subunit of Escherichia coli RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both α and ς70. PMID:9620983

  6. Alphavirus polymerase and RNA replication.

    PubMed

    Pietilä, Maija K; Hellström, Kirsi; Ahola, Tero

    2017-01-16

    Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis. The early replication complex (RC) formed by the polyprotein P123 and nsP4 synthesizes minus RNA strands, and the late RC composed of fully processed nsP1-nsP4 is responsible for the production of genomic and subgenomic plus strands. Different parts of nsP4 recognize the promoters for minus and plus strands but the binding also requires the other nsPs. The alphavirus polymerase has been purified and is capable of de novo RNA synthesis only in the presence of the other nsPs. The purified nsP4 also has terminal adenylyltransferase activity, which may generate the poly(A) tail at the 3' end of the genome. Membrane association of the nsPs is vital for replication, and alphaviruses induce membrane invaginations called spherules, which form a microenvironment for RNA synthesis by concentrating replication components and protecting double-stranded RNA intermediates. The RCs isolated as crude membrane preparations are active in RNA synthesis in vitro, but high-resolution structure of the RC has not been achieved, and thus the arrangement of viral and possible host components remains unknown. For some alphaviruses, Ras-GTPase-activating protein (Src-homology 3 (SH3) domain)-binding proteins (G3BPs) and amphiphysins have been shown to be essential for RNA replication and are present in the RCs. Host factors offer an additional target for antivirals, as only few alphavirus polymerase inhibitors have been described.

  7. A study on the function of the glycine residue in the YGDD motif of the RNA-dependent RNA polymerase beta-subunit from RNA coliphage Q beta 1.

    PubMed

    Inokuchi, Y; Kajitani, M; Hirashima, A

    1994-12-01

    Q beta replicases in which the Gly residue of the beta-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg(2+)-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Q beta phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.

  8. 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.

  9. Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases.

    PubMed

    Thomm, Michael; Reich, Christoph; Grünberg, Sebastian; Naji, Souad

    2009-02-01

    The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure-function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the alpha subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the DeltaH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

  10. Conformational flexibility of bacterial RNA polymerase

    PubMed Central

    Darst, Seth A.; Opalka, Natacha; Chacon, Pablo; Polyakov, Andrey; Richter, Catherine; Zhang, Gongyi; Wriggers, Willy

    2002-01-01

    The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel. PMID:11904365

  11. 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

  12. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay.

    PubMed

    Vassiliou, W; Epp, J B; Wang, B B; Del Vecchio, A M; Widlanski, T; Kao, C C

    2000-09-01

    We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.

  13. 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 Central

    Feklistov, Andrey; Darst, Seth A.

    2013-01-01

    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

  14. 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

  15. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene.

    PubMed

    Coyle, C M; Wittner, M; Kotler, D P; Noyer, C; Orenstein, J M; Tanowitz, H B; Weiss, L M

    1996-11-01

    Microsporidia are emerging as opportunistic pathogens in patients with AIDS. Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis have been implicated in enteric infections in AIDS patients with chronic diarrhea, a wasting syndrome, and malabsorption. We used the polymerase chain reaction (PCR) and primers that amplify the conserved regions of the small-subunit rRNA (SSU-rRNA) gene of E. bieneusi and E. intestinalis in tissue specimens from HIV-infected patients with and without diarrhea to examine the association between microsporidia and diarrhea in patients with AIDS. Tissue specimens were obtained from 68 patients with AIDS and diarrhea (mean CD4 lymphocyte count, 21/mm3) and 43 AIDS patients without diarrhea (mean CD4 lymphocyte count, 60/mm3). By means of PCR with use of the SSU-rRNA primers specific for E. bieneusi and E. intestinalis, we found that 44% of patients with diarrhea were infected with microsporidia, whereas only 2.3% of the patients without diarrhea were infected with microsporidia (P < .001). There was a clear association between the presence of microsporidia and diarrhea. In addition, the SSU-rRNA primers proved to be sensitive and specific when used in this clinical setting.

  16. RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

    PubMed Central

    García-Cuéllar, M P; Esteban, R; Fujimura, T

    1997-01-01

    20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication. PMID:8990396

  17. Transcription by RNA polymerases I and III

    PubMed Central

    Paule, Marvin R.; White, Robert J.

    2000-01-01

    The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesises the large rRNA, pol II synthesises mRNA and pol III synthesises tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences. PMID:10684922

  18. Solving the RNA polymerase I structural puzzle

    PubMed Central

    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

    2014-01-01

    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. PMID:25286842

  19. RNA polymerase IV functions in paramutation in Zea mays.

    PubMed

    Erhard, Karl F; Stonaker, Jennifer L; Parkinson, Susan E; Lim, Jana P; Hale, Christopher J; Hollick, Jay B

    2009-02-27

    Plants have distinct RNA polymerase complexes (Pol IV and Pol V) with largely unknown roles in maintaining small RNA-associated gene silencing. Curiously, the eudicot Arabidopsis thaliana is not affected when either function is lost. By use of mutation selection and positional cloning, we showed that the largest subunit of the presumed maize Pol IV is involved in paramutation, an inherited epigenetic change facilitated by an interaction between two alleles, as well as normal maize development. Bioinformatics analyses and nuclear run-on transcription assays indicate that Pol IV does not engage in the efficient RNA synthesis typical of the three major eukaryotic DNA-dependent RNA polymerases. These results indicate that Pol IV employs abnormal RNA polymerase activities to achieve genome-wide silencing and that its absence affects both maize development and heritable epigenetic changes.

  20. Disengaging polymerase: Terminating RNA polymerase II transcription in budding yeast☆

    PubMed Central

    Mischo, Hannah E.; Proudfoot, Nick J.

    2013-01-01

    Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3′ end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. PMID:23085255

  1. Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

    PubMed Central

    Dove, Simon L.; Hochschild, Ann

    2001-01-01

    A number of transcriptional regulators mediate their effects through direct contact with the ς70 subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς70 that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς70-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς70 and also that amino acid substitution R596H, within region 4 of ς70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς70. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the ς70 subunit of RNAP from this organism. We found that amino acid substitution R600H in ς70 from P. aeruginosa, corresponding to the R596H substitution in E. coli ς70, specifically weakens the interaction between AlgQ and ς70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς70 and probably regulate gene expression through this contact. PMID:11591686

  2. The RNA polymerase II elongation complex.

    PubMed

    Aso, T; Conaway, J W; Conaway, R C

    1995-11-01

    The initiation stage of transcription by RNA polymerase II has long been regarded as the primary site for regulation of eukaryotic gene expression. Nevertheless, a growing body of evidence reveals that the RNA polymerase II elongation complex is also a major target for regulation. Biochemical studies are implicating an increasing number of transcription factors in the regulation of elongation, and these transcription factors are being found to function by a diverse collection of mechanisms. Moreover, unexpected features of the structure and catalytic mechanism of RNA polymerase II are forcing a reconsideration of long-held views on the mechanics of some of the most basic aspects of polymerase function. In this review, we will describe recent insights into the structures and functions of RNA polymerase II and the transcription factors that control its activity during the elongation stage of eukaryotic messenger RNA synthesis.

  3. RNA binding and replication by the poliovirus RNA polymerase

    SciTech Connect

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.

  4. RNase-like domain in DNA-directed RNA polymerase II.

    PubMed Central

    Shirai, T; Go, M

    1991-01-01

    DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function. Images PMID:1924368

  5. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  6. Structural Basis of RNA Polymerase I Transcription Initiation.

    PubMed

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

    2017-03-23

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

  7. 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

  8. 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

  9. Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes.

    PubMed

    Posthuma, Clara C; Te Velthuis, Aartjan J W; Snijder, Eric J

    2017-02-06

    Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26-34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3'-to-5' exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.

  10. 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.

  11. RNA polymerase II transcription: structure and mechanism.

    PubMed

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

    2013-01-01

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

  12. Structural insights into transcription initiation by yeast RNA polymerase I.

    PubMed

    Sadian, Yashar; Tafur, Lucas; Kosinski, Jan; Jakobi, Arjen J; Wetzel, Rene; Buczak, Katarzyna; Hagen, Wim Jh; Beck, Martin; Sachse, Carsten; Müller, Christoph W

    2017-09-15

    In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  13. Identification of previously unrecognized common elements in eukaryotic promoters. A ribosomal RNA gene initiator element for RNA polymerase I.

    PubMed

    Radebaugh, C A; Gong, X; Bartholomew, B; Paule, M R

    1997-02-07

    A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photo-cross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A133) to the rInr sequence. A185 also photo-cross-links when polymerase is stalled at +7.

  14. Immunization of nonautoimmune mice with DNA binding domains of the largest subunit of RNA polymerase I results in production of anti-dsDNA and anti-Sm/RNP antibodies.

    PubMed

    Sciascia, Sandra A; Robson, Kristina; Zhu, Liangjin; Garland, Michael; Grabosch, Shannon; Kelamis, Joseph; Messamore, Will; Bradley, Todd; Sourk, Allison; Westberg, Lindsey; Goodnight, Waco; Tongson, Krystina; Holloway, Naomi; Wardak, Zabi; Sudyka, Michelle; Masrani, Shriti; Chintalapati, Sanket; Cagaanan, Manuel; Brown, John C; Stetler, Dean A

    2007-02-01

    Antibodies against the N-terminal (NT) but not the basic domain (BD), DNA binding regions of the largest subunit (S1) of RNA polymerase I (RNAPI) were detected in the sera of MRL-lpr/lpr lupus mice. Antibodies against both RNAPI(S1)-NT and -BD, as well as other systemic lupus erythematosus (SLE) autoantigens (La, ribosomal P proteins and Sm/RNP) were produced by rabbits immunized with anti-DNA antibodies that had been affinity purified from SLE patients. Immunization of nonautoimmune mice (Balb/c) with RNAPI(S1)-NT, RNAPI(S1)-BD, or La in the form of GST fusion proteins, induced production of anti-double-stranded (ds) DNA and anti-Sm/RNP. GST-P1 did not induce an anti-dsDNA response in these mice. These results demonstrate that RNAPI(S1)-NT, RNAPI(S1)-BD and La can participate in an anti-autoantigen/anti-DNA antibody loop during an SLE-like autoimmune response.

  15. Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase.

    PubMed

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

    2005-02-01

    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.

  16. 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

  17. 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

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

    PubMed

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

    1976-04-01

    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.

  19. Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

    PubMed Central

    Pata, J D; Schultz, S C; Kirkegaard, K

    1995-01-01

    Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 8 PMID:7489508

  20. RNA Polymerase II Transcription: Structure and Mechanism

    PubMed Central

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

    2014-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. PMID:23000482

  1. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  2. RNA-Dependent RNA Polymerase Activity in Influenza Virions

    PubMed Central

    Penhoet, Edward; Miller, Henry; Doyle, Michael; Blatti, Stanley

    1971-01-01

    An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA. PMID:5288388

  3. 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

  4. 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

  5. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  6. Amplification of RNA by an RNA polymerase ribozyme

    PubMed Central

    Horning, David P.; Joyce, Gerald F.

    2016-01-01

    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

  7. FACT facilitates chromatin transcription by RNA polymerases I and III

    PubMed Central

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I; Panova, Tatiana B; Andersen, Jens S; Owen-Hughes, Tom A; Russell, Jackie; Lee, Sheng-Chung; Zomerdijk, Joost C B M

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes. PMID:19214185

  8. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

    PubMed

    Dias, Alexandre; Bouvier, Denis; Crépin, Thibaut; McCarthy, Andrew A; Hart, Darren J; Baudin, Florence; Cusack, Stephen; Ruigrok, Rob W H

    2009-04-16

    The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

  9. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK.

    PubMed

    Feaver, W J; Svejstrup, J Q; Henry, N L; Kornberg, R D

    1994-12-16

    KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

  10. The bacterial enhancer-dependent RNA polymerase

    PubMed Central

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

    2016-01-01

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

  11. Distinct molecular structures of nuclear class I, II, and III DNA-dependent RNA polymerases.

    PubMed

    Sklar, V E; Schwartz, L B; Roeder, R G

    1975-01-01

    Class III RNA polymerases purified from the murine plasmacytoma MOPC 315 and from Xenopus laevis ovaries were compared. The subunit structures of the chromatographically distinct murine enzymes IIIA and IIIB were indistinguishable and were remarkably similar to that of the amphibian enzyme III. The plasmacytoma class III RNA polymerases were also compared with purified plasmacytoma RNA polymerases I and II. Sedimentation studies indicated that RNA polymerase III si significantly larger than RNA polymerase II, which is slightly larger than RNA polymerase I. Structural analyses showed that the molecular weights of the large subunits present in the class III enzymes (138,000 and 155,000) differ from those of the class II enzymes (140,000 and either 170,000, 205,000, or 240,000) and from those of the class I enzymes (117,000 and 195,000). Some low-molecular-weight subunits are also unique to each enzyme class. These results clearly distinguish the class I, II, and III enzymes on a structural basis. In addition, polypeptides of molecular weight 29,000 and 19,000 were found in all enzyme classes, a polypeptide of molecular weight 52,000 was found only in class I and III enzymes, and a polypeptide of molecular weight 41,000 was found only in class II and III enzymes. These findings are discussed in terms of the function and regulation of the RNA polymerases.

  12. 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.

  13. Structural Biology of Bacterial RNA Polymerase

    PubMed Central

    Murakami, Katsuhiko S.

    2015-01-01

    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. PMID:25970587

  14. Interstitial contacts in an RNA-dependent RNA polymerase lattice

    PubMed Central

    Tellez, Andres B.; Wang, Jing; Tanner, Elizabeth J.; Spagnolo, Jeannie F.; Kirkegaard, Karla; Bullitt, Esther

    2011-01-01

    Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the ‘thumb’ domain of one polymerase and the back of the ‘palm’ domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, a hybrid approach of both electron microscopic and biochemical evaluation of wild-type and mutant viral polymerases was used to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality, or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should faciliate antiviral drug design and provide a precedent for other positive-strand RNA viruses. PMID:21839092

  15. Regulation through the RNA Polymerase Secondary Channel

    PubMed Central

    Symersky, Jindrich; Perederina, Anna; Vassylyeva, Marina N.; Svetlov, Vladimir; Artsimovitch, Irina; Vassylyev, Dmitry G.

    2006-01-01

    Gre factors enhance the intrinsic endonucleolytic activity of RNA polymerase to rescue arrested transcription complexes and are thought to confer the high fidelity and processivity of RNA synthesis. The Gre factors insert the extended α-helical coiled-coil domains into the RNA polymerase secondary channel to position two invariant acidic residues at the coiled-coil tip near the active site to stabilize the catalytic metal ion. Gfh1, a GreA homolog from Thermus thermophilus, inhibits rather than activates RNA cleavage. Here we report the structure of the T. thermophilus Gfh1 at 2.4 Å resolution revealing a two-domain architecture closely resembling that of GreA. However, the interdomain orientation is strikingly distinct (~162° rotation) between the two proteins. In contrast to GreA, which has two acidic residues on a well fixed self-stabilized α-turn, the tip of the Gfh1 coiled-coil is flexible and contains four acidic residues. This difference is likely the key to the Gre functional diversity, while Gfh1 inhibits exo- and endonucleolytic cleavage, RNA synthesis, and pyrophosphorolysis, GreA enhances only the endonucleolytic cleavage. We propose that Gfh1 acidic residues stabilize the RNA polymerase active center in a catalytically inactive configuration through Mg2+-mediated interactions. The excess of the acidic residues and inherent flexibility of the coiled-coil tip might allow Gfh1 to adjust its activity to structurally distinct substrates, thereby inhibiting diverse catalytic reactions of RNA polymerase. PMID:16298991

  16. 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

  17. [Procedure for purifying RNA polymerase II from human placenta].

    PubMed

    Kandyba, L V; Matsanova, V R; Shamovskiĭ, I V; Raĭt, V K

    1994-12-01

    DNA-dependent RNA polymerase IIB having a specific activity of 320 u./mg has been isolated from the term placenta homogenate using extraction performed at 4-6 degrees C in the presence of 75 mM ammonium sulfate and 1.5% nonidet P40, fractionation on DEAE-cellulose DE 23, desalting and heparin-agarose chromatography, resulting in 330-fold purification and a 18% yield. Technical details have been determined which are of crucial importance for reproducibility of affinity chromatography. The possibility of proteolysis of the IIc subunit during enzyme purification has been demonstrated.

  18. Giardia lamblia RNA polymerase II: amanitin-resistant transcription.

    PubMed

    Seshadri, Vishwas; McArthur, Andrew G; Sogin, Mitchell L; Adam, Rodney D

    2003-07-25

    Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G. lamblia demonstrate important differences from these processes in higher eukaryotes. The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to alpha-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 microg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template. To better understand transcription in G. lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpb1 from G. lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms. These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G. lamblia.

  19. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  20. 6S RNA-dependent inhibition of RNA polymerase is released by RNA-dependent synthesis of small de novo products.

    PubMed

    Wurm, Reinhild; Neusser, Thomas; Wagner, Rolf

    2010-01-01

    6S RNA from Escherichia coli is known to bind to RNA polymerase, preventing interaction with many promoters during stationary growth. The resulting repression is released under conditions of nutritional upshift, when the growth situation improves. 6S RNA, which binds to the active site of RNA polymerase, has the particularly interesting feature to act as a template, causing the transcription of defined de novo RNAs (dnRNA) that are complementary to a specific sequence region of the 6S RNA. We analyzed the conditions of dnRNA synthesis and determined their effect on the 6S RNA-mediated inhibition of RNA polymerase in vitro and in vivo. Upon nutritional upshift the RNA polymerase/6S RNA complex induces the rapid synthesis of dnRNAs, which form stable hybrids with the 6S RNA template. The resulting structural change destabilizes the inactivated RNA polymerase complex, causing sigma subunit release. Both dnRNA and 6S RNA are rapidly degraded after complex disintegration. Experiments using the transcriptional inhibitor rifampicin demonstrate that active transcription is required for the disintegration of the RNA polymerase/6S RNA complex. Our results support the conclusion that 6S RNA not only inhibits transcription during stationary growth but also enables cells to resume rapid growth after starvation and help to escape from stationary phase.

  1. Fitness-compensatory mutations in rifampicin-resistant RNA polymerase.

    PubMed

    Brandis, Gerrit; Wrande, Marie; Liljas, Lars; Hughes, Diarmaid

    2012-07-01

    Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.

  2. 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.

  3. 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

  4. Origin and Evolution of RNA-Dependent RNA Polymerase.

    PubMed

    de Farias, Savio T; Dos Santos Junior, Ariosvaldo P; Rêgo, Thais G; José, Marco V

    2017-01-01

    RNA-dependent RNA polymerases (RdRp) are very ancient enzymes and are essential for all viruses with RNA genomes. We reconstruct the origin and evolution of this polymerase since the initial stages of the origin of life. The origin of the RdRp was traced back from tRNA ancestors. At the origin of the RdRp the most ancient part of the protein is the cofactor-binding site that had the capacity of binding to simple molecules as magnesium, calcium, and ribonucleotides. Our results suggest that RdRp originated from junctions of proto-tRNAs that worked as the first genes at the emergence of the primitive translation system, where the RNA was the informational molecule. The initial domain, worked as a building block for the emergence of the fingers and thumb domains. From the ancestral RdRp, we could establish the evolutionary stages of viral evolution from a rooted ancestor to modern viruses. It was observed that the selective pressure under the RdRp was the organization and functioning of the genome, where RNA double-stranded and RNA single-stranded virus formed a separate group. We propose an evolutionary route to the polymerases and the results suggest an ancient scenario for the origin of RNA viruses.

  5. RNA Polymerase II Collision Interrupts Convergent Transcription

    PubMed Central

    Hobson, David J.; Wei, Wu; Steinmetz, Lars M.; Svejstrup, Jesper Q.

    2012-01-01

    Summary Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision. PMID:23041286

  6. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. The architecture of RNA polymerase fidelity.

    PubMed

    Kaplan, Craig D

    2010-06-22

    The basis for transcriptional fidelity by RNA polymerase is not understood, but the 'trigger loop', a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A study just published in BMC Biology sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates. See research article http://www.biomedcentral.com/1741-7007/8/54.

  8. RNA polymerase: the vehicle of transcription.

    PubMed

    Borukhov, Sergei; Nudler, Evgeny

    2008-03-01

    RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation for all three divisions of life: Eukaryota, Archaea and Bacteria. Recent progress in the structural and biochemical characterization of RNAP illuminates this enzyme as a flexible, multifunctional molecular machine. During each step of the transcription cycle, RNAP undergoes elaborate conformational changes. As many fundamental and previously mysterious aspects of how RNAP works begin to be understood, this enzyme reveals intriguing similarities to man-made engineered devices. These resemblances can be found in the mechanics of RNAP-DNA complex formation, in RNA chain initiation and in the elongation processes. Here we highlight recent advances in understanding RNAP function and regulation.

  9. Structural basis for transcription elongation by bacterial RNA polymerase.

    PubMed

    Vassylyev, Dmitry G; Vassylyeva, Marina N; Perederina, Anna; Tahirov, Tahir H; Artsimovitch, Irina

    2007-07-12

    The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

  10. The non-coding B2 RNA binds to the DNA cleft and active site region of RNA polymerase II

    PubMed Central

    Ponicsan, Steven L.; Houel, Stephane; Old, William M.; Ahn, Natalie G.; Goodrich, James A.; Kugel, Jennifer F.

    2013-01-01

    The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180 nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is a ~500 kD complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde crosslinking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after crosslinking to B2 RNA mapped to the DNA binding cleft and active site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA. PMID:23416138

  11. The RNA polymerase I transcription machinery.

    PubMed

    Russell, Jackie; Zomerdijk, Joost C B M

    2006-01-01

    The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

  12. Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells.

    PubMed

    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

    2013-11-07

    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.

  13. SAGA Is a General Cofactor for RNA Polymerase II Transcription.

    PubMed

    Baptista, Tiago; Grünberg, Sebastian; Minoungou, Nadège; Koster, Maria J E; Timmers, H T Marc; Hahn, Steve; Devys, Didier; Tora, László

    2017-10-05

    Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Evolution of the RNA polymerase II C-terminal domain

    PubMed Central

    Stiller, John W.; Hall, Benjamin D.

    2002-01-01

    In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms. PMID:11972039

  15. Archaeal RNA polymerase and transcription regulation

    PubMed Central

    Jun, Sung-Hoon; Reichlen, Matthew J.; Tajiri, Momoko; Murakami, Katsuhiko S.

    2010-01-01

    To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high resolution X-ray crystal structures together with structure-guided biochemical, biophysical and genetics studies are essential. The recently-solved X-ray crystal structures of archaeal RNA polymerase (RNAP) allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors, is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all three domains of life. PMID:21250781

  16. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center.

    PubMed

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-11-12

    Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.

  17. Structure-Function Relationships Among RNA-Dependent RNA Polymerases

    PubMed Central

    Ng, Kenneth K.-S.; Arnold, Jamie J.; Cameron, Craig E.

    2008-01-01

    RNA-dependent RNA polymerases (RdRPs) play key roles in viral transcription and genome replication, as well as epigenetic and post-transcriptional control of cellular gene expression. In this article, we review the crystallographic, biochemical, and molecular genetic data available for viral RdRPs that have led to a detailed description of substrate and cofactor binding, fidelity of nucleotide selection and incorporation, and catalysis. It is likely that the cellular RdRPs will share some of the basic structural and mechanistic principles gleaned from studies of viral RdRPs. Therefore, studies of the viral RdRP establish a framework for the study of cellular RdRPs, an important yet understudied class of nucleic acid polymerases. PMID:18268843

  18. Ratcheting of RNA polymerase toward structural principles of RNA polymerase operations

    PubMed Central

    Sekine, Shun-ichi; Murayama, Yuko; Svetlov, Vladimir; Nudler, Evgeny; Yokoyama, Shigeyuki

    2015-01-01

    RNA polymerase (RNAP) performs various tasks during transcription by changing its conformational states, which are gradually becoming clarified. A recent study focusing on the conformational transition of RNAP between the ratcheted and tight forms illuminated the structural principles underlying its functional operations. PMID:26226152

  19. Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase.

    PubMed

    Jaehning, J A; Wiggs, J L; Chamberlin, M J

    1979-11-01

    Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

  20. Molecular phylogeny of Fusarium inferred from partial RNA polymerase II gene sequences

    USDA-ARS?s Scientific Manuscript database

    Currently there are no robust phylogenetic hypotheses for Fusarium based on large-scale sampling across the breadth of this important group of mycotoxigenic phytopathogens. Nucleotide variation within the second largest RNA polymerase subunit (RPB2) protein-coding gene, however, has clearly demonst...

  1. RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

    PubMed Central

    Mizutani, S; Temin, H M

    1976-01-01

    An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions. Images PMID:183017

  2. Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation

    PubMed Central

    Allepuz-Fuster, Paula; Martínez-Fernández, Verónica; Garrido-Godino, Ana I.; Alonso-Aguado, Sergio; Hanes, Steven D.; Navarro, Francisco; Calvo, Olga

    2014-01-01

    The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function. PMID:25416796

  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. Tagetitoxin Inhibits RNA Polymerase through Trapping of the Trigger Loop*

    PubMed Central

    Artsimovitch, Irina; Svetlov, Vladimir; Nemetski, Sondra Maureen; Epshtein, Vitaly; Cardozo, Timothy; Nudler, Evgeny

    2011-01-01

    Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the β′ subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a “slow” enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics. PMID:21976682

  5. Hinge action versus grip in translocation by RNA polymerase.

    PubMed

    Nedialkov, Yuri A; Opron, Kristopher; Caudill, Hailey L; Assaf, Fadi; Anderson, Amanda J; Cukier, Robert I; Wei, Guowei; Burton, Zachary F

    2017-08-30

    Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA-DNA hybrid resulting in opening of the furthest upstream i-8 RNA-DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β' Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ-β-barrel and the associated sandwich barrel hybrid motif (also called the flap domain), the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA-DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K(+) and Mg(2+) and also an RNA 3'-OH or a 3'-H2 affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K(+) and Mg(2+)concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation.

  6. RNA polymerase backtracking in gene regulation and genome instability.

    PubMed

    Nudler, Evgeny

    2012-06-22

    RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. Since its first description 15 years ago, backtracking--the reversible sliding of RNA polymerase along DNA and RNA--has been implicated in many critical processes in bacteria and eukaryotes, including the control of transcription elongation, pausing, termination, fidelity, and genome instability.

  7. RNA Polymerase III Accurately Initiates Transcription from RNA Polymerase II Promoters in Vitro*

    PubMed Central

    Duttke, Sascha H. C.

    2014-01-01

    In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the downstream promoter element (DPE) or the motif ten element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature. PMID:24917680

  8. The punctilious RNA polymerase II core promoter.

    PubMed

    Vo Ngoc, Long; Wang, Yuan-Liang; Kassavetis, George A; Kadonaga, James T

    2017-07-01

    The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter. © 2017 Vo ngoc et al.; Published by Cold Spring Harbor Laboratory Press.

  9. 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

  10. Primary structure of the catalytic subunit of human DNA polymerase. delta. and chromosomal location of the gene

    SciTech Connect

    Chung, D.W.; Davie, E.W. ); Jian Zhang; Chengkeat Tan; So, A.G.; Downey, K.M. )

    1991-12-15

    The catalytic subunit of human DNA polymerase {delta} has been cloned by PCR using poly (A){sup +}RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase {delta}. The human cDNA was 3,443 nucleotides in length and coded for a polypeptide of 1,107 amino acids. The enzyme was 94% identical to bovine DNA polymerase {delta} and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase {delta} was localized to chromosome 19.

  11. Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein-Protein Interaction.

    PubMed

    Banda, Srikanth; Cao, Nan; Tse-Dinh, Yuk-Ching

    2017-09-15

    We report here a distinct mechanism of interaction between topoisomerase I and RNA polymerase in Mycobacterium tuberculosis and Mycobacterium smegmatis that has evolved independently from the previously characterized interaction between bacterial topoisomerase I and RNA polymerase. Bacterial DNA topoisomerase I is responsible for preventing the hyper-negative supercoiling of genomic DNA. The association of topoisomerase I with RNA polymerase during transcription elongation could efficiently relieve transcription-driven negative supercoiling. Our results demonstrate a direct physical interaction between the C-terminal domains of topoisomerase I (TopoI-CTDs) and the β' subunit of RNA polymerase of M. smegmatis in the absence of DNA. The TopoI-CTDs in mycobacteria are evolutionarily unrelated in amino acid sequence and three-dimensional structure to the TopoI-CTD found in the majority of bacterial species outside Actinobacteria, including Escherichia coli. The functional interaction between topoisomerase I and RNA polymerase has evolved independently in mycobacteria and E. coli, with distinctively different structural elements of TopoI-CTD utilized for this protein-protein interaction. Zinc ribbon motifs in E. coli TopoI-CTD are involved in the interaction with RNA polymerase. For M. smegmatis TopoI-CTD, a 27-amino-acid tail that is rich in basic residues at the C-terminal end is responsible for the interaction with RNA polymerase. Overexpression of recombinant TopoI-CTD in M. smegmatis competed with the endogenous topoisomerase I for protein-protein interactions with RNA polymerase. The TopoI-CTD overexpression resulted in decreased survival following treatment with antibiotics and hydrogen peroxide, supporting the importance of the protein-protein interaction between topoisomerase I and RNA polymerase during stress response of mycobacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. 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

  13. Structure of influenza A polymerase bound to the viral RNA promoter.

    PubMed

    Pflug, Alexander; Guilligay, Delphine; Reich, Stefan; Cusack, Stephen

    2014-12-18

    The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3' and 5' extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5' extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design.

  14. 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.

  15. 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

  16. The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila.

    PubMed

    Andrulis, Erik D; Werner, Janis; Nazarian, Arpi; Erdjument-Bromage, Hediye; Tempst, Paul; Lis, John T

    The RNA polymerase II elongation complex contains several factors that facilitate transcription elongation and catalyse the processing of precursor messenger RNAs (pre-mRNAs). The conserved elongation factor Spt6 is recruited rapidly and robustly to sites of active transcription. Here we show that Drosophila Spt6 (dSpt6) co-purifies with the exosome, a complex of 3' to 5' exoribonucleases that is implicated in the processing of structural RNA and in the degradation of improperly processed pre-mRNA. Immunoprecipitation assays of Drosophila nuclear extracts show that the exosome also associates with the elongation factor dSpt5 and RNA polymerase II. In vivo, exosome subunits colocalize with dSpt6 at transcriptionally active loci on polytene chromosomes during normal development and are strongly recruited to heat-shock loci on gene induction. At higher resolution, chromatin immunoprecipitation analysis shows that the exosome is recruited to transcriptionally active units of heat-shock genes. These data provide a physical basis for the hypothesis that exosome-mediated pre-mRNA surveillance accompanies transcription elongation.

  17. 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.

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

    PubMed Central

    Serebryannyy, Leonid A.; Parilla, Megan; Cruz, Christina M.; Laster, Kyle; Kudryashov, Dmitri; Kosak, Steven T.

    2016-01-01

    ABSTRACT 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. PMID:27505898

  19. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  20. Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation

    SciTech Connect

    Frenkel, G.D.; Falvey, D.

    1989-03-01

    We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.

  1. Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25.

    PubMed

    Delgado, M A; Rintoul, M R; Farías, R N; Salomón, R A

    2001-08-01

    Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

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

    PubMed Central

    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. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

  3. 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

    2008-10-04

    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.

  4. Understanding the Molecular Basis of RNA Polymerase II Transcription.

    PubMed

    Zhang, Su; Wang, Dong

    2013-06-01

    Synthetic nucleic acid analogues have profoundly advanced our knowledge of DNA and RNA, as well as the complex biological processes that involve nucleic acids. As a pivotal enzyme, eukaryotic RNA polymerase II (Pol II) is responsible for transcribing DNA into messenger RNA, which serves as a template to direct protein synthesis. Chemically modified nucleic acid analogues have greatly facilitated the structural elucidation of RNA Pol II elongation complex and understanding the key chemical interactions governing RNA Pol II transcriptional fidelity. This review addresses major progress in RNA polymerase II mechanistic studies using modified nucleic acid analogues in recent years.

  5. 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

  6. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    PubMed Central

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco; Haaning, Line Lindegaard; Jensen, Torben Heick

    2014-01-01

    In S. cerevisiae, the 5′-3′ exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood. Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII transcription kinetics, a feature that may contribute to the cellular phenotypes of the mutant. Consistently, the rat1-1 allele is synthetic lethal with the rpb1-E1103G mutation, causing increased RNAPII speed, and is suppressed by the rpb2-10 mutation, causing slowed transcription. Thus, Rat1p plays more complex roles in controlling transcription than previously thought. PMID:24501251

  7. Coliphage HK022 Nun protein inhibits RNA polymerase translocation

    PubMed Central

    Vitiello, Christal L.; Kireeva, Maria L.; Lubkowska, Lucyna; Kashlev, Mikhail; Gottesman, Max

    2014-01-01

    The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA–DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA–DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors. PMID:24853501

  8. Targeting mitochondrial RNA polymerase in acute myeloid leukemia

    PubMed Central

    Bralha, Fernando N.; Liyanage, Sanduni U.; Hurren, Rose; Wang, Xiaoming; Son, Meong Hi; Fung, Thomas A.; Chingcuanco, Francine B.; Tung, Aveline Y. W.; Andreazza, Ana C.; Psarianos, Pamela; Schimmer, Aaron D.; Salmena, Leonardo; Laposa, Rebecca R.

    2015-01-01

    Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 μM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 μMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML. PMID:26484416

  9. 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

  10. RNA polymerase II transcription on the fast lane.

    PubMed

    Marcello, Alessandro

    2012-01-01

    Transcription by RNA polymerase II is the process that copies DNA into RNA leading to the expression of a specific gene. Averaged estimates of polymerase elongation rates in mammalian cells have been shown to vary between 1 and 4 kilobases per minute. However, recent advances in live cell imaging allowed direct measurements of RNA biogenesis from a single gene exceeded 50 kb·min(-1) . This unexpected finding opens novel and intriguing perspectives on the control of metazoan transcription.

  11. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

    PubMed

    Cramer, P; Bushnell, D A; Kornberg, R D

    2001-06-08

    Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

  12. Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7.

    PubMed

    Park, Sung Hee; Nguyen, Tu N; Kirkham, Justin K; Lee, Ju Huck; Günzl, Arthur

    2011-11-01

    Trypanosoma brucei has a multifunctional RNA polymerase (pol) I that transcribes ribosomal gene units (RRNA) and units encoding its major cell surface proteins variant surface glycoprotein (VSG) and procyclin. Previous analysis of tandem affinity-purified, transcriptionally active RNA pol I identified ten subunits including an apparently trypanosomatid-specific protein termed RPA31. Another ortholog was identified in silico. No orthologs of the yeast subunit doublet RPA43/RPA14 have been identified yet. Instead, a recent report presented evidence that RPB7, the RNA pol II paralog of RPA43, is an RNA pol I subunit and essential for RRNA and VSG transcription in bloodstream form trypanosomes [18]. Revisiting this attractive hypothesis, we were unable to detect a stable interaction between RPB7 and RNA pol I in either reciprocal co-immunoprecipitation or tandem affinity purification. Furthermore, immunodepletion of RPB7 from extract virtually abolished RNA pol II transcription in vitro but had no effect on RRNA or VSG ES promoter transcription in the same reactions. Accordingly, chromatin immunoprecipitation analysis revealed cross-linking of RPB7 to known RNA pol II transcription units but not to the VSG ES promoter or to the 18S rRNA coding region. Interestingly, RPB7 did crosslink to the RRNA promoter but so did the RNA pol II-specific subunit RPB9 suggesting that RNA pol II is recruited to this promoter. Overall, our data led to the conclusion that RNA pol I transcription in T. brucei does not require the RNA pol II subunit RPB7.

  13. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. 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

    2016-05-31

    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.

  15. Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

    PubMed

    Tongaonkar, Prasad; French, Sarah L; Oakes, Melanie L; Vu, Loan; Schneider, David A; Beyer, Ann L; Nomura, Masayasu

    2005-07-19

    Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

  16. Human DNA polymerase η accommodates RNA for strand extension.

    PubMed

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2017-09-26

    Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase alpha, telomerase, and the mitochondrial DNA polymerase hpol gamma have been shown to tolerate an entire RNA strand. Y-family DNA polymerase eta] (hpol eta) is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer (CPD) and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol eta is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol eta--as well as another Y-family DNA polymerase, hpol kappa--accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions such as 8-oxo-2'-deoxyguanosine or CPD, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA accommodating ability of hpol eta redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol eta in vivo. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  17. Optical tweezers studies of transcription by eukaryotic RNA polymerases.

    PubMed

    Lisica, Ana; Grill, Stephan W

    2017-02-21

    Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

  18. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

    PubMed Central

    Gibbs, Peter E. M.; McGregor, W. Glenn; Maher, Veronica M.; Nisson, Paul; Lawrence, Christopher W.

    1998-01-01

    To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart. PMID:9618506

  19. 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.

  20. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    PubMed Central

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

  1. Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.

    PubMed

    Bressanin, Daniela; Stefan, Alessandra; Piaz, Fabrizio Dal; Cianchetta, Stefano; Reggiani, Luca; Hochkoeppler, Alejandro

    2009-11-01

    The C-terminal region of the proofreading subunit (epsilon) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (alpha). We also identify two alpha-helices of the polymerase subunit (comprising the residues E311-M335 and G339-D353, respectively) as the determinants of binding to epsilon. The C-terminal region of epsilon is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of epsilon. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of epsilon. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 degrees C, and viability can be restored over-expressing epsilon. Altogether, our observations indicate that the association between epsilon and alpha subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of epsilon. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.

  2. 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

  3. 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.

  4. 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.

  5. relA-dependent RNA polymerase activity in Escherichia coli.

    PubMed Central

    Ryals, J; Bremer, H

    1982-01-01

    Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase. The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total instantaneous rate of RNA synthesis) decreased 1.7-fold in the relA+ strain and increased 1.9-fold in the relA mutant; and (iv) the RNA polymerase activity (measured by the percentage of total RNA polymerase enzyme active in transcription an any instant) decreased from 20 to 3.6% in the relA+ strain and remained unchanged (or increased at most to 22%) in the relA mutant. It is suggested that both rRNA gene activity and the RNA polymerase activity depend on the intracellular concentration of guanosine tetraphosphate, whereas the altered chain elongation rate and stability of rRNA are temperature or amino acid starvation effects, respectively, without involvement of relA function. PMID:6174501

  6. Purification of the DNA-dependent RNA polymerase from the myxobacterium Stigmatella aurantiaca.

    PubMed Central

    Heidelbach, M; Skladny, H; Schairer, H U

    1992-01-01

    The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera. Images PMID:1556092

  7. Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers.

    PubMed

    Mehle, Andrew; Dugan, Vivien G; Taubenberger, Jeffery K; Doudna, Jennifer A

    2012-02-01

    The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.

  8. 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.

  9. DNA wrapping in transcription initiation by RNA polymerase II.

    PubMed

    Coulombe, B

    1999-01-01

    The DNA wrapping model of transcription stipulates that DNA bending and wrapping around RNA polymerase causes an unwinding of the DNA helix at the enzyme catalytic center that stimulates strand separation prior to initiation and during transcript elongation. Recent experiments with mammalian RNA polymerase II indicate the significance of DNA bending and wrapping in transcriptional mechanisms. These findings have important implications in our understanding of the role of the general transcription factors in transcriptional initiation and the mechanisms underlying transcriptional regulation.

  10. X-ray crystal structures of the Escherichia coli RNA polymerase in complex with Benzoxazinorifamycins

    PubMed Central

    Molodtsov, Vadim; Nawarathne, Irosha N.; Scharf, Nathan T.; Kirchhoff, Paul D.; Hollis Showalter, H. D.; Garcia, George A.; Murakami, Katsuhiko S.

    2013-01-01

    Rifampin, a semi-synthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semi-synthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases, and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the β subunit, blocking the path of the RNA transcript. The C3′-tail of benzoxazinorifamycin fits a cavity between the β subunit and σ factor. We propose that, in addition to blocking RNA exit, the benzoxazinorifamycin C3′-tail changes the σ region3.2 loop position, which influences the template DNA at the active site thereby reducing the efficiency of transcription initiation. This study supports expansion of structure–activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential. PMID:23679862

  11. 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.

  12. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    PubMed Central

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  13. 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.

  14. A protein-protein interaction map of yeast RNA polymerase III.

    PubMed

    Flores, A; Briand, J F; Gadal, O; Andrau, J C; Rubbi, L; Van Mullem, V; Boschiero, C; Goussot, M; Marck, C; Carles, C; Thuriaux, P; Sentenac, A; Werner, M

    1999-07-06

    The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

  15. Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

    PubMed

    Mirzakhanyan, Yeva; Gershon, Paul D

    2017-09-01

    The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

  16. 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

  17. Visualization of Single Molecules of RNA Polymerase Sliding along DNA

    NASA Astrophysics Data System (ADS)

    Kabata, Hiroyuki; Kurosawa, Osamu; Arai, Ichiro; Washizu, Masao; Margarson, Stefanie A.; Glass, Robert E.; Shimamoto, Nobuo

    1993-12-01

    Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage λ and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

  18. CDK9-dependent RNA polymerase II pausing controls transcription initiation.

    PubMed

    Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

    2017-10-10

    Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

  19. Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids.

    PubMed Central

    Bischler, N; Balavoine, F; Milkereit, P; Tschochner, H; Mioskowski, C; Schultz, P

    1998-01-01

    Nickel-chelating lipid monolayers were used to generate two-dimensional crystals from yeast RNA polymerase I that was histidine-tagged on one of its subunits. The interaction of the enzyme with the spread lipid layers was found to be imidazole dependent, and the formation of two-dimensional crystals required small amounts of imidazole, probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms, indicating that the position of the tag determines the crystallization process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a three-dimensional model which shows that the tagged subunits are in contact with the lipid layer. PMID:9512048

  20. 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.

  1. The non-coding B2 RNA binds to the DNA cleft and active-site region of RNA polymerase II.

    PubMed

    Ponicsan, Steven L; Houel, Stephane; Old, William M; Ahn, Natalie G; Goodrich, James A; Kugel, Jennifer F

    2013-10-09

    The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription

    PubMed Central

    Clemente-Blanco, Andres; Sen, Nicholas; Mayan-Santos, Maria; Sacristán, Maria P.; Graham, Bryony; Jarmuz, Adam; Giess, Adam; Webb, Elizabeth; Game, Laurence; Eick, Dirk; Bueno, Avelino; Merkenschlager, Matthias; Aragón, Luis

    2011-01-01

    Kinases and phosphatases regulate mRNA synthesis through post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II 1. In yeast, the phosphatase Cdc14 is required for mitotic exit 2,3 and for segregation of repetitive regions 4. Cdc14 is also a subunit of the silencing complex RENT 5,6, but no roles in transcription repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences (IGS) of ribosomal genes during interphase and at Y’ repeats in sub-telomeric regions during mitosis. We show that Cdc14 role in silencing is independent from the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA Polymerase II by targeting CTD phosphorylation at S2 and S5. We also find that Cdc14 role as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants 4 correlate with the presence of sub-telomeric Y’ elements and can be rescued by transcriptional inhibition of RNA Pol II. PMID:22020438

  3. Evolution of tertiary structure of viral RNA dependent polymerases.

    PubMed

    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.

  4. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    SciTech Connect

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  5. 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.

  6. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24614752

  8. 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.

  9. Backtracking dynamics of RNA polymerase: pausing and error correction

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2013-09-01

    Transcription by RNA polymerases is frequently interrupted by pauses. One mechanism of such pauses is backtracking, where the RNA polymerase translocates backward with respect to both the DNA template and the RNA transcript, without shortening the transcript. Backtracked RNA polymerases move in a diffusive fashion and can return to active transcription either by diffusive return to the position where backtracking was initiated or by cleaving the transcript. The latter process also provides a mechanism for proofreading. Here we present some exact results for a kinetic model of backtracking and analyse its impact on the speed and the accuracy of transcription. We show that proofreading through backtracking is different from the classical (Hopfield-Ninio) scheme of kinetic proofreading. Our analysis also suggests that, in addition to contributing to the accuracy of transcription, backtracking may have a second effect: it attenuates the slow down of transcription that arises as a side effect of discriminating between correct and incorrect nucleotides based on the stepping rates.

  10. Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

    PubMed Central

    Liu, B; Troxler, R F

    1996-01-01

    We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level. Images Fig. 1 Fig. 4 Fig. 6 PMID:8622935

  11. The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

    PubMed

    Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

    2011-01-01

    The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

  12. Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome.

    PubMed Central

    Pühler, G; Leffers, H; Gropp, F; Palm, P; Klenk, H P; Lottspeich, F; Garrett, R A; Zillig, W

    1989-01-01

    Genes for DNA-dependent RNA polymerase components B, A, and C from the archaebacterium Sulfolobus acidocaldarius and for components B", B', A, and C from the archaebacterium Halobacterium halobium were cloned and sequenced. They are organized in gene clusters in the order above, which corresponds to the order of the homologous rpoB and rpoC genes in the corresponding operon of the Escherichia coli genome. Derived amino acid sequences of archaebacterial components A and C were aligned with each other and with the sequences of corresponding (largest) subunits from the archaebacterium Methanobacterium thermoautotrophicum, with sequences of various eukaryotic nuclear RNA polymerases I, II, and III, and with the sequence of the beta' component from E. coli polymerase. The archaebacterial genes for component A are homologous to about the first two-thirds of genes for the eukaryotic component A and the eubacterial component beta', and the archaebacterial genes for component C are homologous to the last third of the genes for the eukaryotic component A and the eubacterial component beta'. Unrooted phylogenetic dendrograms derived from both distance matrix and parsimony analyses show the archaebacteria are a coherent group closely related to the eukaryotic nuclear RNA polymerase II and/or III lineages. The eukaryotic polymerase I lineage appears to arise separately from a bifurcation with the eubacterial beta' component lineage. PMID:2499884

  13. RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.

    PubMed Central

    Brabant, F; Acheson, N H

    1995-01-01

    RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion. PMID:7769704

  14. A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain.

    PubMed

    Liu, B; Wong, M L; Alberts, B

    1994-10-25

    We have demonstrated elsewhere that a precisely placed, stalled Escherichia coli RNA polymerase ternary transcription complex (polymerase-RNA-DNA) stays on the DNA template after passage of a DNA replication fork. Moreover, the bypassed complex remains competent to resume elongation of its bound RNA chain. But the simplicity of our experimental system left several important questions unresolved: in particular, might the observation be relevant only to the particular ternary complex that we studied, and can the finding be generalized to a transcribing instead of a stalled RNA polymerase? To address these issues, we have created three additional ternary transcription complexes and examined their fates after passage of a replication fork. In addition, we have examined the fate of moving RNA polymerase molecules during DNA replication. The results suggest that our previous finding applies to all transcription intermediates of the E. coli RNA polymerase.

  15. The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site

    PubMed Central

    Tupin, Audrey; Gualtieri, Maxime; Leonetti, Jean-Paul; Brodolin, Konstantin

    2010-01-01

    Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the σ70 subunit region 3.2 and the RNAP β′ subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the ‘closed'-promoter complex to the transcriptionally competent ‘open' complex and blocks σ70-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the β′ subunit switch-2 element and the σ70 subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process. PMID:20562828

  16. The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site.

    PubMed

    Tupin, Audrey; Gualtieri, Maxime; Leonetti, Jean-Paul; Brodolin, Konstantin

    2010-08-04

    Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the sigma(70) subunit region 3.2 and the RNAP beta' subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks sigma(70)-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the beta' subunit switch-2 element and the sigma(70) subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

  17. Molecular architecture of the vesicular stomatitis virus RNA polymerase

    PubMed Central

    Rahmeh, Amal A.; Schenk, Andreas D.; Danek, Eric I.; Kranzusch, Philip J.; Liang, Bo; Walz, Thomas; Whelan, Sean P. J.

    2010-01-01

    Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein–RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses. PMID:21041632

  18. Molecular architecture of the vesicular stomatitis virus RNA polymerase.

    PubMed

    Rahmeh, Amal A; Schenk, Andreas D; Danek, Eric I; Kranzusch, Philip J; Liang, Bo; Walz, Thomas; Whelan, Sean P J

    2010-11-16

    Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

  19. The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

    PubMed Central

    Wu-Baer, F; Lane, W S; Gaynor, R B

    1995-01-01

    Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation. Images PMID:8846792

  20. A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

    PubMed Central

    Suh, Man-Hee; Meyer, Peter A.; Gu, Meigang; Ye, Ping; Zhang, Mincheng; Kaplan, Craig D.; Lima, Christopher D.; Fu, Jianhua

    2010-01-01

    RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes. PMID:20720002

  1. Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

    PubMed Central

    Zhu, Bin; Hernandez, Alfredo; Tan, Min; Wollenhaupt, Jan; Tabor, Stanley; Richardson, Charles C.

    2015-01-01

    The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified. PMID:25897116

  2. Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II.

    PubMed

    Cline, Susan D; Riggins, James N; Tornaletti, Silvia; Marnett, Lawrence J; Hanawalt, Philip C

    2004-05-11

    Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

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

    PubMed

    Theis, Karsten

    2013-12-01

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

  4. Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex

    NASA Astrophysics Data System (ADS)

    Murakami, Katsuhiko S.; Masuda, Shoko; Campbell, Elizabeth A.; Muzzin, Oriana; Darst, Seth A.

    2002-05-01

    The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (α2ββ'ωσA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the σ subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the σ subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

  5. 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.

  6. A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases

    PubMed Central

    Bruenn, Jeremy A.

    2003-01-01

    A systematic bioinformatic approach to identifying the evolutionarily conserved regions of proteins has verified the universality of a newly described conserved motif in RNA-dependent RNA polymerases (motif F). In combination with structural comparisons, this approach has defined two regions that may be involved in unwinding double-stranded RNA (dsRNA) for transcription. One of these is the N-terminal portion of motif F and the second is a large insertion in motif F present in the RNA-dependent RNA polymerases of some dsRNA viruses. PMID:12654997

  7. Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

    PubMed Central

    Gulati, S. C.; Kacian, D. L.; Spiegelman, S.

    1974-01-01

    Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture. PMID:4133845

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria

    PubMed Central

    Dey, Abhinav; Adithi, V.R.; Chatterji, Dipankar

    2012-01-01

    The role of RbpA in the backdrop of M. smegmatis showed that it rescues mycobacterial RNA polymerase from rifampicin-mediated inhibition (Dey et al., 2010; Dey et al., 2011). Paget and co-workers (Paget et al., 2001; Newell et al., 2006) have revealed that RbpA homologs occur exclusively in actinobacteria. Newell et al. (2006) showed that MtbRbpA, when complemented in a ∆rbpA mutant of S. coelicolor, showed a low recovery of MIC (from 0.75 to 2 μg/ml) as compared to complementation by native RbpA of S. coelicolor (MIC increases from 0.75 to 11 μg/ml). Our studies on MsRbpA show that it is a differential marker for M. smegmatis RNA polymerase as compared to E. coli RNA polymerase at IC50 levels of rifampicin. A recent sequence-based analysis by Lane and Darst (2010) has shown that RNA polymerases from Proteobacteria and Actinobacteria have had a divergent evolution. E. coli is a representative of Proteobacteria and M. smegmatis is an Actinobacterium. RbpA has an exclusive occurrence in Actinobacteria. Since protein–protein interactions might not be conserved across different species, therefore, the probable reason for the indifference of MsRbpA toward E. coli RNA polymerase could be the lineage-specific differences between actinobacterial and proteobacterial RNA polymerases. These observations led us to ask the question as to whether the evolution of RbpA in Actinobacteria followed the same route as that of RNA polymerase subunits from actinobacterial species. We show that the exclusivity of RbpA in Actinobacteria and the unique evolution of RNA polymerase in this phylum share a co-evolutionary link. We have addressed this issue by a blending of experimental and bioinformatics based approaches. They comprise of induction of bacterial cultures coupled to rifampicin-tolerance, transcription assays and statistical comparison of phylogenetic trees for different pairs of proteins in actinobacteria. PMID:27896048

  14. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    PubMed Central

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  16. 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.

  17. A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.

    PubMed

    Yakunina, Maria; Artamonova, Tatyana; Borukhov, Sergei; Makarova, Kira S; Severinov, Konstantin; Minakhin, Leonid

    2015-12-02

    The infection of Pseudomonas aeruginosa by the giant bacteriophage phiKZ is resistant to host RNA polymerase (RNAP) inhibitor rifampicin. phiKZ encodes two sets of polypeptides that are distantly related to fragments of the two largest subunits of cellular multisubunit RNAPs. Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions. Polypeptides of the second set are encoded by early phage genes and are absent from virions. Here, we report isolation of a five-subunit RNAP from phiKZ-infected cells. Four subunits of this enzyme are cellular RNAP subunits homologs of the non-virion set; the fifth subunit is a protein of unknown function. In vitro, this complex initiates transcription from late phiKZ promoters in rifampicin-resistant manner. Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes. The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.

  18. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

    PubMed Central

    te Velthuis, Aartjan J.W.; Fodor, Ervin

    2016-01-01

    The genome of influenza viruses consists of multiple segments of single stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, forming viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, how it carries out transcription and replication, and how its activities are modulated by viral and host factors. Furthermore, we discuss how advances in our understanding of polymerase function could help identifying new antiviral targets. PMID:27396566

  19. Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

    PubMed Central

    Spagnolo, Jeannie F.; Rossignol, Evan; Bullitt, Esther; Kirkegaard, Karla

    2010-01-01

    Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells. PMID:20051491

  20. Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

    PubMed

    Yu, Hsiang-Ping; Hsiao, Yi-Ling; Pan, Hung-Yin; Huang, Chih-Hung; Hou, Shao-Yi

    2011-12-15

    A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.

  1. Recent highlights of RNA-polymerase-II-mediated transcription.

    PubMed

    Sims, Robert J; Mandal, Subhrangsu S; Reinberg, Danny

    2004-06-01

    Considerable advances into the basis of RNA-polymerase-II-mediated transcriptional regulation have recently emerged. Biochemical, genetic and structural studies have contributed to novel insights into transcription, as well as the functional significance of covalent histone modifications. New details regarding transcription elongation through chromatin have further defined the mechanism behind this action, and identified how chromatin structure may be maintained after RNAP II traverses a nucleosome. ATP-dependent chromatin remodeling complexes, along with histone chaperone complexes, were recently discovered to facilitate histone exchange. In addition, it has become increasingly clear that transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm.

  2. 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

  3. The catalytic subunit of DNA polymerase δ inhibits γTuRC activity and regulates Golgi-derived microtubules.

    PubMed

    Shen, Yuehong; Liu, Pengfei; Jiang, Taolue; Hu, Yu; Au, Franco K C; Qi, Robert Z

    2017-09-15

    γ-Tubulin ring complexes (γTuRCs) initiate microtubule growth and mediate microtubule attachment at microtubule-organizing centers, such as centrosomes and the Golgi complex. However, the mechanisms that control γTuRC-mediated microtubule nucleation have remained mostly unknown. Here, we show that the DNA polymerase δ catalytic subunit (PolD1) binds directly to γTuRCs and potently inhibits γTuRC-mediated microtubule nucleation. Whereas PolD1 depletion through RNA interference does not influence centrosome-based microtubule growth, the depletion augments microtubule nucleation at the Golgi complex. Conversely, PolD1 overexpression inhibits Golgi-based microtubule nucleation. Golgi-derived microtubules are required for the assembly and maintenance of the proper Golgi structure, and we found that alteration of PolD1 levels affects Golgi structural organization. Moreover, suppression of PolD1 expression impairs Golgi reassembly after nocodazole-induced disassembly and causes defects in Golgi reorientation and directional cell migration. Collectively, these results reveal a mechanism that controls noncentrosomal γTuRC activity and regulates the organization of Golgi-derived microtubules.Microtubule organization requires γ-tubulin ring complexes (γTuRCs), but the mechanisms that control γTuRC-mediated microtubule nucleation are unclear. Here the authors show that the DNA polymerase δ catalytic subunit controls noncentrosomal γTuRC activity and regulates the organization of Golgi-derived microtubules.

  4. Single-molecule tracking of mRNA exiting from RNA polymerase II.

    PubMed

    Andrecka, Joanna; Lewis, Robert; Brückner, Florian; Lehmann, Elisabeth; Cramer, Patrick; Michaelis, Jens

    2008-01-08

    Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5' end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5'-capping enzyme and other RNA processing factors.

  5. The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

    PubMed Central

    te Velthuis, Aartjan J. W.; Arnold, Jamie J.; Cameron, Craig E.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

    2010-01-01

    An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the ∼30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8. PMID:19875418

  6. Structural basis of RNA polymerase II backtracking, arrest and reactivation.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2011-03-10

    During gene transcription, RNA polymerase (Pol) II moves forwards along DNA and synthesizes messenger RNA. However, at certain DNA sequences, Pol II moves backwards, and such backtracking can arrest transcription. Arrested Pol II is reactivated by transcription factor IIS (TFIIS), which induces RNA cleavage that is required for cell viability. Pol II arrest and reactivation are involved in transcription through nucleosomes and in promoter-proximal gene regulation. Here we present X-ray structures at 3.3 Å resolution of an arrested Saccharomyces cerevisiae Pol II complex with DNA and RNA, and of a reactivation intermediate that additionally contains TFIIS. In the arrested complex, eight nucleotides of backtracked RNA bind a conserved 'backtrack site' in the Pol II pore and funnel, trapping the active centre trigger loop and inhibiting mRNA elongation. In the reactivation intermediate, TFIIS locks the trigger loop away from backtracked RNA, displaces RNA from the backtrack site, and complements the polymerase active site with a basic and two acidic residues that may catalyse proton transfers during RNA cleavage. The active site is demarcated from the backtrack site by a 'gating tyrosine' residue that probably delimits backtracking. These results establish the structural basis of Pol II backtracking, arrest and reactivation, and provide a framework for analysing gene regulation during transcription elongation.

  7. DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons.

    PubMed

    Zhong, Xuehua; Hale, Christopher J; Law, Julie A; Johnson, Lianna M; Feng, Suhua; Tu, Andy; Jacobsen, Steven E

    2012-09-01

    The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution.

  8. RNA-dependent DNA-polymerase activity in human milk.

    PubMed

    McFarlane, E S; Ryan, S M; Mann, E

    1975-08-01

    A simple method is described for testing milk specimens from nursing mothers for the presence of RNA-dependent DNA-polymerase activity. Positive results were obtained in five of 137 women (3.6%) without a family history of breast cancer, and in six of 31 women (19.3%) with a family history of breast cancer.

  9. Transcription rate of RNA polymerase under rotary torque

    NASA Astrophysics Data System (ADS)

    Kiryu, H.

    2004-04-01

    We investigated the transcription rates of RNA polymerases that were subjected to rotational drag. By combining chemical kinetics with mechanical equations, we derived formulas for the transcription rate in the case where the torque was caused by the hydrodynamic drag to DNA rotation.

  10. The Continuing SAGA of TFIID and RNA Polymerase II Transcription.

    PubMed

    Taatjes, Dylan J

    2017-10-05

    Using improved techniques, Baptista et al. (2017) and Warfield et al. (2017) revisit fundamental questions about SAGA and TFIID function in yeast. They conclude that each complex independently contributes to the expression of all genes transcribed by RNA polymerase II. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. The Ribosome Holds the RNA Polymerase on Track in Bacteria.

    PubMed

    Klaholz, Bruno P

    2017-09-01

    The central dogma of molecular biology comprises two fundamental mechanistic steps of gene expression (transcription and translation), which, in bacteria, are coupled. A recent study provides structural insights into a supercomplex between the RNA polymerase and the ribosome, thus highlighting the synergy between two key macromolecular machineries in the cell. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Effects of anti-alpha monoclonal antibodies on initiation and elongation by the Escherichia coli RNA polymerase.

    PubMed

    Venezia, N D; Krakow, J S

    1990-05-15

    Anti-alpha monoclonal antibodies have been used to investigate the role of the alpha subunit of RNA polymerase in initiation and elongation. The four inhibitory monoclonal antibodies studied strongly inhibit cAMP receptor protein-dependent initiation with lac P+ and partially inhibit initiation directed by the lac UV5 promoter. The data suggest that the epitopes to which each of the monoclonal antibodies bind may be proximal to the contact domain between cAMP receptor protein and RNA polymerase. Recycling of RNA polymerase through the initiation process is slower in the presence of an inhibitory monoclonal antibody. Once a 9-nucleotide-long transcript has formed, incubation with the anti-alpha monoclonal antibody does not affect subsequent elongation. The monoclonal antibodies still bind to the elongation complex as indicated by sedimentation of the complex formed after incubation with Staphylococcus aureus cells (immunoprecipitin). These results suggest that the resistance of the elongation complex to these antibodies is not a consequence of their inability to bind to RNA polymerase. Only one of the alpha subunits may be involved in the initial process of transcription, and the antigenic domain of this subunit appears to be occluded by the nascent transcript present in the elongation complex.

  13. Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

    PubMed Central

    Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

    2014-01-01

    The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

  14. Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules.

    PubMed

    Chetverin, Alexander B; Kopein, Damir S; Chetverina, Helena V; Demidenko, Alexander A; Ugarov, Victor I

    2005-03-11

    An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

  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

    SciTech Connect

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

    2015-03-12

    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. Finally, evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.

  17. Ancient origin and recent innovations of RNA polymerase IV and V

    DOE PAGES

    Huang, Yi; Kendall, Timmy; Forsythe, Evan S.; ...

    2015-03-12

    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 highlymore » 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. Finally, evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.« less

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

    PubMed

    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

    2015-07-01

    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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. 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

  20. Influenza virion RNA-dependent RNA polymerase: stimulation by guanosine and related compounds.

    PubMed Central

    McGeoch, D; Kitron, N

    1975-01-01

    The activity of RNA-dependent RNA polymerase of several influenza viruses is stimulated by guanosine. Depending upon the virus strain used, the stimulation of initial reaction rate is up to 10-fold. 5'-GMP, 3',5'-cyclic GMP, and 5'-GDP show lesser stimulation effects. No other nucleosides of 5'-NMPs stimulate, but the dinucleoside monophosphates GpG and GpC show large stimulations. We present evidence that the stimulation represents preferential initiation of genome complementary RNA chains with guanosine: (i) [3-H] guanosine is incorporated specifically at the 5'terminus of RNA in polymerase reaction mixes in vitro. (ii) This incorporation reaction has several properties similar to those of the virion polymerase elongation reaction. (iii) RNA made in the stimulated reaction behaves as complementary RNA in annealing kinetic studies, as does RNA labeled with [3-H]guanosine. PMID:163915

  1. 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

  2. Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

    PubMed

    Miyake, Yukari; Ishii, Kosuke; Honda, Ayae

    2017-01-01

    Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

  3. Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase

    PubMed Central

    Miyake, Yukari; Ishii, Kosuke; Honda, Ayae

    2017-01-01

    Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase. PMID:28232820

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

    PubMed Central

    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. PMID:25764112

  5. 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.

  6. A movie of the RNA polymerase nucleotide addition cycle.

    PubMed

    Brueckner, Florian; Ortiz, Julio; Cramer, Patrick

    2009-06-01

    During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop. The movie facilitates teaching and a mechanistic analysis of transcription and can be downloaded from http://www.lmb.uni-muenchen.de/cramer/pr-materials.

  7. Fast transcription rates of RNA polymerase II in human cells

    PubMed Central

    Maiuri, Paolo; Knezevich, Anna; De Marco, Alex; Mazza, Davide; Kula, Anna; McNally, Jim G; Marcello, Alessandro

    2011-01-01

    Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1-derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells. PMID:22015688

  8. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

    PubMed Central

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-01-01

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene. PMID:28289142

  9. The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain.

    PubMed

    Harlen, Kevin M; Churchman, L Stirling

    2017-04-01

    The carboxy-terminal domain (CTD) extends from the largest subunit of RNA polymerase II (Pol II) as a long, repetitive and largely unstructured polypeptide chain. Throughout the transcription process, the CTD is dynamically modified by post-translational modifications, many of which facilitate or hinder the recruitment of key regulatory factors of Pol II that collectively constitute the 'CTD code'. Recent studies have revealed how the physicochemical properties of the CTD promote phase separation in the presence of other low-complexity domains. Here, we discuss the intricacies of the CTD code and how the newly characterized physicochemical properties of the CTD expand the function of the CTD beyond the code.

  10. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene.

    PubMed

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-02-15

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene.

  11. 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.

  12. RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization.

    PubMed

    Hizi, A; Yaniv, A

    1980-06-01

    An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus.

  13. RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization.

    PubMed Central

    Hizi, A; Yaniv, A

    1980-01-01

    An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus. Images PMID:6155478

  14. Insights into the Mechanism of Initial Transcription in Escherichia coli RNA Polymerase*

    PubMed Central

    Samanta, Satamita; Martin, Craig T.

    2013-01-01

    It has long been known that during initial transcription of the first 8–10 bases of RNA, complexes are relatively unstable, leading to the release of short abortive RNA transcripts. An early “stressed intermediate” model led to a more specific mechanistic model proposing “scrunching” stress as the basis for the instability. Recent studies in the single subunit T7 RNA polymerase have argued against scrunching as the energetic driving force and instead argue for a model in which pushing of the RNA-DNA hybrid against a protein element associated with promoter binding, while likely driving promoter release, reciprocally leads to instability of the hybrid. In this study, we test these models in the structurally unrelated multisubunit bacterial RNA polymerase. Via the targeted introduction of mismatches and nicks in the DNA, we demonstrate that neither downstream bubble collapse nor compaction/scrunching of either the single-stranded template or nontemplate strands is a major force driving abortive instability (although collapse from the downstream end of the bubble does contribute significantly to the instability of artificially halted complexes). In contrast, pushing of the hybrid against a mobile protein element (σ3.2 in the bacterial enzyme) results in substantially increased abortive instability and is likely the primary energetic contributor to abortive cycling. The results suggest that abortive instability is a by-product of the mechanistic need to couple the energy of nucleotide addition (RNA chain growth) to driving the timed release of promoter contacts during initial transcription. PMID:24047893

  15. Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

    PubMed

    Herrera-Asmat, Omar; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos J; Guerra, Daniel G; Kireeva, Maria L

    2017-03-18

    Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α2ββ'ω·σ(A) can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD.

  16. A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions.

    PubMed

    Chen, Han; Coseno, Molly; Ficarro, Scott B; Mansueto, My Sam; Komazin-Meredith, Gloria; Boissel, Sandrine; Filman, David J; Marto, Jarrod A; Hogle, James M; Coen, Donald M

    2017-02-10

    Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

  17. Different pathways for the nuclear import of yeast RNA polymerase II.

    PubMed

    Gómez-Navarro, Natalia; Estruch, Francisco

    2015-11-01

    Recent studies suggest that RNA polymerase II (Pol II) has to be fully assembled before being imported into the nucleus, while other reports indicate a distinct mechanism to import large and small subunits. In yeast, Iwr1 binds to the holoenzyme assembled in the cytoplasm and directs its nuclear entry. However, as IWR1 is not an essential gene, Iwr1-independent pathway(s) for the nuclear import of Pol II must exist. In this paper, we investigate the transport into the nucleus of several large and small Pol II subunits in the mutants of genes involved in Pol II biogenesis. We also analyse subcellular localization in the presence of drugs that can potentially affect Pol II nuclear import. Our results show differences in the cellular distribution between large and small subunits when Pol II biogenesis was impaired. Our data suggest that, in addition to the fully assembled holoenzyme, Pol II subunits can be imported to the nucleus, either independently or as partial assemblies, through different pathways, including passive diffusion for the small subunits.

  18. 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

  19. 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.

  20. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis.

    PubMed

    Wiedermannová, Jana; Sudzinová, Petra; Kovaľ, Tomaš; Rabatinová, Alžbeta; Šanderova, Hana; Ramaniuk, Olga; Rittich, Šimon; Dohnálek, Jan; Fu, Zhihui; Halada, Petr; Lewis, Peter; Krásny, Libor

    2014-04-01

    Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

  1. Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

    PubMed Central

    Kim, Tae-Kyung; Lagrange, Thierry; Wang, Yuh-Hwa; Griffith, Jack D.; Reinberg, Danny; Ebright, Richard H.

    1997-01-01

    By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, ≈240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of ≈50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA. PMID:9356438

  2. Rtp1p is a karyopherin-like protein required for RNA polymerase II biogenesis.

    PubMed

    Gómez-Navarro, Natalia; Peiró-Chova, Lorena; Rodriguez-Navarro, Susana; Polaina, Julio; Estruch, Francisco

    2013-05-01

    The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex.

  3. Recruitment of RED-SMU1 Complex by Influenza A Virus RNA Polymerase to Control Viral mRNA Splicing

    PubMed Central

    Munier, Sandie; Tomoiu, Andru; Demeret, Caroline; Vidalain, Pierre-Olivier; Jacob, Yves; Naffakh, Nadia

    2014-01-01

    Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs. PMID:24945353

  4. Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

    PubMed

    Fournier, Guillaume; Chiang, Chiayn; Munier, Sandie; Tomoiu, Andru; Demeret, Caroline; Vidalain, Pierre-Olivier; Jacob, Yves; Naffakh, Nadia

    2014-06-01

    Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

  5. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair.

    PubMed

    Brocas, Clémentine; Charbonnier, Jean-Baptiste; Dhérin, Claudine; Gangloff, Serge; Maloisel, Laurent

    2010-10-05

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair.

  6. Cloning, expression, and functional characterization of the equine herpesvirus 1 DNA polymerase and its accessory subunit.

    PubMed

    Loregian, Arianna; Case, Alessandro; Cancellotti, Enrico; Valente, Carlo; Marsden, Howard S; Palù, Giorgio

    2006-07-01

    We report the expression and characterization of the putative catalytic subunit (pORF30) and accessory protein (pORF18) of equine herpesvirus 1 DNA polymerase, which are encoded by open reading frames 30 and 18 and are homologous to herpes simplex virus type 1 UL30 and UL42, respectively. In vitro transcription-translation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitro-expressed pORF30 possessed basal DNA polymerase activity that was stimulated by pORF18, as measured by DNA polymerase assays in vitro. Purified baculovirus-expressed pORF30 exhibited DNA polymerase activity similar to that of the in vitro-expressed protein, and baculovirus-expressed pORF18 could stimulate both nucleotide incorporation and long-chain DNA synthesis by pORF30 in a dose- and time-dependent manner. The salt optima for activity of both pORF30 and the holoenzyme were substantially different from those for other herpesvirus DNA polymerases. As demonstrated by yeast two-hybrid assays, pORF30 and pORF18 could physically interact, most likely with a 1:1 stoichiometry. Finally, by mutational analysis of the 1,220-residue pORF30, we demonstrated that the extreme C terminus of pORF30 is important for physical and functional interaction with the accessory protein, as reported for UL30 and other herpesvirus DNA polymerases. In addition, a C-proximal region of pORF30, corresponding to residues 1114 to 1172, is involved in binding to, and stimulation by, pORF18. Taken together, the results indicate that pORF30 and pORF18 are the equine herpesvirus 1 counterparts of herpes simplex virus type 1 UL30 and UL42 and share many, but not all, of their characteristics.

  7. Human Polymerase-Associated Factor complex (PAFc) connects the Super Elongation Complex (SEC) to RNA polymerase II on chromatin.

    PubMed

    He, Nanhai; Chan, Caleb K; Sobhian, Bijan; Chou, Seemay; Xue, Yuhua; Liu, Min; Alber, Tom; Benkirane, Monsef; Zhou, Qiang

    2011-09-06

    The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.

  8. Architecture of the RNA polymerase II-Mediator core initiation complex.

    PubMed

    Plaschka, C; Larivière, L; Wenzeck, L; Seizl, M; Hemann, M; Tegunov, D; Petrotchenko, E V; Borchers, C H; Baumeister, W; Herzog, F; Villa, E; Cramer, P

    2015-02-19

    The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

  9. 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

  10. 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

  11. Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme*

    PubMed Central

    Basu, Ritwika S.; Warner, Brittany A.; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S.

    2014-01-01

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5′-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. PMID:24973216

  12. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme.

    PubMed

    Basu, Ritwika S; Warner, Brittany A; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S

    2014-08-29

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Functional state of rat liver RNA polymerase IA and IB.

    PubMed

    Zoncheddu, A; Accomando, R; Pertica, M; Orunesu, M

    1979-01-01

    Phosphocellulose chromatography has been employed to characterize RNA polymerase I present in two different functional states in rat liver cells. The actively transcribing enzyme solubilized from nuclei appears to belong both to the IA and IB classes, whereas the non-transcribing enzyme present in the cytoplasmic fraction has been found to belong only to the IA class. Indirect and direct evidence indicates, however, that in isolated nuclei only the IB form is to be regarded as the physiological form of the enzyme, the IA form arising as a procedural artefact during the extraction process. It may, therefore, be concluded that rat liver IA and IB RNA polymerase are to be strictly regarded as the non-transcribing and transcribing form of the enzyme, respectively.

  14. 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

  15. Structure and function of the bacteriophage T7 RNA polymerase (or, the virtues of simplicity).

    PubMed

    McAllister, W T

    1993-01-01

    A consideration of the properties of a number of mutants of T7 RNA polymerase, together with emerging structural information (Sousa et al., 1993) allows an interpretation of the the mechanics of transcription by this relatively simple RNA polymerase. Evidence indicating features in common with other nucleotide polymerases (such as DNA polymerases and reverse transcriptases) is reviewed.

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

    PubMed Central

    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 K354, R355, and K367 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 system

  17. The RNA-dependent RNA polymerase of the influenza A virus

    PubMed Central

    Stubbs, Thomas M; te Velthuis, Aartjan JW

    2014-01-01

    The influenza A virus causes a highly contagious respiratory disease that significantly impacts our economy and health. Its replication and transcription is catalyzed by the viral RNA polymerase. This enzyme is also crucial for the virus, because it is involved in the adaptation of zoonotic strains. It is thus of major interest for the development of antiviral therapies and is being intensively studied. In this article, we will discuss recent advances that have improved our knowledge of the structure of the RNA polymerase and how mutations in the polymerase help the virus to spread effectively among new hosts. PMID:25431616

  18. 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

  19. 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.

  20. Transcription by RNA polymerase III: insights into mechanism and regulation

    PubMed Central

    Turowski, Tomasz W.; Tollervey, David

    2016-01-01

    The highly abundant, small stable RNAs that are synthesized by RNA polymerase III (RNAPIII) have key functional roles, particularly in the protein synthesis apparatus. Their expression is metabolically demanding, and is therefore coupled to changing demands for protein synthesis during cell growth and division. Here, we review the regulatory mechanisms that control the levels of RNAPIII transcripts and discuss their potential physiological relevance. Recent analyses have revealed differential regulation of tRNA expression at all steps on its biogenesis, with significant deregulation of mature tRNAs in cancer cells. PMID:27911719

  1. Structural basis of transcription initiation by RNA polymerase II.

    PubMed

    Sainsbury, Sarah; Bernecky, Carrie; Cramer, Patrick

    2015-03-01

    Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained. Although mechanistic details await elucidation, available data outline how Pol II cooperates with the general transcription factors to bind to and open promoter DNA, and how Pol II directs RNA synthesis and escapes from the promoter.

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

    PubMed

    Toulokhonov, Innokenti; Landick, Robert

    2006-08-25

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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

    PubMed Central

    Han, Jungwon; Lee, Jong-Hyuk; Park, Sunyoung; Yoon, Soomin; Yoon, Aerin; Hwang, Do B; Lee, Hwa K; Kim, Min S; Lee, Yujean; Yang, Won J; Youn, Hong-Duk; Kim, Hyori; Chung, Junho

    2016-01-01

    The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition. PMID:27857068

  9. Quantifying the influence of 5'-RNA modifications on RNA polymerase I activity.

    PubMed

    Appling, Francis D; Lucius, Aaron L; Schneider, David A

    2017-09-01

    For ensemble and single-molecule analyses of transcription, the use of synthetic transcription elongation complexes has been a versatile and powerful tool. However, structural analyses demonstrate that short RNA substrates, often employed in these assays, would occupy space within the RNA polymerase. Most commercial RNA oligonucleotides do not carry a 5'-triphosphate as would be present on a natural, de novo synthesized RNA. To examine the effects of 5'-moities on transcription kinetics, we measured nucleotide addition and 3'-dinucleotide cleavage by eukaryotic RNA polymerase I using 5'-hydroxyl and 5'-triphosphate RNA substrates. We found that 5' modifications had no discernable effect on the kinetics of nucleotide addition; however, we observed clear, but modest, effects on the rate of backtracking and/or dinucleotide cleavage. These data suggest that the 5'-end may influence RNA polymerase translocation, consistent with previous prokaryotic studies, and these findings may have implications on kinetic barriers that confront RNA polymerases during the transition from initiation to elongation. Copyright © 2017. Published by Elsevier B.V.

  10. Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

    PubMed

    Walker, Scott S; Degen, David; Nickbarg, Elliott; Carr, Donna; Soriano, Aileen; Mandal, Mihir; Painter, Ronald E; Sheth, Payal; Xiao, Li; Sher, Xinwei; Murgolo, Nicholas; Su, Jing; Olsen, David B; Ebright, Richard H; Young, Katherine

    2017-03-31

    The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP β' subunit or a null mutation in the rpoZ gene encoding the RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP β' subunit residue 622 and the RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.

  11. 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.

  12. Two isoforms of human RNA polymerase III with specific functions in cell growth and transformation

    PubMed Central

    Dumay-Odelot, Hélène; Da Silva, Daniel; Rey, Christophe; Prochazkova, Martina; Roeder, Robert G.; Besser, Daniel; Teichmann, Martin

    2010-01-01

    Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32β) that led to the demonstration of two human Pol III isoforms (Pol IIIα and Pol IIIβ). RPC32β-containing Pol IIIβ is ubiquitously expressed and essential for growth of human cells. RPC32α-containing Pol IIIα is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32α expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32α in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation. PMID:20154270

  13. 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.

  14. Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment.

    PubMed

    Janke, Abigail M; Seo, Da Hee; Rahmanian, Vahid; Conicella, Alexander E; Mathews, Kaylee L; Burke, Kathleen A; Mittal, Jeetain; Fawzi, Nicolas L

    2017-10-11

    Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. We report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.

  15. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  16. RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography.

    PubMed Central

    Plevan, P; Albertini, A M; Galizzi, A; Adamoli, A; Mastromei, G; Riva, S; Cassani, G

    1977-01-01

    A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands. Images PMID:405660

  17. Regulation of mammalian ribosomal gene transcription by RNA polymerase I.

    PubMed

    Grummt, I

    1999-01-01

    All cells, from prokaryotes to vertebrates, synthesize vast amounts of ribosomal RNA to produce the several million new ribosomes per generation that are required to maintain the protein synthetic capacity of the daughter cells. Ribosomal gene (rDNA) transcription is governed by RNA polymerase I (Pol I) assisted by a dedicated set of transcription factors that mediate the specificity of transcription and are the targets of the pleiotrophic pathways the cell uses to adapt rRNA synthesis to cell growth. In the past few years we have begun to understand the specific functions of individual factors involved in rDNA transcription and to elucidate on a molecular level how transcriptional regulation is achieved. This article reviews our present knowledge of the molecular mechanism of rDNA transcriptional regulation.

  18. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

    PubMed Central

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-01-01

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association. DOI: http://dx.doi.org/10.7554/eLife.20832.001 PMID:28262097

  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. 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.

  1. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

    PubMed

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-03-06

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

  2. 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 Central

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

    2012-01-01

    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. PMID:22546509

  3. Dynamics of bridge helix bending in RNA polymerase II.

    PubMed

    Wang, Zhan-Feng; Fu, Yi-Ben; Wang, Peng-Ye; Xie, Ping

    2017-04-01

    One of critical issues for RNA polymerase is how the enzyme translocates along the DNA substrate during transcription elongation cycle. Comparisons of the structure of RNA polymerase II (Pol II) with that of bacterial enzyme have suggested that the transition of the bridge helix (BH) from straight to flipped-out conformations facilitates the translocation of upstream DNA-RNA hybrid. However, the flipped-out conformation of BH in Pol II has not been observed up to now and the detailed mechanism of how the BH facilitating upstream hybrid translocation still remains obscure. Here we use all-atom molecular dynamics simulations to study the transition dynamics of BH in Pol II. Two different flipped-out conformations (termed as F1 and F2) are derived from our simulation trajectories, both of which could contribute to upstream hybrid translocation. In particular, the structure of BH in F2 conformation shows nearly identical to that observed in free bacterial enzyme, showing the existence of the flipped-out conformation in Pol II. Analysis of hydrogen bonds and salt bridge formed intra BH in different conformations indicates that the flipped-out conformations are more unstable than the straight conformation. Moreover, a detailed understanding of how the transition of BH conformations facilitating upstream hybrid translocation is given. Proteins 2017; 85:614-629. © 2016 Wiley Periodicals, Inc.

  4. 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

  5. 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

  6. 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

  7. 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

  8. RNA polymerase and the ribosome: the close relationship.

    PubMed

    McGary, Katelyn; Nudler, Evgeny

    2013-04-01

    In bacteria transcription and translation are linked in time and space. When coupled to RNA polymerase (RNAP), the translating ribosome ensures transcriptional processivity by preventing RNAP backtracking. Recent advances in the field have characterized important linker proteins that bridge the gap between transcription and translation: In particular, the NusE(S10):NusG complex and the NusG homolog, RfaH. The direct link between the moving ribosome and RNAP provides a basis for maintaining genomic integrity while enabling efficient transcription and timely translation of various genes within the bacterial cell. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Design, Synthesis, and Structure–Activity Relationships of Pyridoquinazolinecarboxamides as RNA Polymerase I Inhibitors

    PubMed Central

    2015-01-01

    RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Pol I large catalytic subunit protein. Structure–activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxicity. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies. PMID:24847734

  10. Human GTPases associate with RNA polymerase II to mediate its nuclear import.

    PubMed

    Carré, Clément; Shiekhattar, Ramin

    2011-10-01

    Small GTPases share a biochemical mechanism and act as binary molecular switches. One important function of small GTPases in the cell is nucleocytoplasmic transport of both proteins and RNA. Here, we show the stable association of human GPN1 and GPN3, small GTPases related to Ran, with RNA polymerase II (RNAPII) isolated from either the cytoplasmic or nuclear fraction. GPN1 and GPN3 directly interact with RNAPII subunit 7 (RPB7)/RPB4 and the C-terminal domain (CTD) of RNAPII. Depletion of GPN1 or GPN3 using small interfering RNAs led to decreased RNAPII levels in the nucleus and an accumulation of this enzyme in the cytoplasm of human cells. Furthermore, isolation of a GPN1/GPN3/RNAPII complex from stable cell lines expressing a dominant negative GPN1 harboring mutations in the GTP-binding pocket demonstrated a role for these proteins in nuclear import of RNAPII. Thus, GPN1/GPN3 define a new family of small GTPases that are specialized for the transport of RNA polymerase II into the nucleus.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. RNA polymerase active center: the molecular engine of transcription.

    PubMed

    Nudler, Evgeny

    2009-01-01

    RNA polymerase (RNAP) is a complex molecular machine that governs gene expression and its regulation in all cellular organisms. To accomplish its function of accurately producing a full-length RNA copy of a gene, RNAP performs a plethora of chemical reactions and undergoes multiple conformational changes in response to cellular conditions. At the heart of this machine is the active center, the engine, which is composed of distinct fixed and moving parts that serve as the ultimate acceptor of regulatory signals and as the target of inhibitory drugs. Recent advances in the structural and biochemical characterization of RNAP explain the active center at the atomic level and enable new approaches to understanding the entire transcription mechanism, its exceptional fidelity and control.

  18. Prokaryotic histone-like protein interacting with RNA polymerase.

    PubMed Central

    Lathe, R; Buc, H; Lecocq, J P; Bautz, E K

    1980-01-01

    firA mutation of Escherichia coli can render RNA synthesis thermosensitive and confer abnormal sensitivity to rifampicin, an antibiotic that specifically inhibits the activity of RNA polymerase. We previously described the cloning of a chromosomal HindIII fragment containing the firA gene, and we now present strong evidence that the product of this gene is a 17,000-dalton polypeptide which, by various criteria, closely resembles the eukaryotic histones. This protein forms the largest of a unique set of three abundant histone-like proteins (HLP) found in E. coli and is hence referred to as HLPI. We discuss possible routes by which these proteins might affect transcription. Images PMID:6447875

  19. RNA polymerase III under control: repression and de-repression.

    PubMed

    Boguta, Magdalena; Graczyk, Damian

    2011-09-01

    The synthesis of tRNA by yeast RNA polymerase III (Pol III) is regulated in response to changing environmental conditions. This control is mediated by Maf1, the global negative regulator of Pol III transcription conserved from yeast to humans. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from recently reported structural and biochemical data on Pol III and Maf1. Efficient Pol III transcription, following the shift of cells from a non-fermentable carbon source to glucose, requires phosphorylation of Maf1. One of the newly identified Maf1 kinases is the chromatin-bound casein kinase II (CK2). Current studies have allowed us to propose an innovative mechanism of Pol III regulation. We suggest that CK2-mediated phosphorylation of Maf1, occurring directly on tDNA chromatin, controls Pol III recycling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. RNA polymerase structure and function at lac operon.

    PubMed

    Borukhov, Sergei; Lee, Jookyung

    2005-06-01

    Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind promoters, melt the DNA and initiate RNA synthesis, is positively regulated by transcription activators, such as cyclic AMP-receptor protein, CRP, and negatively regulated by lac-repressor, LacI. In this review, we discuss the recent progress in structural and biochemical studies of RNAP and its binary and ternary complexes with CRP and lac promoter. With structural information now available for RNAP and models of binary and ternary elongation complexes, the interaction between these factors and RNAP can be modeled, and possible molecular mechanisms of their action can be inferred.

  1. RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase

    PubMed Central

    STEIN, PAULA; SVOBODA, PETR; ANGER, MARTIN; SCHULTZ, RICHARD M.

    2003-01-01

    Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos–EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation. PMID:12554861

  2. Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse.

    PubMed Central

    Shibata, S; Muryoi, T; Saitoh, Y; Brumeanu, T D; Bona, C A; Kasturi, K N

    1993-01-01

    Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases. Production of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis. Previous investigations have shown that the tight skin (TSK) mouse is an experimental model for systemic sclerosis. In the present study we show that the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes. To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from these mice. Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mice that in the controls. mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Escherichia coli and phage T7 RNA polymerases (155-, 150-, and 107-kD polypeptides). We have also demonstrated that these antibodies bind better to the phosphorylated enzymes. The anti-RNA pol I mAbs were divided into three groups in terms of their functional property. The first group of antibodies increased the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity. Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epitopes. The third group of antibodies was neutral and had no activity on the enzyme function. These results suggest that TSK mouse anti-RNA pol I antibodies recognize three or more conserved epitopes. To understand the molecular basis of the generation of such autoreactive antibodies we analyzed their V gene repertoire. Northern analysis of RNAs of 14 TSK hybridomas showed that the VH genes encoding these antibodies were mainly from VH J558 family. It is possible that these genes were derived from a single germline gene or from a set of related genes of a single subgroup. Images PMID:8349828

  3. 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.

  4. 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.

  5. 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.

  6. Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing.

    PubMed

    Saldi, Tassa; Cortazar, Michael A; Sheridan, Ryan M; Bentley, David L

    2016-06-19

    Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II. The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes the current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. High Fidelity of Yellow Fever Virus RNA Polymerase

    PubMed Central

    Pugachev, Konstantin V.; Guirakhoo, Farshad; Ocran, Simeon W.; Mitchell, Fred; Parsons, Megan; Penal, Caroline; Girakhoo, Soheila; Pougatcheva, Svetlana O.; Arroyo, Juan; Trent, Dennis W.; Monath, Thomas P.

    2004-01-01

    Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10−4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10−7 to 2.3 × 10−7. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly. PMID:14694136

  8. Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing.

    PubMed

    Bremer, H; Ehrenberg, M

    1995-05-17

    A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

    PubMed Central

    Fong, Baley A; Gillies, Alison R; Ghazi, Iraj; LeRoy, Gary; Lee, Kathleen C; Westblade, Lars F; Wood, David W

    2010-01-01

    A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target. PMID:20512976

  14. The X-ray crystal structure of RNA polymerase from Archaea.

    PubMed

    Hirata, Akira; Klein, Brianna J; Murakami, Katsuhiko S

    2008-02-14

    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 subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) 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.

  15. 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.

  16. Novel layers of RNA polymerase III control affecting tRNA gene transcription in eukaryotes

    PubMed Central

    Leśniewska, Ewa

    2017-01-01

    RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency. PMID:28228471

  17. Novel layers of RNA polymerase III control affecting tRNA gene transcription in eukaryotes.

    PubMed

    Leśniewska, Ewa; Boguta, Magdalena

    2017-02-01

    RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency.

  18. The uncoupling of catalysis and translocation in the viral RNA-dependent RNA polymerase.

    PubMed

    Shu, Bo; Gong, Peng

    2017-03-01

    The nucleotide addition cycle of nucleic acid polymerases includes two major events: the pre-chemistry active site closure leading to the addition of one nucleotide to the product chain; the post-chemistry translocation step moving the polymerase active site one position downstream on its template. In viral RNA-dependent RNA polymerases (RdRPs), structural and biochemical evidences suggest that these two events are not tightly coupled, unlike the situation observed in A-family polymerases such as the bacteriophage T7 RNA polymerase. Recently, an RdRP translocation intermediate crystal structure of enterovirus 71 shed light on how translocation may be controlled by elements within RdRP catalytic motifs, and a series of poliovirus apo RdRP crystal structures explicitly suggest that a motif B loop may assist the movement of the template strand in late stages of transcription. Implications of RdRP catalysis-translocation uncoupling and the remaining challenges to further elucidate RdRP translocation mechanism are also discussed.

  19. Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter.

    PubMed

    Palmer, T D; Miller, A D; Reeder, R H; McStay, B

    1993-07-25

    In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.

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

    PubMed Central

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

    2013-01-01

    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. PMID:25764110

  1. Function and Control of RNA Polymerase II C-Terminal Domain Phosphorylation in Vertebrate Transcription and RNA Processing

    PubMed Central

    Hsin, Jing-Ping; Xiang, Kehui

    2014-01-01

    The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y1S2P3T4S5P6S7. We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3′-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3′-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1. PMID:24752900

  2. 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

  3. Structural basis for promoter specificity switching of RNA polymerase by a phage factor.

    PubMed

    Tagami, Shunsuke; Sekine, Shun-ichi; Minakhin, Leonid; Esyunina, Daria; Akasaka, Ryogo; Shirouzu, Mikako; Kulbachinskiy, Andrey; Severinov, Konstantin; Yokoyama, Shigeyuki

    2014-03-01

    Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major "-10/-35" class promoters. Phage promoters belonging to the minor "extended -10" class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP β-flap domain and the C-terminal domain of the σ subunit (region 4 of the σ subunit [σ4]), thus relocating the β-flap tip and σ4. The ~45 Å displacement of σ4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.

  4. 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.

  5. Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase

    PubMed Central

    Gleghorn, Michael L.; Davydova, Elena K.; Rothman-Denes, Lucia B.; Murakami, Katsuhiko S.

    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 five- to seven-base pair stem and three-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. PMID:19061645

  6. Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure.

    PubMed Central

    Wilson, D W; Meacock, P A

    1988-01-01

    Strains of the yeast Kluyveromyces lactis that produce killer-toxin have been found to contain two linear dsDNA plasmids, k1 (8.9 Kb) and k2 (13.4 Kb). The four transcribed open reading frames of plasmid k1 contain no recognisable yeast nuclear expression signals. Moreover, a toxin subunit gene fused with the lacZ gene of Escherichia coli is not detectably expressed when introduced to K.lactis or Saccharomyces cerevisiae on a nuclear vector, even when native k1 and k2 are present in the cell. This and other evidence is consistent with the hypothesis that k1 and k2 reside in an extranuclear location, and do not utilise the nuclear RNA polymerases I, II or III for transcription of their genes. Sequencing of plasmid k2, which is thought to encode factors necessary for the maintenance or expression of k1, reveals an open reading frame predicted to encode a 974 amino acid polypeptide with homology to several DNA-directed RNA polymerases. We suggest that this is a component of a novel plasmid-specific extranuclear gene expression system. PMID:3138657

  7. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  8. 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

  9. Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control.

    PubMed

    Oelgeschläger, Thomas

    2002-02-01

    The carboxyl-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (RNAP II) consists of 52 repeats of a consensus heptapeptide and is subject to phosphorylation and dephosphorylation events during each round of transcription. RNAP II activity is regulated during the cell cycle and cell cycle-dependend changes in RNAP II activity correlate well with CTD phosphorylation. In addition, global changes in the CTD phosphorylation status are observed in response to mitogenic or cytostatic signals such as growth factors, mitogens and DNA-damaging agents. Several CTD kinases are members of the cyclin-dependent kinase (CDK) superfamily and associate with transcription initiation complexes. Other CTD kinases implicated in cell cycle regulation include the mitogen-activated protein kinases ERK-1/2 and the c-Abl tyrosine kinase. These observations suggest that reversible RNAP II CTD phosphorylation may play a key role in linking cell cycle regulatory events to coordinated changes in transcription.

  10. 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

  11. Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilizes RNA polymerase I and III complex integrity.

    PubMed

    Walker-Kopp, Nancy; Jackobel, Ashleigh J; Pannafino, Gianno N; Morocho, Paola A; Xu, Xia; Knutson, Bruce A

    2017-08-14

    Treacher Collins syndrome (TCS) is a craniofacial disorder that is characterized by the malformation of the facial bones. Mutations in three genes (TCOF1, POLR1C, and POLR1D) involved in RNA polymerase I (Pol I) transcription account for more than 90% of disease cases. Two of these TCS-associated genes, POLR1C and POLR1D, encode for essential Pol I/III subunits that form a heterodimer necessary for Pol I/III assembly, and many TCS mutations lie along their evolutionarily conserved dimerization interface. Here we elucidate the molecular basis of TCS mutations in S. cerevisiae, and present a new model for how TCS mutations may disrupt Pol I and III complex integrity. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. 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

  13. E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells.

    PubMed

    Sun, Jianbo; Keim, Celia D; Wang, Jiguang; Kazadi, David; Oliver, Paula M; Rabadan, Raul; Basu, Uttiya

    2013-08-15

    Programmed mutagenesis of the immunoglobulin locus of B lymphocytes during class switch recombination (CSR) and somatic hypermutation requires RNA polymerase II (polII) transcription complex-dependent targeting of the DNA mutator activation-induced cytidine deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism, and this activity is stimulated by the RNA polII stalling cofactor Spt5 and the 11-subunit cellular noncoding RNA 3'-5' exonucleolytic processing complex RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event, leading to generation of 3' end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. We found that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes and defective CSR. Taken together, our study links noncoding RNA processing following RNA polII pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of noncoding RNAs that are generated by stalled RNA polII genome-wide.

  14. 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.

  15. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    DOE PAGES

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

    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

  16. The RNA polymerase flow model of gene transcription.

    PubMed

    Edri, Shlomit; Gazit, Eran; Cohen, Eyal; Tuller, Tamir

    2014-02-01

    Gene expression is a fundamental cellular process by which proteins are synthesized based on the information coded in the genes. The two major steps of this process are the transcription of the DNA segment corresponding to a gene to mRNA molecules and the translation of the mRNA molecules to proteins by the ribosome. Thus, understanding, modeling and engineering the different stages of this process have both important biotechnological applications and contributions to basic life science. In previous studies we have introduced the Homogenous Ribosome Flow Model (HRFM) and demonstrated its advantages in analyses of the translation process. In this study we introduce the RNA Polymerase Flow Model (RPFM), a non trivial extension of the HRFM, which also includes a backward flow and can be used for modeling transcription and maybe other similar processes. We compare the HRFM and the RPFM in the three regimes of the transcription process: rate limiting initiation, rate limiting elongation and rate limiting termination via a simulative and analytical analysis. In addition, based on experimental data, we show that RPFM is a better choice for modeling transcription process.

  17. Millisecond dynamics of RNA polymerase II translocation at atomic resolution

    PubMed Central

    Silva, Daniel-Adriano; Weiss, Dahlia R.; Pardo Avila, Fátima; Da, Lin-Tai; Levitt, Michael; Wang, Dong; Huang, Xuhui

    2014-01-01

    Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level. PMID:24753580

  18. Impact of template backbone heterogeneity on RNA polymerase II transcription

    PubMed Central

    Xu, Liang; Wang, Wei; Zhang, Lu; Chong, Jenny; Huang, Xuhui; Wang, Dong

    2015-01-01

    Variations in the sugar component (ribose or deoxyribose) and the nature of the phosphodiester linkage (3′-5′ or 2′-5′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eukaryotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity remain elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quantitatively investigated pol II transcriptional behaviors through different template backbone variants. We revealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phosphodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA. PMID:25662224

  19. 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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. VHL-dependent patterns of RNA polymerase II hydroxylation in human renal clear cell carcinomas

    PubMed Central

    Yi, Ying; Mikhaylova, Olga; Mamedova, Aygun; Bastola, Prabhat; Biesiada, Jacek; Alshaikh, Enas; Levin, Linda; Sheridan, Rachel M.; Meller, Jarek; Czyzyk-Krzeska, Maria F.

    2010-01-01

    Purpose We have previously shown that VHL regulates ubiquitylation and Proline P1465 hydroxylation of the large subunit of RNA polymerase II, Rpb1, in human RCC cell lines. Here, our goal was to determine the impact of this VHL function and the status of P1465 hydroxylation in human RCC tumors. Experimental Design Primary human tumors and matched normal kidney samples were probed for expression levels of the large subunit of RNA polymerase II (Rpb1), Rpb1 hydroxylated on P1465 (Rpb1(OH)), Rpb1 phosphorylated on Ser5 (Rpb1(S5P)), and proline hydroxylases PHD1, PHD2, and PHD3. Results from RCC tumors were categorized according to the status of VHL gene. Mechanistic analysis was performed in orthotopic xenograft model using 786-O RCC cells with wild-type VHL and knockdown of PHD2, characterized by high levels of Rpb1(OH) and PHD1. Results Levels of Rpb1(OH), PHD1, and PHD2 were significantly higher in RCC tumors as compared with normal kidneys. RCC tumors with wild-type VHL had higher levels of Rpb1(OH) and PHD1 and lower levels of PHD2 than tumors with VHL gene alterations. Levels of Rpb1(OH) significantly correlated with levels of PHD1 in tumors and in normal kidneys. Knockdown of PHD2 in 786-O VHL(+) cells resulted in a more malignant phenotype in orthotopic xenografts and higher expression of specific cell cycle regulators (CDC25A, CDK2, CCNA2), as compared with VHL(−) RCC cells. Conclusions Elevated PHD1 concomitant with decreased PHD2 are causatively related to Rpb1 hydroxylation and oncogenesis in human RCC tumors with wild-type VHL gene. Thus, P1465-hydroxylated Rpb1 and PHD1 represent attractive drug targets for new RCC treatments. PMID:20978146

  1. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.

    PubMed

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-02-13

    The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.

  2. Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase.

    PubMed

    Godoy, Andre S; Lima, Gustavo M A; Oliveira, Ketllyn I Z; Torres, Naiara U; Maluf, Fernando V; Guido, Rafael V C; Oliva, Glaucius

    2017-03-27

    The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution.

  3. Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase

    PubMed Central

    Godoy, Andre S.; Lima, Gustavo M. A.; Oliveira, Ketllyn I. Z.; Torres, Naiara U.; Maluf, Fernando V.; Guido, Rafael V. C.; Oliva, Glaucius

    2017-01-01

    The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution. PMID:28345596

  4. Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase epsilon.

    PubMed Central

    Huang, D; Pospiech, H; Kesti, T; Syväoja, J E

    1999-01-01

    The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters. PMID:10215605

  5. Roles of POLD4, smallest subunit of DNA polymerase {delta}, in nuclear structures and genomic stability of human cells

    SciTech Connect

    Huang, Qin Miao; Akashi, Tomohiro; Masuda, Yuji; Kamiya, Kenji; Takahashi, Takashi; Suzuki, Motoshi

    2010-01-01

    Mammalian DNA polymerase {delta} (pol {delta}) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol {delta} activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol {delta} activity, and that it functions in cell proliferation and maintenance of genomic stability of human cells.

  6. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function

    NASA Astrophysics Data System (ADS)

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-03-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  7. A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

    PubMed Central

    Aitken, Stuart; Alexander, Ross D.; Beggs, Jean D.

    2013-01-01

    The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3′ end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics. PMID:23804443

  8. RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumors

    PubMed Central

    Winter, Andrew G.; Sourvinos, George; Allison, Simon J.; Tosh, Kerrie; Scott, Pamela H.; Spandidos, Demetrios A.; White, Robert J.

    2000-01-01

    Most transformed cells display abnormally high levels of RNA polymerase (pol) III transcripts. Although the full significance of this is unclear, it may be fundamental because healthy cells use two key tumor suppressors to restrain pol III activity. We present the first evidence that a pol III transcription factor is overexpressed in tumors. This factor, TFIIIC2, is a histone acetyltransferase that is required for synthesis of most pol III products, including tRNA and 5S rRNA. TFIIIC2 is a complex of five polypeptides, and mRNAs encoding each of these subunits are overexpressed in human ovarian carcinomas; this may explain the elevated TFIIIC2 activity that is found consistently in the tumors. Deregulation in these cancers is unlikely to be a secondary response to rapid proliferation, because there is little or no change in TFIIIC2 mRNA levels when actively cycling cells are compared with growth-arrested cells in culture. Using purified factors, we show that raising the level of TFIIIC2 is sufficient to stimulate pol III transcription in ovarian cell extracts. The data suggest that overexpression of TFIIIC2 contributes to the abnormal abundance of pol III transcripts in ovarian tumors. PMID:11058163

  9. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

    PubMed

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-04-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg(2+) concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  10. 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.

  11. How slow RNA polymerase II elongation favors alternative exon skipping.

    PubMed

    Dujardin, Gwendal; Lafaille, Celina; de la Mata, Manuel; Marasco, Luciano E; Muñoz, Manuel J; Le Jossic-C