1887

Abstract

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core , and ′ subunits. The subunit is conserved between Gram-positive and Gram-negative bacteria, while the subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via assays, very little is known about their roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the and subunits in with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed to be present at equimolar levels with RNAP and to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP ′ and subunits in was also investigated. Similar to , ′ and closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike in , levels in were close to parity with those of ′. These results indicate that is likely to be an integral RNAP subunit in Gram-positives, whereas levels differ substantially between Gram-positives and -negatives. The subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

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2010-12-01
2020-12-02
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References

  1. Albertazzi L., Arosio D., Marchetti L., Ricci F., Beltram F. 2009; Quantitative FRET analysis with the EGFP–mCherry fluorescent protein pair. Photochem Photobiol 85:287–297
    [Google Scholar]
  2. Andersen J. B., Sternberg C., Poulsen L. K., Bjorn S. P., Givskov M., Molin S. 1998; New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64:2240–2246
    [Google Scholar]
  3. Botella E., Fogg M. J., Jules M., Piersma S., Doherty G. P., Hansen A., Denham E. L., Le Chat L., Veiga P. other authors 2010; pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis . Microbiology 156:1600–1608
    [Google Scholar]
  4. Cabrera J. E., Jin D. J. 2003; The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues. Mol Microbiol 50:1493–1505
    [Google Scholar]
  5. Cashel M., Gentry D. R., Hernandez V. H., Vinella D. 1996; The stringent response. In Escherichia coli and Salmonella typhimurium Edited by Neidhardt F. C. pp 1458–1496 Washington, DC: American Society for Microbiology;
    [Google Scholar]
  6. Cheng C. Y., Yu Y. J., Yang M. T. 2010; Coexpression of ω subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase. Protein Expr Purif 69:91–98
    [Google Scholar]
  7. Cormack B. P., Valdivia R. H., Falkow S. 1996; FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33–38
    [Google Scholar]
  8. Datsenko K. A., Wanner B. L. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
    [Google Scholar]
  9. Davies K. M., Lewis P. J. 2003; Localization of rRNA synthesis in Bacillus subtilis : characterization of loci involved in transcription focus formation. J Bacteriol 185:2346–2353
    [Google Scholar]
  10. Davies K. M., Dedman A. J., van Hork S., Lewis P. J. 2005; The NusA : RNA polymerase ratio is increased at sites of rRNA synthesis in Bacillus subtilis . Mol Microbiol 57:366–379
    [Google Scholar]
  11. Doherty G. P., Meredith D. H., Lewis P. J. 2006; Subcellular partitioning of transcription factors in Bacillus subtilis . J Bacteriol 188:4101–4110
    [Google Scholar]
  12. Gao H., Aronson A. I. 2004; The delta subunit of RNA polymerase functions in sporulation. Curr Microbiol 48:401–404
    [Google Scholar]
  13. Gentry D. R., Burgess R. R. 1986; The cloning and sequence of the gene encoding the omega ( ω ) subunit of Escherichia coli RNA polymerase. Gene 48:33–40
    [Google Scholar]
  14. Gentry D. R., Burgess R. R. 1989; rpoZ , encoding the omega subunit of Escherichia coli RNA polymerase is in the same operon as spoT . J Bacteriol 171:1271–1277
    [Google Scholar]
  15. Gentry D. R., Burgess R. R. 1990; Overproduction and purification of the ω subunit of Escherichia coli RNA polymerase. Protein Expr Purif 1:81–86
    [Google Scholar]
  16. Gentry D., Xiao H., Burgess R., Cashel M. 1991; The ω subunit of Escherichia coli K-12 RNA polymerase is not required for stringent RNA control in vivo . J Bacteriol 173:3901–3903
    [Google Scholar]
  17. Ghosh P., Ishihama A., Chatterji D. 2001; Escherichia coli RNA polymerase subunit ω and its N-terminal domain bind full-length β ′ to facilitate incorporation into the α 2 β subassembly. Eur J Biochem 268:4621–4627
    [Google Scholar]
  18. Glaser P., Sharpe M. E., Raether B., Perego M., Ohlsen K., Errington J. 1997; Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev 11:1160–1168
    [Google Scholar]
  19. Gross C. A., Chan C., Dombroski A., Gruber T., Sharp M., Tupy J., Young B. 1998; The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb Symp Quant Biol 63:141–155
    [Google Scholar]
  20. Hyde E. I., Hilton M. D., Whiteley H. R. 1986; Interactions of Bacillus subtilis RNA polymerase with subunits determining the specificity of initiation: sigma and delta peptides can bind simultaneously to core. J Biol Chem 261:16565–16570
    [Google Scholar]
  21. Igarashi K., Fujita N., Ishihama A. 1989; Promoter selectivity of Escherichia coli RNA polymerase: omega factor is responsible for the ppGpp sensitivity. Nucleic Acids Res 17:8755–8765
    [Google Scholar]
  22. Jones A. L., Needham R. H., Rubens C. E. 2003; The delta subunit of RNA polymerase is required for virulence of Streptococcus agalactiae . Infect Immun 71:4011–4017
    [Google Scholar]
  23. Juang Y.-L., Helmann J. D. 1994; The δ subunit of Bacillus subtilis RNA polymerase: an allosteric effector of the initiation and corerecycling phases of transcription. J Mol Biol 239:1–14
    [Google Scholar]
  24. Kojima I., Kasuga K., Kobayashi M., Fukasawa A., Mizuno S., Arisawa A., Akagawa H. 2002; The rpoZ gene, encoding the RNA polymerase omega subunit, is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis . J Bacteriol 184:6417–6423
    [Google Scholar]
  25. Krásný L., Gourse R. L. 2004; An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J 23:4473–4483
    [Google Scholar]
  26. Kunst F., Rapoport G. 1995; Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis . J Bacteriol 177:2403–2407
    [Google Scholar]
  27. Lampe M., Binnie C., Schmidt R., Losick R. 1988; Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase. Gene 67:13–19
    [Google Scholar]
  28. Lewis P. J., Marston A. L. 1999; GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis . Gene 227:101–110
    [Google Scholar]
  29. Lewis P. J., Thaker S. D., Errington J. 2000; Compartmentalization of transcription and translation in Bacillus subtilis . EMBO J 19:710–718
    [Google Scholar]
  30. López de Saro F. J., Woody A.-Y. M., Helmann J. D. 1995; Structural analysis of the Bacillus subtilis δ factor: a protein polyanion which displaces RNA from RNA polymerase. J Mol Biol 252:189–202
    [Google Scholar]
  31. López de Saro F. J., Yoshikawa F. N., Helmann J. D. 1999; Expression, abundance, and RNA polymerase binding properties of the δ factor of Bacillus subtilis . J Biol Chem 274:15953–15958
    [Google Scholar]
  32. Minakhin L., Bhagat S., Brunning A., Campbell E. A., Darst S. A., Ebright R. H., Severinov K. 2001; Bacterial RNA polymerase subunit ω and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly. Proc Natl Acad Sci U S A 98:892–897
    [Google Scholar]
  33. Motáčková V., Sanderová H., Zídek L., Nováček J., Padrta P., Svenková A., Korelusová J., Jonák J., Krásný L., Sklenář V. 2010; Solution structure of the N-terminal domain of Bacillus subtilis δ subunit of RNA polymerase and its classification based on structural homologs. Proteins 78:1807–1810
    [Google Scholar]
  34. Mukherjee K., Nagai H., Shimamoto N., Chatterji D. 1999; GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the omega subunit in vivo . Eur J Biochem 266:228–235
    [Google Scholar]
  35. Neylon C., Brown S. E., Kralicek A. V., Hill T. M., Dixon N. E. 2000; Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance. Biochemistry 39:11989–11999
    [Google Scholar]
  36. Pero J., Nelson J., Fox T. D. 1975; Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein. Proc Natl Acad Sci U S A 72:1589–1593
    [Google Scholar]
  37. Seepersaud R., Needham R. H. V., Kim C. S., Jones A. L. 2006; Abundance of the δ subunit of RNA polymerase is linked to the virulence of Streptococcus agalactiae . J Bacteriol 188:2096–2105
    [Google Scholar]
  38. Shaner N. C., Campbell R. E., Steinbach P. A., Giepmans B. N., Palmer A. E., Tsien R. Y. 2004; Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572
    [Google Scholar]
  39. Studier F. W., Moffatt B. A. 1986; Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
    [Google Scholar]
  40. Vassylyev D. G., Sekine S.-I., Laptenko O., Lee J., Vassylyeva M., Borukhov S., Yokohama S. 2002; Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417:712–719
    [Google Scholar]
  41. Vrentas C. E., Gaal T., Ross W., Ebright R. H., Gourse R. L. 2005; Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA. Genes Dev 19:2378–2387
    [Google Scholar]
  42. Woldringh C. L., Jensen P. R., Westerfho H. V. 1995; Structure and partitioning of bacterial DNA: determined by a balance of compaction and expansion forces?. FEMS Microbiol Lett 131:235–242
    [Google Scholar]
  43. Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M., Errington J. 1995; A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis . Genes Dev 9:1316–1326
    [Google Scholar]
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