Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Oct;172(10):5732–5741. doi: 10.1128/jb.172.10.5732-5741.1990

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

J N Engel 1, J Pollack 1, F Malik 1, D Ganem 1
PMCID: PMC526889  PMID: 2211507

Abstract

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.

Full text

PDF
5732

Images in this article

5736Image
on p.5736
5738Image
on p.5738
5738Image
on p.5738
5739Image
on p.5739

Selected References

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

  1. Allison L. A., Moyle M., Shales M., Ingles C. J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985 Sep;42(2):599–610. doi: 10.1016/0092-8674(85)90117-5. [DOI] [PubMed] [Google Scholar]
  2. Baehr W., Devlin M. J., Applebury M. L. Isolation and characterization of cGMP phosphodiesterase from bovine rod outer segments. J Biol Chem. 1979 Nov 25;254(22):11669–11677. [PubMed] [Google Scholar]
  3. Becker Y. The chlamydia: molecular biology of procaryotic obligate parasites of eucaryocytes. Microbiol Rev. 1978 Jun;42(2):274–306. doi: 10.1128/mr.42.2.274-306.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biggs J., Searles L. L., Greenleaf A. L. Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II. Cell. 1985 Sep;42(2):611–621. doi: 10.1016/0092-8674(85)90118-7. [DOI] [PubMed] [Google Scholar]
  5. Boylan S. A., Suh J. W., Thomas S. M., Price C. W. Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17. J Bacteriol. 1989 May;171(5):2553–2562. doi: 10.1128/jb.171.5.2553-2562.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Delcuve G., Downing W., Lewis H., Dennis P. P. Nucleotide sequence of the proximal portion of the RNA polymerase beta subunit gene of Escherichia coli. Gene. 1980 Nov;11(3-4):367–373. doi: 10.1016/0378-1119(80)90076-1. [DOI] [PubMed] [Google Scholar]
  7. Earl P. L., Jones E. V., Moss B. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659–3663. doi: 10.1073/pnas.83.11.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engel J. N., Ganem D. A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis. J Bacteriol. 1990 May;172(5):2447–2455. doi: 10.1128/jb.172.5.2447-2455.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engel J. N., Ganem D. Chlamydial rRNA operons: gene organization and identification of putative tandem promoters. J Bacteriol. 1987 Dec;169(12):5678–5685. doi: 10.1128/jb.169.12.5678-5685.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Lee J. Y., Zalenskaya K., Shin Y. K., McKinney J. D., Park J. H., Goldfarb A. Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase. J Bacteriol. 1989 Jun;171(6):3002–3007. doi: 10.1128/jb.171.6.3002-3007.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Losick R., Youngman P., Piggot P. J. Genetics of endospore formation in Bacillus subtilis. Annu Rev Genet. 1986;20:625–669. doi: 10.1146/annurev.ge.20.120186.003205. [DOI] [PubMed] [Google Scholar]
  13. Ohme M., Tanaka M., Chunwongse J., Shinozaki K., Sugiura M. A tobacco chloroplast DNA sequence possibly coding for a polypeptide similar to E. coli RNA polymerase beta-subunit. FEBS Lett. 1986 May 5;200(1):87–90. doi: 10.1016/0014-5793(86)80516-6. [DOI] [PubMed] [Google Scholar]
  14. Ovchinnikov YuA, Monastyrskaya G. S., Gubanov V. V., Guryev S. O., Salomatina I. S., Shuvaeva T. M., Lipkin V. M., Sverdlov E. D. The primary structure of E. coli RNA polymerase, Nucleotide sequence of the rpoC gene and amino acid sequence of the beta'-subunit. Nucleic Acids Res. 1982 Jul 10;10(13):4035–4044. doi: 10.1093/nar/10.13.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Post L. E., Strycharz G. D., Nomura M., Lewis H., Dennis P. P. Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1697–1701. doi: 10.1073/pnas.76.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Reuter S. H., Shapiro L. Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus. J Mol Biol. 1987 Apr 20;194(4):653–662. doi: 10.1016/0022-2836(87)90242-7. [DOI] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sardinia L. M., Engel J. N., Ganem D. Chlamydial gene encoding a 70-kilodalton antigen in Escherichia coli: analysis of expression signals and identification of the gene product. J Bacteriol. 1989 Jan;171(1):335–341. doi: 10.1128/jb.171.1.335-341.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sardinia L. M., Segal E., Ganem D. Developmental regulation of the cysteine-rich outer-membrane proteins of murine Chlamydia trachomatis. J Gen Microbiol. 1988 Apr;134(4):997–1004. doi: 10.1099/00221287-134-4-997. [DOI] [PubMed] [Google Scholar]
  20. Schachter J., Caldwell H. D. Chlamydiae. Annu Rev Microbiol. 1980;34:285–309. doi: 10.1146/annurev.mi.34.100180.001441. [DOI] [PubMed] [Google Scholar]
  21. Schachter J. The intracellular life of Chlamydia. Curr Top Microbiol Immunol. 1988;138:109–139. [PubMed] [Google Scholar]
  22. Shinozaki K., Ohme M., Tanaka M., Wakasugi T., Hayashida N., Matsubayashi T., Zaita N., Chunwongse J., Obokata J., Yamaguchi-Shinozaki K. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986 Sep;5(9):2043–2049. doi: 10.1002/j.1460-2075.1986.tb04464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stephens R. S., Wagar E. A., Edman U. Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis. J Bacteriol. 1988 Feb;170(2):744–750. doi: 10.1128/jb.170.2.744-750.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES