1887

Abstract

The promoter selectivity of RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening , we identified the whole set of ‘constitutive’ promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ), RpoS (σ), RpoH (σ), RpoF (σ) or RpoE (σ), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61–81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the ‘repressive’ promoter. The presence of high-level RpoN sigma in growing K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into survival under nitrogen-depleted conditions in nature.

Funding
This study was supported by the:
  • japan society for the promotion of science (Award 25430173)
    • Principle Award Recipient: AkiraIshihama
  • japan society for the promotion of science (Award 18310133)
    • Principle Award Recipient: AkiraIshihama
  • japan society for the promotion of science (Award 19K06618)
    • Principle Award Recipient: TomohiroShimada
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2021-11-17
2024-04-12
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References

  1. Ishihama A. Molecular assembly and functional modulation of Escherichia coli RNA polymerase. Adv Biophys 1990; 26:19–31 [View Article] [PubMed]
    [Google Scholar]
  2. Ishihama A. Functional modulation of Escherichia coli RNA polymerase. Annu Rev Microbiol 2000; 54:499–518 [View Article] [PubMed]
    [Google Scholar]
  3. Hirschman J, Wong PK, Sei K, Keener J, Kustu S. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci USA 1985; 82:7525–7529 [View Article] [PubMed]
    [Google Scholar]
  4. Kustu SG, Santero E, Keener J, Popham D, Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev 1989; 53:367–376 [View Article] [PubMed]
    [Google Scholar]
  5. Studholme DJ, Buck M. The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiol Lett 2000; 186:1–9 [View Article] [PubMed]
    [Google Scholar]
  6. Reitzer L, Schneider BL. Metabolic context and possible physiological themes of σ54-dependent genes in Escherichia coli. Microbiol Mol Biol Rev 2001; 65:422–444 [View Article] [PubMed]
    [Google Scholar]
  7. Reitzer LJ. Sources of nitrogen and their utilization. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd. edn Washington, DC: American Society for Microbiology; 1996 pp 380–390
    [Google Scholar]
  8. Weiss V, Claverie-Martin F, Magasanic B. Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription. Proc Natl Acad Sci USA 1992; 89:5088–5092 [View Article] [PubMed]
    [Google Scholar]
  9. Reitzer LJ, Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 1986; 45:785–792 [View Article] [PubMed]
    [Google Scholar]
  10. Wigneshweraraj SR, Chaney MK, Ishihama A, Buck M. Regulatory sequences in sigma 54 localise near the start of DNA melting. J Mol Biol 2001; 306:681–701 [View Article] [PubMed]
    [Google Scholar]
  11. Carlo SD, Chen B, Hoover TR, Kondrashkina E, Nogales E et al. The structural basis for regulated assembly and function of the transcriptional activator NtrC. Genes Dev 2006; 20:1485–1495 [View Article] [PubMed]
    [Google Scholar]
  12. Zimmer DP, Soupene E, Lee HL, Wendisch VF, Khodursky AB et al. Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation. Proc Natl Acad Sci USA 2000; 97:14674–14679 [View Article] [PubMed]
    [Google Scholar]
  13. Buck M, Miller S, Drummond M, Dixon R. Upstream ativatior sequences are present in the promoters of nitrogen fixation genes. Nature 1986; 320:374–378
    [Google Scholar]
  14. Buck M, Gallegos MT, Studholme DJ, Guo Y, Gralla JD. The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor. J Bacteriol 2000; 182:4129–4136 [View Article] [PubMed]
    [Google Scholar]
  15. Danson AE, Jovanovic M, Buck M, Zhang X. Mechanisms of σ54-dependent transcription initiation and regulation. J Mol Biol 2019; 431:3960–3974 [View Article] [PubMed]
    [Google Scholar]
  16. Ishihama A, Shimada T, Yamazaki Y. Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors. Nucleic Acids Res 2016; 44:2058–2074 [View Article] [PubMed]
    [Google Scholar]
  17. Bonocora RP, Smith C, Lapierre P, Wade JT. Genome-scale mapping of Escherichia coli σ54 reveals widespread, conserved intragenic binding. PLoS Genet 2015; 11:e1005552 [View Article] [PubMed]
    [Google Scholar]
  18. Schaefer J, Engl C, Zhang N, Lawton E, Buck M. Genome wide interactions of wild-type and activator bypass forms of σ54. Nucleic Acids Res 2015; 43:7280–7291 [View Article] [PubMed]
    [Google Scholar]
  19. Ishihama A. Prokaryotic genome regulation: multifactor promoters, multitarget regulators and hierarchic networks. FEMS Microbiol Rev 2010; 34:628–645 [View Article] [PubMed]
    [Google Scholar]
  20. Ishihama A. Prokaryotic genome regulation: a revolutionary paradigm. Proc Jpn Acad Ser B 2012; 88:485–508 [View Article]
    [Google Scholar]
  21. Gyaneshwar P, Paliy O, McAuliffe J, Popham DL, Jordan MI et al. Sulfur and nitrogen limitation in Escherichia coli K-12: specific homeostatic responses. J Bacteriol 2005; 187:1074–1090 [View Article] [PubMed]
    [Google Scholar]
  22. Brown DR, Barton G, Pan Z, Buck M, Wigneshweraraj S. Nitrogen stress response are coupled in Escherichia coli. Nat Commun 2014; 5:4115 [View Article] [PubMed]
    [Google Scholar]
  23. Shimada T, Fujita N, Maeda M, Ishihama A. Systematic search for the Cra-binding promoters using genomic SELEX system. Genes Cells 2005; 10:907–918 [View Article] [PubMed]
    [Google Scholar]
  24. Shimada T, Ogasawara H, Ishihama A. Genomic SELEX screening of regulatory targets of Escherichia coli transcription factors. Meth Mol Biol 2018; 1837:49–69
    [Google Scholar]
  25. Shimada T, Ogasawara H, Ishihama A. Single-target regulators form a minor group of transcription factors in Escherichia coli K-12. Nucleic Acids Res 2018; 46:3921–3936 [View Article] [PubMed]
    [Google Scholar]
  26. Shimada T, Yamazaki Y, Tanaka K, Ishihama A. The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS One 2014; 9:e90447 [View Article]
    [Google Scholar]
  27. Shimada T, Tanaka K, Ishihama A. The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase. PLoS One 2017; 12:e0179181
    [Google Scholar]
  28. Ishihama A. Building a complete image of genome regulation in the model organism Escherichia coli. J Gen Appl Microbiol 2018; 63:311–324 [View Article] [PubMed]
    [Google Scholar]
  29. Jishage M, Ishihama A. Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110. J Bacteriol 1997; 179:959–963 [View Article] [PubMed]
    [Google Scholar]
  30. Maeda H, Ishihama A. Competition among seven Escherichia coli s subunits: relative binding affinity to the core RNA polymerase. Nucleic Acids Res 2000; 28:3467–3503
    [Google Scholar]
  31. Igarashi K, Ishihama A. Bipartite functional map of the E. coli RNA polymerase a subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell 1991; 65:1015–1022 [View Article] [PubMed]
    [Google Scholar]
  32. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000; 97:6640–6645 [View Article] [PubMed]
    [Google Scholar]
  33. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y et al. Construction of Escherichia coli in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2:2006.0008
    [Google Scholar]
  34. Gutnick D, Calvo JM, Klopotowski T, Ames BN. Compounds which serve as the source of carbon or nitrogen for Salmonella typhimurium LT-2. J Bacteriol 1969; 1:215–219
    [Google Scholar]
  35. Fujita N, Ishihama A. Reconstitution of RNA polymerase. Meth Enzymol 1996; 273:121–130
    [Google Scholar]
  36. Jishage M, Ishihama A. Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of σ70 and σ38. J Bacteriol 1995; 177:6832–6835 [View Article] [PubMed]
    [Google Scholar]
  37. Jishage M, Iwata A, Ueda S, Ishihama A. Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions. J Bacteriol 1996; 178:5447–5451 [View Article] [PubMed]
    [Google Scholar]
  38. Bailey TL, Boden M, Buske FA, Frith M, Grant CE et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 2009; 37:W202–W208
    [Google Scholar]
  39. Shimada T, Hirao K, Kori A, Yamamoto K, Ishihama A. RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines. Mol Microbiol 2007; 66:744–757 [View Article] [PubMed]
    [Google Scholar]
  40. Anzai T, Imamura S, Ishihama A, Shimada T. Expanded roles of pyruvate-sensing PdhR in transcription regulation of the Escherichia coli genome: fatty acid catabolism and cell motility. Microb Genom 2020; 6:mgen000442 [View Article] [PubMed]
    [Google Scholar]
  41. Singer BS, Shtatland T, Brown D, Gold L. Libraries for genomic SELEX. Nucleic Acids Res 1997; 25:781–786 [View Article] [PubMed]
    [Google Scholar]
  42. Ellington AD, Szostak JW. In vitro selection of DNA molecules that bind specific ligands. Nature 1990; 346:818–822 [View Article] [PubMed]
    [Google Scholar]
  43. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990; 249:505–510 [View Article] [PubMed]
    [Google Scholar]
  44. Shimada T, Ishihama A, Busby SJW, Grainger DC. The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions. Nucleic Acids Res 2008; 36:3950–3955 [View Article] [PubMed]
    [Google Scholar]
  45. Yamamoto K, Hirao K, Oshima T, Aiba H, Utsumi R et al. Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J Biol Chem 2005; 280:1448–1456 [View Article] [PubMed]
    [Google Scholar]
  46. Reitzer LJ, Magasanik B. Isolation of the nitrogen assimilation regulator NRI, the product of the glnG gene of Escherichia coli. Proc Natl Acad Sci USA 1983; 80:5554–5558 [View Article] [PubMed]
    [Google Scholar]
  47. Reitzer L. Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Micribiol 2003; 57:155–176
    [Google Scholar]
  48. Wigneshweraraj SR, Ishihama A, Buck M. In vitro roles of invariant helix-turn-helix motif residue R383 in σ54 (σN). Nucleic Acids Res 2001; 5:1163–1174
    [Google Scholar]
  49. Burrows PC, Severinov K, Ishihama A, Buck M, Wigneshweraraj SR. Mapping σ54-RNA polymerase interactions at the -24 consensus promoter element. J Biol Chem 2003; 278:29728–29743 [View Article] [PubMed]
    [Google Scholar]
  50. Santos-Zavaleta A, Salgado H, Gama-Castro S, Sanchez-Perez M, Gomez-Romero L et al. RegulonDB v10.5: tackling challenges to unify classic and high throughput knowledge of gene regulation in E. coli K-12. Nucleic Acids Res 2019; 47:D212–D220 [View Article] [PubMed]
    [Google Scholar]
  51. Lee SJ, Xie A, Jiang W, Etchegaray JP, Jones PG et al. Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins. Mol Microbiol 1994; 11:833–839 [View Article] [PubMed]
    [Google Scholar]
  52. Jovanovic G, Dworkin J, Model P. Autogenous control of PspF, a constitutively active enhancer-binding protein of Escherichia coli. J Bacteriol 1997; 179:5232–5237 [View Article] [PubMed]
    [Google Scholar]
  53. Vanderpool CK, Gottesman S. Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system. Mol Microbiol 2004; 54:1076–1089 [View Article] [PubMed]
    [Google Scholar]
  54. Raina M, Storz G. SgrT, a small protein that packs a sweet punch. J Bacteriol 2017; 199:e00130-17 [View Article] [PubMed]
    [Google Scholar]
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