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Journal of Biosciences

, Volume 36, Issue 1, pp 43–54 | Cite as

Effects of substitutions at position 180 in the Escherichia coli RNA polymerase σ70 subunit

  • Olga N KorolevaEmail author
  • Stephen JW Busby
  • Valeriy L Drutsa
Article

Abstract

In order to investigate the role of His180 residue, located in the non-conserved region of the σ70 subunit of Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic acid were constructed and purified using the IMPACT system. The ability of mutant σ70 subunits to interact with core RNA polymerase was investigated using native gel-electrophoresis. The properties of the corresponding reconstituted holoenzymes, as provided by gel shift analysis of their complexes with single- and double-stranded promoter-like DNA and by in vitro transcription experiments, allowed one to deduce that His180 influences several steps of transcription initiation, including core binding, promoter DNA recognition and open complex formation.

Keywords

Non-conserved region RNA polymerase σ subunit site-specific mutagenesis transcription 

Abbreviations

IMPACT

Intein-Mediated Purification with an Affinity Chitin-binding Tag

NCR

non-conserved region

RNAP

RNA polymerase

References

  1. Bradford M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 248–254PubMedCrossRefGoogle Scholar
  2. Burgess RR and Anthony L 2001 How sigma docks to RNA polymerase and what sigma does. Curr. Opin. Microbiol. 4 126–131PubMedCrossRefGoogle Scholar
  3. Burton Z, Burgess RR, Lin J, Moore D, Holder S and Gross CA 1981 The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coli K12. Nucleic Acids Res. 9 2889–2903PubMedCrossRefGoogle Scholar
  4. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, et al. 1997 Single-column purification of free recombinant protein using a self-cleavable affinity tag derived from a protein splicing element. Gene 192 271–281PubMedCrossRefGoogle Scholar
  5. DeLano WL 2002 The PyMOL molecular graphics system (San Carlos: DeLano Scientific)Google Scholar
  6. Dombroski AJ, Walter WA, Record MT Jr, Siegele DA and Gross CA 1992 Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Cell 70 501–512PubMedCrossRefGoogle Scholar
  7. Dombroski AJ, Walter WA and Gross CA 1993 Amino-terminal amino acids modulate sigma-factor DNA-binding activity. Gene. Dev. 7 2446–2455PubMedCrossRefGoogle Scholar
  8. Drutsa VL and Kaberdin VR 1992 Use of oligonucleotides and nick translation for site-directed mutagenesis in plasmids. Nucleic Acids Res. 20 922PubMedCrossRefGoogle Scholar
  9. Fedoriw AM, Liu H, Anderson VE and deHaseth PL 1998 Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides. Biochemistry 37 11971–11979PubMedCrossRefGoogle Scholar
  10. Fenton MS, Lee SJ and Gralla JD 2000 Escherichia coli promoter opening and −10 recognition: mutational analysis of sigma 70. EMBO J. 19 1130–1137PubMedCrossRefGoogle Scholar
  11. Gardella T, Moyle H and Susskind MM 1989 A mutant Escherichia coli σ70 subunit of RNA polymerase with altered promoter specificity. J. Mol. Biol. 206 579–590PubMedCrossRefGoogle Scholar
  12. Gopal V, Ma HW, Kumaran MK and Chatterji D 1994 A point mutation at the junction of domain 2.3/2.4 of transcription factor sigma 70 abrogates productive transcription and restores its expected mobility on a denaturing gel. J. Mol. Biol. 242 9–22PubMedCrossRefGoogle Scholar
  13. Gopal V and Chatterji D 1997 Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma70 and disruption of its overall structure. Eur. J. Biochem. 244 613–618PubMedCrossRefGoogle Scholar
  14. Helmann JD and Chamberlin MJ 1988 Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57 839–872PubMedCrossRefGoogle Scholar
  15. Higuchi R, Krummel B and Saiki RK 1988 A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16 7351–7367PubMedCrossRefGoogle Scholar
  16. Huang X, Lopez de Saro FJ and Helmann JD 1997 σ factor mutations affecting the sequence-selective interaction of RNA polymerase with −10 region single-stranded DNA. Nucleic Acids Res. 25 2603–2609PubMedCrossRefGoogle Scholar
  17. Hudson BP, Quispe J, Lara-González S, Kim Y, Berman HM, Arnold E, Ebright RH and Lawson CL 2009 Three-dimensional EM structure of an intact activator-dependent transcription initiation complex. Proc. Natl. Acad. Sci. USA 106 19830–19835PubMedGoogle Scholar
  18. Ilag LL, Westblade LF, Deshayes C, Kolb A, Busby SJW and Robinson CV 2004 Mass spectrometry of Escherichia coli RNA polymerase: interactions of the core enzyme with σ70 and Rsd protein. Structure 12 269–275PubMedGoogle Scholar
  19. Khodak Yu A, Koroleva ON and Drutsa VL 2007 A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components. Biochemistry (Moscow) 72 178–187CrossRefGoogle Scholar
  20. Ko DC, Marr MT, Guo J and Roberts JW 1998 A surface of Escherichia coli sigma 70 required for promoter function and antitermination by phage lambda Q protein. Gene. Dev. 12 3276–3285PubMedCrossRefGoogle Scholar
  21. Koroleva ON and Drutsa VL 1991 In vivo promoter activity of the synthetic Pribnow box. FEBS Lett. 278 207–210PubMedCrossRefGoogle Scholar
  22. Koroleva ON, Drutsa VL and Busby SJ 1997 Model of the prokaryotic promoter. VI. Characteristics of the constructions with the overlapped promoters. Mol. Biol. (Moscow) 31 935–944Google Scholar
  23. Kumar A, Williamson HS, Fujita N, Ishihama A and Hayward RS 1995 A partially functional 245-amino-acid internal deletion derivative of Escherichia coli σ70. J. Bacteriol. 177 5193–5196PubMedGoogle Scholar
  24. Leibman M and Hochschild A 2007 A σ-core interaction of the RNA polymerase holoenzyme that enhances promoter escape. EMBO J. 26 1579–1590PubMedCrossRefGoogle Scholar
  25. Lesley SR and Burgess RR 1989 Characterization of the Escherichia coli transcription factor σ70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry 28 7728–7734PubMedCrossRefGoogle Scholar
  26. Lonetto M., Gribskov M and Gross CA 1992 The σ70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174 3843–3849PubMedGoogle Scholar
  27. Lowe PA, Hager DA and Burgess RR 1979 Purification and properties of the σ subunit of E. coli DNA dependent RNA polymerase. Biochemistry 19 1344–1352CrossRefGoogle Scholar
  28. Malhotra A, Severinova E and Darst SA 1996 Crystal structure of a σ70 subunit fragment from E. coli RNA polymerase. Cell 87 127–136PubMedCrossRefGoogle Scholar
  29. Marr M and Roberts J 1997 Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide. Science 276 258–260CrossRefGoogle Scholar
  30. Maxam AM and Gilbert W 1977 A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74 560–564PubMedCrossRefGoogle Scholar
  31. Panaghie G, Aiyar SE, Bobb KL, Hayward RS and deHaseth PL 2000 Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex. J. Mol. Biol. 299 1217–1230PubMedCrossRefGoogle Scholar
  32. Rudakova EA, Ivanovskaya MG, Kozlov MV, Khoretonenko MV, Oretskaya TS and Nikiforov VG 2000 Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E. coli RNA polymerase using regioselective cross-linking. Biochemistry (Moscow) 65 640–650Google Scholar
  33. Sasse-Dwight S and Gralla JD 1989 KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J. Biol. Chem. 264 8074–8081PubMedGoogle Scholar
  34. Savinkova LK, Baranova LV, Knorre VL and Salganik RI 1988 Binding of RNA-polymerase from Escherichia coli with oligodeoxyribonucleotides homologous to transcribed and non-transcribed DNA strands in the “-10”-promoter region of bacterial genes. Mol. Biol. (Moscow) 22 807–812Google Scholar
  35. Siegele DA, Hu JC, Walter WA and Gross CA 1989 Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206 591–603PubMedCrossRefGoogle Scholar
  36. Tomsic M, Tsujikawa L, Pahagie G, Azok YJ and deHaseth PL 2001 Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes. J. Biol. Chem. 276 31891–31896PubMedCrossRefGoogle Scholar
  37. Von Hippel PH, Bear DG, Morgan WD and McSwiggen JA 1984 Protein-nucleic acid interactions in transcription: a molecular analysis. Annu. Rev. Biochem. 53 389–446CrossRefGoogle Scholar
  38. Waldburger C and Susskind MM 1994 Probing the informational content of Escherichia coli σ70 region 2.3 by combinatorial cassette mutagenesis. J. Mol. Biol. 235 1489–1500PubMedCrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2011

Authors and Affiliations

  • Olga N Koroleva
    • 1
    Email author
  • Stephen JW Busby
    • 2
  • Valeriy L Drutsa
    • 3
  1. 1.Chemical FacultyLomonosov Moscow State UniversityMoscowRussia
  2. 2.School of BiosciencesThe University of BirminghamBirminghamUK
  3. 3.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

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