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. 1996 Jul 23;93(15):7794–7799. doi: 10.1073/pnas.93.15.7794

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

B A Bensing 1, B J Meyer 1, G M Dunny 1
PMCID: PMC38827  PMID: 8755555

Abstract

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

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Selected References

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

  1. Alifano P., Bruni C. B., Carlomagno M. S. Control of mRNA processing and decay in prokaryotes. Genetica. 1994;94(2-3):157–172. doi: 10.1007/BF01443430. [DOI] [PubMed] [Google Scholar]
  2. Bechhofer D. H. Triple post-transcriptional control. Mol Microbiol. 1990 Sep;4(9):1419–1423. doi: 10.1111/j.1365-2958.1990.tb02051.x. [DOI] [PubMed] [Google Scholar]
  3. Belasco J. G., Higgins C. F. Mechanisms of mRNA decay in bacteria: a perspective. Gene. 1988 Dec 10;72(1-2):15–23. doi: 10.1016/0378-1119(88)90123-0. [DOI] [PubMed] [Google Scholar]
  4. Chung J. W., Bensing B. A., Dunny G. M. Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions. J Bacteriol. 1995 Apr;177(8):2107–2117. doi: 10.1128/jb.177.8.2107-2117.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung J. W., Dunny G. M. Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9020–9024. doi: 10.1073/pnas.89.19.9020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung J. W., Dunny G. M. Transcriptional analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions. J Bacteriol. 1995 Apr;177(8):2118–2124. doi: 10.1128/jb.177.8.2118-2124.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunny G. M., Leonard B. A., Hedberg P. J. Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication. J Bacteriol. 1995 Feb;177(4):871–876. doi: 10.1128/jb.177.4.871-876.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunny G. M., Zimmerman D. L., Tortorello M. L. Induction of surface exclusion (entry exclusion) by Streptococcus faecalis sex pheromones: use of monoclonal antibodies to identify an inducible surface antigen involved in the exclusion process. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8582–8586. doi: 10.1073/pnas.82.24.8582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fromont-Racine M., Bertrand E., Pictet R., Grange T. A highly sensitive method for mapping the 5' termini of mRNAs. Nucleic Acids Res. 1993 Apr 11;21(7):1683–1684. doi: 10.1093/nar/21.7.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haynes L. L., Rothman-Denes L. B. N4 virion RNA polymerase sites of transcription initiation. Cell. 1985 Jun;41(2):597–605. doi: 10.1016/s0092-8674(85)80032-5. [DOI] [PubMed] [Google Scholar]
  11. Hedberg P. J., Leonard B. A., Ruhfel R. E., Dunny G. M. Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation. Plasmid. 1996 Jan;35(1):46–57. doi: 10.1006/plas.1996.0005. [DOI] [PubMed] [Google Scholar]
  12. Kao S. M., Olmsted S. B., Viksnins A. S., Gallo J. C., Dunny G. M. Molecular and genetic analysis of a region of plasmid pCF10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis. J Bacteriol. 1991 Dec;173(23):7650–7664. doi: 10.1128/jb.173.23.7650-7664.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leonard B. A., Podbielski A., Hedberg P. J., Dunny G. M. Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):260–264. doi: 10.1073/pnas.93.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meyer B. J., Southern P. J. Concurrent sequence analysis of 5' and 3' RNA termini by intramolecular circularization reveals 5' nontemplated bases and 3' terminal heterogeneity for lymphocytic choriomeningitis virus mRNAs. J Virol. 1993 May;67(5):2621–2627. doi: 10.1128/jvi.67.5.2621-2627.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Meyer B. J., Southern P. J. Sequence heterogeneity in the termini of lymphocytic choriomeningitis virus genomic and antigenomic RNAs. J Virol. 1994 Nov;68(11):7659–7664. doi: 10.1128/jvi.68.11.7659-7664.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nakayama J., Ruhfel R. E., Dunny G. M., Isogai A., Suzuki A. The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10. J Bacteriol. 1994 Dec;176(23):7405–7408. doi: 10.1128/jb.176.23.7405-7408.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaw J. H., Clewell D. B. Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. J Bacteriol. 1985 Nov;164(2):782–796. doi: 10.1128/jb.164.2.782-796.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Silber R., Malathi V. G., Hurwitz J. Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3009–3013. doi: 10.1073/pnas.69.10.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]

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