1887

Abstract

6S RNA from acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with To test for potential structural and functional conservation, we selected four predicted cyanobacterial 6S RNAs (, , and ), which we compared with their counterpart. Temperature-gradient gel electrophoresis revealed similar thermodynamic transition profiles for all 6S RNAs, indicating basically similar secondary structures. Subtle differences in melting behaviour of the different RNAs point to minor structural variations possibly linked to differences in optimal growth temperature. Secondary structural analysis of three cyanobacterial 6S RNAs employing limited enzymic hydrolysis and in-line probing supported the predicted high degree of secondary structure conservation. Testing for functional homology we found that all cyanobacterial 6S RNAs were active in binding RNA polymerase and transcriptional inhibition, and had the ability to act as template for transcription of product RNAs (pRNAs). Deletion of the 6S RNA gene in did not significantly affect cell growth in liquid media but reduced fitness during growth on solid agar. While our study shows that basic 6S RNA functions are conserved in species as distantly related as and cyanobacteria, we also noted a subtle degree of divergence, which might reflect fundamental differences in transcriptional regulation and lifestyle, thus providing the first evidence for a possible physiological role in cyanobacteria.

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2012-10-01
2024-04-25
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References

  1. Akinyanju J. A., Smith R. J. ( 1982). Energy deprivation and guanosine 5′-diphosphate 3′-diphosphate synthesis in cyanobacteria. J Bacteriol 149:681–684[PubMed]
    [Google Scholar]
  2. Axmann I. M., Kensche P., Vogel J., Kohl S., Herzel H., Hess W. R. ( 2005). Identification of cyanobacterial non-coding RNAs by comparative genome analysis. Genome Biol 6:R73 [View Article][PubMed]
    [Google Scholar]
  3. Axmann I. M., Holtzendorff J., Voß B., Kensche P., Hess W. R. ( 2007). Two distinct types of 6S RNA in Prochlorococcus . Gene 406:69–78 [View Article][PubMed]
    [Google Scholar]
  4. Barrick J. E., Sudarsan N., Weinberg Z., Ruzzo W. L., Breaker R. R. ( 2005). 6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter. RNA 11:774–784 [View Article][PubMed]
    [Google Scholar]
  5. Beckmann B. M., Burenina O. Y., Hoch P. G., Kubareva E. A., Sharma C. M., Hartmann R. K. ( 2011). In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis . RNA Biol 8:839–849 [View Article][PubMed]
    [Google Scholar]
  6. Beidler J. L., Hilliard P. R., Rill R. L. ( 1982). Ultrasensitive staining of nucleic acids with silver. Anal Biochem 126:374–380 [View Article][PubMed]
    [Google Scholar]
  7. Brow J. W., Ellis J. C. ( 2005). Comparative analysis of RNA secondary structures: 6S RNA. Handbook of RNA Biochemistry Hartmann R. K., Bindereif A., Schön A., Westhof E. Weinheim: Wiley-VCH Verlag; [View Article]
    [Google Scholar]
  8. Burgess R. R., Jendrisak J. J. ( 1975). Procedure for the rapid, large-scale purification of Escherichia coli DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14:4634–4638 [View Article]
    [Google Scholar]
  9. Cavanagh A. T., Klocko A. D., Liu X., Wassarman K. M. ( 2008). Promoter specificity for 6S RNA regulation of transcription is determined by core promoter sequences and competition for region 4.2 of σ70 . Mol Microbiol 67:1242–1256 [View Article][PubMed]
    [Google Scholar]
  10. Cavanagh A. T., Chandrangsu P., Wassarman K. M. ( 2010). 6S RNA regulation of relA alters ppGpp levels in early stationary phase. Microbiology 156:3791–3800 [View Article][PubMed]
    [Google Scholar]
  11. 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 [View Article][PubMed]
    [Google Scholar]
  12. Ermakova S. Y., Elanskaya I. V., Kallies K. U., Weihe A., Börner T., Shestakov S. V. ( 1993). Cloning and sequencing of mutant psbB genes of the cyanobacterium Synechocystis PCC 6803. Photosynth Res 37:139–146 [View Article]
    [Google Scholar]
  13. Faucher S. P., Friedlander G., Livny J., Margalit H., Shuman H. A. ( 2010). Legionella pneumophila 6S RNA optimizes intracellular multiplication. Proc Natl Acad Sci U S A 107:7533–7538 [View Article][PubMed]
    [Google Scholar]
  14. Geißen R., Steuten B., Polen T., Wagner R. ( 2010). E. coli 6S RNA: a universal transcriptional regulator within the centre of growth adaptation. RNA Biol 7:564–568 [View Article][PubMed]
    [Google Scholar]
  15. Gildehaus N., Neusser T., Wurm R., Wagner R. ( 2007). Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts. Nucleic Acids Res 35:1885–1896 [View Article][PubMed]
    [Google Scholar]
  16. Gonzalez N., Wiggs J., Chamberlin M. J. ( 1977). A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase. Arch Biochem Biophys 182:404–408 [View Article][PubMed]
    [Google Scholar]
  17. Hindley J. ( 1967). Fractionation of 32P-labelled ribonucleic acids on polyacrylamide gels and their characterization by fingerprinting. J Mol Biol 30:125–136 [View Article][PubMed]
    [Google Scholar]
  18. Irnov I., Sharma C. M., Vogel J., Winkler W. C. ( 2010). Identification of regulatory RNAs in Bacillus subtilis . Nucleic Acids Res 38:6637–6651 [View Article][PubMed]
    [Google Scholar]
  19. Jeanguenin L., Lara-Núñez A., Pribat A., Mageroy M. H., Gregory J. F. III, Rice K. C., de Crécy-Lagard V., Hanson A. D. ( 2010). Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase. J Biol Chem 285:41557–41566 [View Article][PubMed]
    [Google Scholar]
  20. Klocko A. D., Wassarman K. M. ( 2009). 6S RNA binding to Eσ70 requires a positively charged surface of σ70 region 4.2. Mol Microbiol 73:152–164 [View Article][PubMed]
    [Google Scholar]
  21. Kugel J. F., Goodrich J. A. ( 2007). An RNA transcriptional regulator templates its own regulatory RNA. Nat Chem Biol 3:89–90 [View Article][PubMed]
    [Google Scholar]
  22. Lee S. Y., Bailey S. C., Apirion D. ( 1978). Small stable RNAs from Escherichia coli: evidence for the existence of new molecules and for a new ribonucleoprotein particle containing 6S RNA. J Bacteriol 133:1015–1023[PubMed]
    [Google Scholar]
  23. Mitschke J., Georg J., Scholz I., Sharma C. M., Dienst D., Bantscheff J., Voss B., Steglich C., Wilde A. & other authors ( 2011). An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A 108:2124–2129 [View Article][PubMed]
    [Google Scholar]
  24. Neußer T., Polen T., Geissen R., Wagner R. ( 2010). Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery. BMC Genomics 11:165–178 [View Article][PubMed]
    [Google Scholar]
  25. Pánek J., Krásny L., Bobek J., Jezková E., Korelusová J., Vohradsky J. ( 2011). The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAs using suboptimal RNA structures. Nucleic Acids Res 39:3418–3426 [View Article][PubMed]
    [Google Scholar]
  26. Rippka R., Deruelles J., Waterbury J., Herdman M., Stanier R. ( 1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61 [View Article]
    [Google Scholar]
  27. Rosenbaum V., Riesner D. ( 1987). Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts. Biophys Chem 26:235–246 [View Article][PubMed]
    [Google Scholar]
  28. Schneider G. J., Tumer N. E., Richaud C., Borbely G., Haselkorn R. ( 1987). Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120. J Biol Chem 262:14633–14639[PubMed]
    [Google Scholar]
  29. Schopf J. W. ( 1993). Microfossils of the Early Archean Apex chert: new evidence of the antiquity of life. Science 260:640–646 [View Article][PubMed]
    [Google Scholar]
  30. Schyns G., Jia L., Coursin T., Tandeau de Marsac N., Houmard J. ( 1998). Promoter recognition by a cyanobacterial RNA polymerase: in vitro studies with the Calothrix sp. PCC 7601 transcriptional factors RcaA and RcaD. Plant Mol Biol 36:649–659 [View Article][PubMed]
    [Google Scholar]
  31. Sharma C. M., Hoffmann S., Darfeuille F., Reignier J., Findeiß S., Sittka A., Chabas S., Reiche K., Hackermüller J. & other authors ( 2010). The primary transcriptome of the major human pathogen Helicobacter pylori . Nature 464:250–255 [View Article][PubMed]
    [Google Scholar]
  32. Soukup G. A., Breaker R. R. ( 1999). Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5:1308–1325 [View Article][PubMed]
    [Google Scholar]
  33. Trotochaud A. E., Wassarman K. M. ( 2004). 6S RNA function enhances long-term cell survival. J Bacteriol 186:4978–4985 [View Article][PubMed]
    [Google Scholar]
  34. Trotochaud A. E., Wassarman K. M. ( 2005). A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol 12:313–319 [View Article][PubMed]
    [Google Scholar]
  35. Trotochaud A. E., Wassarman K. M. ( 2006). 6S RNA regulation of pspF transcription leads to altered cell survival at high pH. J Bacteriol 188:3936–3943 [View Article][PubMed]
    [Google Scholar]
  36. Vogel J., Axmann I. M., Herzel H., Hess W. R. ( 2003). Experimental and computational analysis of transcriptional start sites in the cyanobacterium Prochlorococcus MED4. Nucleic Acids Res 31:2890–2899 [View Article][PubMed]
    [Google Scholar]
  37. Wagner R. ( 2006). Protein-Nucleinsäure Wechselwirkungen. Bioanalytik Lottspeich F., Engels J. W. Heidelberg: Elsevier GmbH, Spektrum-Akademischer Verlag;
    [Google Scholar]
  38. Wassarman K. M. ( 2007). 6S RNA: a small RNA regulator of transcription. Curr Opin Microbiol 10:164–168 [View Article][PubMed]
    [Google Scholar]
  39. Wassarman K. M., Saecker R. M. ( 2006). Synthesis-mediated release of a small RNA inhibitor of RNA polymerase. Science 314:1601–1603 [View Article][PubMed]
    [Google Scholar]
  40. Wassarman K. M., Storz G. ( 2000). 6S RNA regulates E. coli RNA polymerase activity. Cell 101:613–623 [View Article][PubMed]
    [Google Scholar]
  41. Watanabe T., Sugiura M., Sugita M. ( 1997). A novel small stable RNA, 6Sa RNA, from the cyanobacterium Synechococcus sp. strain PCC6301. FEBS Lett 416:302–306 [View Article][PubMed]
    [Google Scholar]
  42. Wurm R., Neußer T., Wagner R. ( 2010). 6S RNA-dependent inhibition of RNA polymerase is released by RNA-dependent synthesis of small de novo products. Biol Chem 391:187–196 [View Article][PubMed]
    [Google Scholar]
  43. Xie W. Q., Jäger K., Potts M. ( 1989). Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli . J Bacteriol 171:1967–1973[PubMed]
    [Google Scholar]
  44. Zuker M. ( 2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415 [View Article][PubMed]
    [Google Scholar]
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