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. 1990 Jun;172(6):3257–3263. doi: 10.1128/jb.172.6.3257-3263.1990

Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor.

B Y Chang 1, R H Doi 1
PMCID: PMC209133  PMID: 2111806

Abstract

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43–46. doi: 10.1038/221043a0. [DOI] [PubMed] [Google Scholar]
  2. Chamberlin M. J. The selectivity of transcription. Annu Rev Biochem. 1974;43(0):721–775. doi: 10.1146/annurev.bi.43.070174.003445. [DOI] [PubMed] [Google Scholar]
  3. Davison B. L., Leighton T., Rabinowitz J. C. Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA. J Biol Chem. 1979 Sep 25;254(18):9220–9226. [PubMed] [Google Scholar]
  4. Doi R. H., Wang L. F. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol Rev. 1986 Sep;50(3):227–243. doi: 10.1128/mr.50.3.227-243.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fallon A. M., Jinks C. S., Yamamoto M., Nomura M. Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid. J Bacteriol. 1979 May;138(2):383–396. doi: 10.1128/jb.138.2.383-396.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fisher R., Blumenthal T. Analysis of RNA polymerase by trypsin cleavage. Evidence for a specific association between subunits sigma and beta involved in the closed to open complex transition. J Biol Chem. 1980 Nov 25;255(22):11056–11062. [PubMed] [Google Scholar]
  7. Fukuda R., Doi R. H. Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation. J Bacteriol. 1977 Jan;129(1):422–432. doi: 10.1128/jb.129.1.422-432.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gardella T., Moyle H., Susskind M. M. A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. J Mol Biol. 1989 Apr 20;206(4):579–590. doi: 10.1016/0022-2836(89)90567-6. [DOI] [PubMed] [Google Scholar]
  9. Gitt M. A., Wang L. F., Doi R. H. A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli. J Biol Chem. 1985 Jun 25;260(12):7178–7185. [PubMed] [Google Scholar]
  10. Gribskov M., Burgess R. R. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene. 1983 Dec;26(2-3):109–118. doi: 10.1016/0378-1119(83)90180-4. [DOI] [PubMed] [Google Scholar]
  11. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  12. Halling S. M., Sanchez-Anzaldo F. J., Fukuda R., Doi R. H., Meares C. F. Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis. Biochemistry. 1977 Jun 28;16(13):2880–2884. doi: 10.1021/bi00632a012. [DOI] [PubMed] [Google Scholar]
  13. Hayward R. S., Austin S. J., Scaife J. G. The effect of gene dosage on the synthesis and stability of RNA polymerase subunits in Escherichia coli. Mol Gen Genet. 1974;131(2):173–180. doi: 10.1007/BF00266152. [DOI] [PubMed] [Google Scholar]
  14. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lesley S. A., Burgess R. R. Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry. 1989 Sep 19;28(19):7728–7734. doi: 10.1021/bi00445a031. [DOI] [PubMed] [Google Scholar]
  17. Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
  18. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  19. Reznikoff W. S., Siegele D. A., Cowing D. W., Gross C. A. The regulation of transcription initiation in bacteria. Annu Rev Genet. 1985;19:355–387. doi: 10.1146/annurev.ge.19.120185.002035. [DOI] [PubMed] [Google Scholar]
  20. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  21. Scheinker V. S., Beresten S. F., Degtyarev S. K., Kisselev L. L. The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase. Nucleic Acids Res. 1979 Oct 10;7(3):625–637. doi: 10.1093/nar/7.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Siegele D. A., Hu J. C., Walter W. A., Gross C. A. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol. 1989 Apr 20;206(4):591–603. doi: 10.1016/0022-2836(89)90568-8. [DOI] [PubMed] [Google Scholar]
  23. Strickland M. S., Thompson N. E., Burgess R. R. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry. 1988 Jul 26;27(15):5755–5762. doi: 10.1021/bi00415a054. [DOI] [PubMed] [Google Scholar]
  24. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  25. Zuber P., Healy J., Carter H. L., 3rd, Cutting S., Moran C. P., Jr, Losick R. Mutation changing the specificity of an RNA polymerase sigma factor. J Mol Biol. 1989 Apr 20;206(4):605–614. doi: 10.1016/0022-2836(89)90569-x. [DOI] [PubMed] [Google Scholar]

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