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. 1995 Aug;177(16):4730–4741. doi: 10.1128/jb.177.16.4730-4741.1995

The replication of an IncL/M plasmid is subject to antisense control.

V Athanasopoulos 1, J Praszkier 1, A J Pittard 1
PMCID: PMC177239  PMID: 7543895

Abstract

A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnal gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.

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Selected References

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  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Aksoy S., Squires C. L., Squires C. Translational coupling of the trpB and trpA genes in the Escherichia coli tryptophan operon. J Bacteriol. 1984 Feb;157(2):363–367. doi: 10.1128/jb.157.2.363-367.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asano K., Kato A., Moriwaki H., Hama C., Shiba K., Mizobuchi K. Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level. J Biol Chem. 1991 Feb 25;266(6):3774–3781. [PubMed] [Google Scholar]
  4. Asano K., Moriwaki H., Mizobuchi K. An induced mRNA secondary structure enhances repZ translation in plasmid ColIb-P9. J Biol Chem. 1991 Dec 25;266(36):24549–24556. [PubMed] [Google Scholar]
  5. Blomberg P., Nordström K., Wagner E. G. Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. EMBO J. 1992 Jul;11(7):2675–2683. doi: 10.1002/j.1460-2075.1992.tb05333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brady G., Frey J., Danbara H., Timmis K. N. Replication control mutations of plasmid R6-5 and their effects on interactions of the RNA-I control element with its target. J Bacteriol. 1983 Apr;154(1):429–436. doi: 10.1128/jb.154.1.429-436.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Das A., Yanofsky C. A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair. Nucleic Acids Res. 1984 Jun 11;12(11):4757–4768. doi: 10.1093/nar/12.11.4757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davey R. B., Bird P. I., Nikoletti S. M., Praszkier J., Pittard J. The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids. Plasmid. 1984 May;11(3):234–242. doi: 10.1016/0147-619x(84)90029-5. [DOI] [PubMed] [Google Scholar]
  10. Gerhart E., Wagner H., Nordström K. Structural analysis of an RNA molecule involved in replication control of plasmid R1. Nucleic Acids Res. 1986 Mar 25;14(6):2523–2538. doi: 10.1093/nar/14.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Givskov M., Molin S. Copy mutants of plasmid R1: effects of base pair substitutions in the copA gene on the replication control system. Mol Gen Genet. 1984;194(1-2):286–292. doi: 10.1007/BF00383529. [DOI] [PubMed] [Google Scholar]
  12. Goodrich J. A., Schwartz M. L., McClure W. R. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 1990 Sep 11;18(17):4993–5000. doi: 10.1093/nar/18.17.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hama C., Takizawa T., Moriwaki H., Mizobuchi K. Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid. J Biol Chem. 1990 Jun 25;265(18):10666–10673. [PubMed] [Google Scholar]
  14. Hama C., Takizawa T., Moriwaki H., Urasaki Y., Mizobuchi K. Organization of the replication control region of plasmid ColIb-P9. J Bacteriol. 1990 Apr;172(4):1983–1991. doi: 10.1128/jb.172.4.1983-1991.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higgins D. G., Sharp P. M. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15;73(1):237–244. doi: 10.1016/0378-1119(88)90330-7. [DOI] [PubMed] [Google Scholar]
  16. Higgins D. G., Sharp P. M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr;5(2):151–153. doi: 10.1093/bioinformatics/5.2.151. [DOI] [PubMed] [Google Scholar]
  17. Jaeger J. A., Turner D. H., Zuker M. Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706–7710. doi: 10.1073/pnas.86.20.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jaeger J. A., Turner D. H., Zuker M. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 1990;183:281–306. doi: 10.1016/0076-6879(90)83019-6. [DOI] [PubMed] [Google Scholar]
  19. Kieny M. P., Lathe R., Lecocq J. P. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene. 1983 Dec;26(1):91–99. doi: 10.1016/0378-1119(83)90039-2. [DOI] [PubMed] [Google Scholar]
  20. Lacatena R. M., Cesareni G. Base pairing of RNA I with its complementary sequence in the primer precursor inhibits ColE1 replication. Nature. 1981 Dec 17;294(5842):623–626. doi: 10.1038/294623a0. [DOI] [PubMed] [Google Scholar]
  21. Lawley B., Pittard A. J. Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12. J Bacteriol. 1994 Nov;176(22):6921–6930. doi: 10.1128/jb.176.22.6921-6930.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Light J., Molin S. Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule. EMBO J. 1983;2(1):93–98. doi: 10.1002/j.1460-2075.1983.tb01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Light J., Molin S. The sites of action of the two copy number control functions of plasmid R1. Mol Gen Genet. 1982;187(3):486–493. doi: 10.1007/BF00332633. [DOI] [PubMed] [Google Scholar]
  24. MONOD J., COHEN-BAZIRE G., COHN M. Sur la biosynthèse de la beta-galactosidase (lactase) chez Escherichia coli; la spécificité de l'induction. Biochim Biophys Acta. 1951 Nov;7(4):585–599. doi: 10.1016/0006-3002(51)90072-8. [DOI] [PubMed] [Google Scholar]
  25. Maas R., Oppenheim J., Saadi S., Fuchs T., Maas W. K. Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC. Mol Microbiol. 1991 Apr;5(4):927–932. doi: 10.1111/j.1365-2958.1991.tb00767.x. [DOI] [PubMed] [Google Scholar]
  26. Masukata H., Tomizawa J. Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript. Cell. 1986 Jan 17;44(1):125–136. doi: 10.1016/0092-8674(86)90491-5. [DOI] [PubMed] [Google Scholar]
  27. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  28. Nikoletti S., Bird P., Praszkier J., Pittard J. Analysis of the incompatibility determinants of I-complex plasmids. J Bacteriol. 1988 Mar;170(3):1311–1318. doi: 10.1128/jb.170.3.1311-1318.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Novick R. P., Adler G. K., Majumder S., Khan S. A., Carleton S., Rosenblum W. D., Iordanescu S. Coding sequence for the pT181 repC product: a plasmid-coded protein uniquely required for replication. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4108–4112. doi: 10.1073/pnas.79.13.4108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Novick R. P., Iordanescu S., Projan S. J., Kornblum J., Edelman I. pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator. Cell. 1989 Oct 20;59(2):395–404. doi: 10.1016/0092-8674(89)90300-0. [DOI] [PubMed] [Google Scholar]
  31. Novick R. P. Plasmid incompatibility. Microbiol Rev. 1987 Dec;51(4):381–395. doi: 10.1128/mr.51.4.381-395.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ohman M., Wagner E. G. Secondary structure analysis of the RepA mRNA leader transcript involved in control of replication of plasmid R1. Nucleic Acids Res. 1989 Apr 11;17(7):2557–2579. doi: 10.1093/nar/17.7.2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oppenheim D. S., Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. doi: 10.1093/genetics/95.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Persson C., Wagner E. G., Nordström K. Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT. EMBO J. 1990 Nov;9(11):3767–3775. doi: 10.1002/j.1460-2075.1990.tb07590.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Praszkier J., Bird P., Nikoletti S., Pittard J. Role of countertranscript RNA in the copy number control system of an IncB miniplasmid. J Bacteriol. 1989 Sep;171(9):5056–5064. doi: 10.1128/jb.171.9.5056-5064.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Praszkier J., Wei T., Siemering K., Pittard J. Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids. J Bacteriol. 1991 Apr;173(7):2393–2397. doi: 10.1128/jb.173.7.2393-2397.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Praszkier J., Wilson I. W., Pittard A. J. Mutations affecting translational coupling between the rep genes of an IncB miniplasmid. J Bacteriol. 1992 Apr;174(7):2376–2383. doi: 10.1128/jb.174.7.2376-2383.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ringquist S., Shinedling S., Barrick D., Green L., Binkley J., Stormo G. D., Gold L. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol. 1992 May;6(9):1219–1229. doi: 10.1111/j.1365-2958.1992.tb01561.x. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schümperli D., McKenney K., Sobieski D. A., Rosenberg M. Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon. Cell. 1982 Oct;30(3):865–871. doi: 10.1016/0092-8674(82)90291-4. [DOI] [PubMed] [Google Scholar]
  41. Shiba K., Mizobuchi K. Posttranscriptional control of plasmid ColIb-P9 repZ gene expression by a small RNA. J Bacteriol. 1990 Apr;172(4):1992–1997. doi: 10.1128/jb.172.4.1992-1997.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Siemering K. R., Praszkier J., Pittard A. J. Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication. J Bacteriol. 1993 May;175(10):2895–2906. doi: 10.1128/jb.175.10.2895-2906.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Staden R. Further procedures for sequence analysis by computer. Nucleic Acids Res. 1978 Mar;5(3):1013–1016. doi: 10.1093/nar/5.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Staden R. Sequence data handling by computer. Nucleic Acids Res. 1977 Nov;4(11):4037–4051. doi: 10.1093/nar/4.11.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stougaard P., Molin S., Nordström K. RNAs involved in copy-number control and incompatibility of plasmid R1. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6008–6012. doi: 10.1073/pnas.78.10.6008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tamm J., Polisky B. Structural analysis of RNA molecules involved in plasmid copy number control. Nucleic Acids Res. 1983 Sep 24;11(18):6381–6397. doi: 10.1093/nar/11.18.6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Theisen M., Neuhard J. Translational coupling in the pyrF operon of Salmonella typhimurium. Mol Gen Genet. 1990 Jul;222(2-3):345–352. doi: 10.1007/BF00633839. [DOI] [PubMed] [Google Scholar]
  48. Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984 Oct;38(3):861–870. doi: 10.1016/0092-8674(84)90281-2. [DOI] [PubMed] [Google Scholar]
  49. Tomizawa J., Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6096–6100. doi: 10.1073/pnas.78.10.6096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vandeyar M. A., Weiner M. P., Hutton C. J., Batt C. A. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene. 1988 May 15;65(1):129–133. doi: 10.1016/0378-1119(88)90425-8. [DOI] [PubMed] [Google Scholar]
  51. Wilson I. W., Praszkier J., Pittard A. J. Molecular analysis of RNAI control of repB translation in IncB plasmids. J Bacteriol. 1994 Nov;176(21):6497–6508. doi: 10.1128/jb.176.21.6497-6508.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilson I. W., Praszkier J., Pittard A. J. Mutations affecting pseudoknot control of the replication of B group plasmids. J Bacteriol. 1993 Oct;175(20):6476–6483. doi: 10.1128/jb.175.20.6476-6483.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Womble D. D., Dong X., Wu R. P., Luckow V. A., Martinez A. F., Rownd R. H. IncFII plasmid incompatibility product and its target are both RNA transcripts. J Bacteriol. 1984 Oct;160(1):28–35. doi: 10.1128/jb.160.1.28-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu R., Wang X., Womble D. D., Rownd R. H. Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide. J Bacteriol. 1992 Dec;174(23):7620–7628. doi: 10.1128/jb.174.23.7620-7628.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]

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