Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Dec;175(23):7629–7638. doi: 10.1128/jb.175.23.7629-7638.1993

Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides.

R K Karls 1, D J Jin 1, T J Donohue 1
PMCID: PMC206919  PMID: 8244932

Abstract

We have been characterizing RNA polymerase holoenzymes from Rhodobacter sphaeroides. RNA polymerase purified from R. sphaeroides transcribed from promoters recognized by Escherichia coli E sigma 32 or E sigma 70 holoenzyme. Antisera to E. coli sigma 32 or sigma 70 indicated that related polypeptides of approximately 37 kDa (sigma 37) and 93 kDa (sigma 93), respectively, are present in this preparation. Transcription of sigma 32-dependent promoters was observed in a further fractionated R. sphaeroides holoenzyme containing the sigma 37 polypeptide, while a preparation enriched in sigma 93 transcribed sigma 70-dependent promoters. To demonstrate further that the sigma 93 polypeptide functions like E. coli sigma 70, we obtained an R. sphaeroides E sigma 93 holoenzyme capable of transcription from sigma 70-dependent promoters by combining sigma 93 with (i) an E sigma 37 fraction with diminished sigma 93 polypeptide content or (ii) E. coli core RNA polymerase. The generation of analogous DNase I footprints on the lacUV5 promoter by R. sphaeroides E sigma 93 and by E. coli E sigma 70 suggests that the overall structures of these two holoenzymes are similar. However, some differences in promoter specificity between R. sphaeroides E sigma 93 and E. coli E sigma 70 exist because transcription of an R. sphaeroides rRNA promoter was detected only with E sigma 93.

Full text

PDF
7629

Images in this article

7631Image
on p.7631
7632Image
on p.7632
7633Image
on p.7633
7633Image
on p.7633
7634Image
on p.7634
7635Image
on p.7635
7636Image
on p.7636
7636Image
on p.7636

Selected References

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

  1. Armstrong G. A., Alberti M., Leach F., Hearst J. E. Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet. 1989 Apr;216(2-3):254–268. doi: 10.1007/BF00334364. [DOI] [PubMed] [Google Scholar]
  2. Balbás P., Soberón X., Merino E., Zurita M., Lomeli H., Valle F., Flores N., Bolivar F. Plasmid vector pBR322 and its special-purpose derivatives--a review. Gene. 1986;50(1-3):3–40. doi: 10.1016/0378-1119(86)90307-0. [DOI] [PubMed] [Google Scholar]
  3. Brandner J. P., McEwan A. G., Kaplan S., Donohue T. J. Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene. J Bacteriol. 1989 Jan;171(1):360–368. doi: 10.1128/jb.171.1.360-368.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  5. Chen C. Y., Galluppi G. R., Richardson J. P. Transcription termination at lambda tR1 is mediated by interaction of rho with specific single-stranded domains near the 3' end of cro mRNA. Cell. 1986 Sep 26;46(7):1023–1028. doi: 10.1016/0092-8674(86)90701-4. [DOI] [PubMed] [Google Scholar]
  6. Covarrubias L., Cervantes L., Covarrubias A., Soberón X., Vichido I., Blanco A., Kupersztoch-Portnoy Y. M., Bolivar F. Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene. 1981 Jan-Feb;13(1):25–35. doi: 10.1016/0378-1119(81)90040-8. [DOI] [PubMed] [Google Scholar]
  7. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cowing D. W., Gross C. A. Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection. J Mol Biol. 1989 Dec 5;210(3):513–520. doi: 10.1016/0022-2836(89)90127-7. [DOI] [PubMed] [Google Scholar]
  9. Donohue T. J., Kaplan S. Genetic techniques in rhodospirillaceae. Methods Enzymol. 1991;204:459–485. doi: 10.1016/0076-6879(91)04024-i. [DOI] [PubMed] [Google Scholar]
  10. Donohue T. J., McEwan A. G., Van Doren S., Crofts A. R., Kaplan S. Phenotypic and genetic characterization of cytochrome c2 deficient mutants of Rhodobacter sphaeroides. Biochemistry. 1988 Mar 22;27(6):1918–1925. doi: 10.1021/bi00406a018. [DOI] [PubMed] [Google Scholar]
  11. Dryden S. C., Kaplan S. Identification of cis-acting regulatory regions upstream of the rRNA operons of Rhodobacter sphaeroides. J Bacteriol. 1993 Oct;175(20):6392–6402. doi: 10.1128/jb.175.20.6392-6402.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dryden S. C., Kaplan S. Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. Nucleic Acids Res. 1990 Dec 25;18(24):7267–7277. doi: 10.1093/nar/18.24.7267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Erickson J. W., Vaughn V., Walter W. A., Neidhardt F. C., Gross C. A. Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene. Genes Dev. 1987 Jul;1(5):419–432. doi: 10.1101/gad.1.5.419. [DOI] [PubMed] [Google Scholar]
  14. Gao J. G., Gussin G. N. Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro. J Bacteriol. 1991 Jun;173(12):3763–3769. doi: 10.1128/jb.173.12.3763-3769.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao J. G., Gussin G. N. RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins. J Bacteriol. 1991 Jan;173(1):394–397. doi: 10.1128/jb.173.1.394-397.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  17. Hager D. A., Jin D. J., Burgess R. R. Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase. Biochemistry. 1990 Aug 28;29(34):7890–7894. doi: 10.1021/bi00486a016. [DOI] [PubMed] [Google Scholar]
  18. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kansy J. W., Kaplan S. Purification, characterization, and transcriptional analyses of RNA polymerases from Rhodobacter sphaeroides cells grown chemoheterotrophically and photoheterotrophically. J Biol Chem. 1989 Aug 15;264(23):13751–13759. [PubMed] [Google Scholar]
  21. Kiley P. J., Kaplan S. Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol Rev. 1988 Mar;52(1):50–69. doi: 10.1128/mr.52.1.50-69.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee J. K., Kiley P. J., Kaplan S. Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides. J Bacteriol. 1989 Jun;171(6):3391–3405. doi: 10.1128/jb.171.6.3391-3405.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Ma D., Cook D. N., O'Brien D. A., Hearst J. E. Analysis of the promoter and regulatory sequences of an oxygen-regulated bch operon in Rhodobacter capsulatus by site-directed mutagenesis. J Bacteriol. 1993 Apr;175(7):2037–2045. doi: 10.1128/jb.175.7.2037-2045.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MacGregor B. J., Donohue T. J. Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides. J Bacteriol. 1991 Jul;173(13):3949–3957. doi: 10.1128/jb.173.13.3949-3957.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  27. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  28. Newlands J. T., Ross W., Gosink K. K., Gourse R. L. Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. J Mol Biol. 1991 Aug 5;220(3):569–583. doi: 10.1016/0022-2836(91)90101-b. [DOI] [PubMed] [Google Scholar]
  29. Ross W., Thompson J. F., Newlands J. T., Gourse R. L. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. doi: 10.1002/j.1460-2075.1990.tb07586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. SISTROM W. R. A requirement for sodium in the growth of Rhodopseudomonas spheroides. J Gen Microbiol. 1960 Jun;22:778–785. doi: 10.1099/00221287-22-3-778. [DOI] [PubMed] [Google Scholar]
  31. Sheen J. Y., Seed B. Electrolyte gradient gels for DNA sequencing. Biotechniques. 1988 Nov-Dec;6(10):942–944. [PubMed] [Google Scholar]
  32. 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]
  33. Strohl W. R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992 Mar 11;20(5):961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Taylor W. E., Straus D. B., Grossman A. D., Burton Z. F., Gross C. A., Burgess R. R. Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase. Cell. 1984 Sep;38(2):371–381. doi: 10.1016/0092-8674(84)90492-6. [DOI] [PubMed] [Google Scholar]
  35. Wellington C. L., Beatty J. T. Promoter mapping and nucleotide sequence of the bchC bacteriochlorophyll biosynthesis gene from Rhodobacter capsulatus. Gene. 1989 Nov 30;83(2):251–261. doi: 10.1016/0378-1119(89)90111-x. [DOI] [PubMed] [Google Scholar]
  36. Wiggs J. L., Bush J. W., Chamberlin M. J. Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders. Cell. 1979 Jan;16(1):97–109. doi: 10.1016/0092-8674(79)90191-0. [DOI] [PubMed] [Google Scholar]
  37. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES