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. 1993 Jul 15;90(14):6756–6760. doi: 10.1073/pnas.90.14.6756

The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates.

F Xu 1, S Lin-Chao 1, S N Cohen 1
PMCID: PMC47011  PMID: 7688127

Abstract

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.

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Selected References

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

  1. Bassett C. L., Kushner S. R. Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli. J Bacteriol. 1984 Feb;157(2):661–664. doi: 10.1128/jb.157.2.661-664.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bouvet P., Belasco J. G. Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli. Nature. 1992 Dec 3;360(6403):488–491. doi: 10.1038/360488a0. [DOI] [PubMed] [Google Scholar]
  3. Cao G. J., Sarkar N. Identification of the gene for an Escherichia coli poly(A) polymerase. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10380–10384. doi: 10.1073/pnas.89.21.10380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao G. J., Sarkar N. Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7546–7550. doi: 10.1073/pnas.89.16.7546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiang C. S., Xu Y. C., Bremer H. Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli. Mol Gen Genet. 1991 Mar;225(3):435–442. doi: 10.1007/BF00261684. [DOI] [PubMed] [Google Scholar]
  6. Cudny H., Lupski J. R., Godson G. N., Deutscher M. P. Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase. J Biol Chem. 1986 May 15;261(14):6444–6449. [PubMed] [Google Scholar]
  7. Eguchi Y., Itoh T., Tomizawa J. Antisense RNA. Annu Rev Biochem. 1991;60:631–652. doi: 10.1146/annurev.bi.60.070191.003215. [DOI] [PubMed] [Google Scholar]
  8. Gopalakrishna Y., Sarkar N. Selective resistance of bacterial polyadenylate-containing RNA to hydrolysis by guanosine 3'-5'-monophosphate-sensitive nuclease of Bacillus brevis. Biochem Biophys Res Commun. 1981 Nov 30;103(2):454–460. doi: 10.1016/0006-291x(81)90474-5. [DOI] [PubMed] [Google Scholar]
  9. Helmer-Citterich M., Anceschi M. M., Banner D. W., Cesareni G. Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII. EMBO J. 1988 Feb;7(2):557–566. doi: 10.1002/j.1460-2075.1988.tb02845.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kuwano M., Ono M., Endo H., Hori K., Nakamura K., Hirota Y., Ohnishi Y. Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability. Mol Gen Genet. 1977 Sep 9;154(3):279–285. doi: 10.1007/BF00571283. [DOI] [PubMed] [Google Scholar]
  12. Lin-Chao S., Chen W. T., Wong T. T. High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol. 1992 Nov;6(22):3385–3393. doi: 10.1111/j.1365-2958.1992.tb02206.x. [DOI] [PubMed] [Google Scholar]
  13. Liu J. D., Parkinson J. S. Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control. J Bacteriol. 1989 Mar;171(3):1254–1261. doi: 10.1128/jb.171.3.1254-1261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lopilato J., Bortner S., Beckwith J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet. 1986 Nov;205(2):285–290. doi: 10.1007/BF00430440. [DOI] [PubMed] [Google Scholar]
  15. March J. B., Colloms M. D., Hart-Davis D., Oliver I. R., Masters M. Cloning and characterization of an Escherichia coli gene, pcnB, affecting plasmid copy number. Mol Microbiol. 1989 Jul;3(7):903–910. doi: 10.1111/j.1365-2958.1989.tb00239.x. [DOI] [PubMed] [Google Scholar]
  16. Masters M., March J. B., Oliver I. R., Collins J. F. A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions. Mol Gen Genet. 1990 Jan;220(2):341–344. doi: 10.1007/BF00260507. [DOI] [PubMed] [Google Scholar]
  17. Ohmori H., Murakami Y., Nagata T. Nucleotide sequences required for a ColE1-type plasmid to replicate in Escherichia coli cells with or without RNase H. J Mol Biol. 1987 Nov 20;198(2):223–234. doi: 10.1016/0022-2836(87)90308-1. [DOI] [PubMed] [Google Scholar]
  18. Ray B. K., Singh B., Roy M. K., Apirion D. Ribonuclease E is involved in the processing of 5-S rRNA from a number of rRNA transcription units. Eur J Biochem. 1982 Jul;125(2):283–289. doi: 10.1111/j.1432-1033.1982.tb06680.x. [DOI] [PubMed] [Google Scholar]
  19. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  20. Tomcsányi T., Apirion D. Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis. J Mol Biol. 1985 Oct 20;185(4):713–720. doi: 10.1016/0022-2836(85)90056-7. [DOI] [PubMed] [Google Scholar]

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