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. 1987 Feb;6(2):515–522. doi: 10.1002/j.1460-2075.1987.tb04783.x

Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA.

E G Wagner, J von Heijne, K Nordström
PMCID: PMC553424  PMID: 3556167

Abstract

Replication of IncFII plasmids is regulated through the expression of a gene, repA. The RepA protein is rate-limiting for initiation of replication. The main negative control is exerted by a countertranscript, CopA RNA, that binds to the complementary region of the RepA mRNA, thereby inhibiting the formation of the RepA protein. The target region for CopA RNA, CopT, is located upstream of the RepA coding region. An open reading frame for a putative 7k protein overlaps the CopT sequence. Here we show by using lacZ fusions that the 7k gene is expressed. We constructed a translation start mutation in order to abolish formation of the 7k protein. This resulted in a 10-fold decrease in repA expression. The 7k protein produced in trans did not reverse this effect, so the 7k protein per se does not control expression of repA. However, translation of the 7k coding sequence must influence CopA/CopT RNA recognition, since ribosomes will transiently disrupt the target hairpin. We propose here a novel mechanism that affects the level of gene expression: the 7k region of the RepA mRNA is a leader sequence that is involved in expression of the downstream gene; translation of the 7k region competes with a negative control system involving RNA-RNA interaction.

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Selected References

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  1. BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. doi: 10.1128/jb.62.3.293-300.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkhout B., Kastelein R. A., van Duin J. Translational interference at overlapping reading frames in prokaryotic messenger RNA. Gene. 1985;37(1-3):171–179. doi: 10.1016/0378-1119(85)90270-7. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Bonekamp F., Clemmesen K., Karlström O., Jensen K. F. Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription. EMBO J. 1984 Dec 1;3(12):2857–2861. doi: 10.1002/j.1460-2075.1984.tb02220.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Brawner M. E., Jaskunas S. R. Identification of polypeptides encoded by the replication of resistance factor R100. J Mol Biol. 1982 Jul 25;159(1):35–55. doi: 10.1016/0022-2836(82)90030-4. [DOI] [PubMed] [Google Scholar]
  9. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Collins J. Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3. Gene. 1979 May;6(1):29–42. doi: 10.1016/0378-1119(79)90083-0. [DOI] [PubMed] [Google Scholar]
  12. Danbara H., Brady G., Timmis J. K., Timmis K. N. Regulation of DNA replication: "target" determinant of the replication control elements of plasmid R6-5 lies within a control element gene. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4699–4703. doi: 10.1073/pnas.78.8.4699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DeLeon D. V., Cox K. H., Angerer L. M., Angerer R. C. Most early-variant histone mRNA is contained in the pronucleus of sea urchin eggs. Dev Biol. 1983 Nov;100(1):197–206. doi: 10.1016/0012-1606(83)90211-7. [DOI] [PubMed] [Google Scholar]
  14. Dong X., Womble D. D., Luckow V. A., Rownd R. H. Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein. J Bacteriol. 1985 Feb;161(2):544–551. doi: 10.1128/jb.161.2.544-551.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fickett J. W. Recognition of protein coding regions in DNA sequences. Nucleic Acids Res. 1982 Sep 11;10(17):5303–5318. doi: 10.1093/nar/10.17.5303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerdes K., Larsen J. E., Molin S. Stable inheritance of plasmid R1 requires two different loci. J Bacteriol. 1985 Jan;161(1):292–298. doi: 10.1128/jb.161.1.292-298.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Gryczan T. J., Grandi G., Hahn J., Grandi R., Dubnau D. Conformational alteration of mRNA structure and the posttranscriptional regulation of erythromycin-induced drug resistance. Nucleic Acids Res. 1980 Dec 20;8(24):6081–6097. doi: 10.1093/nar/8.24.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horinouchi S., Weisblum B. Posttranscriptional modification of mRNA conformation: mechanism that regulates erythromycin-induced resistance. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7079–7083. doi: 10.1073/pnas.77.12.7079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kollek R., Oertel W., Goebel W. Isolation and characterization of the minimal fragment required for autonomous replication ("basic replicon") of a copy mutant (pKN102) of the antibiotic resistance factor R1. Mol Gen Genet. 1978 Jun 1;162(1):51–57. doi: 10.1007/BF00333850. [DOI] [PubMed] [Google Scholar]
  22. Kolter R., Yanofsky C. Attenuation in amino acid biosynthetic operons. Annu Rev Genet. 1982;16:113–134. doi: 10.1146/annurev.ge.16.120182.000553. [DOI] [PubMed] [Google Scholar]
  23. Larsen J. E., Gerdes K., Light J., Molin S. Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter. Gene. 1984 Apr;28(1):45–54. doi: 10.1016/0378-1119(84)90086-6. [DOI] [PubMed] [Google Scholar]
  24. Light J., Molin S. Expression of a copy number control gene (copB) of plasmid R1 is constitutive and growth rate dependent. J Bacteriol. 1982 Sep;151(3):1129–1135. doi: 10.1128/jb.151.3.1129-1135.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Light J., Molin S. Replication control functions of plasmid R1 act as inhibitors of expression of a gene required for replication. Mol Gen Genet. 1981;184(1):56–61. doi: 10.1007/BF00271195. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Looman A. C., de Gruyter M., Vogelaar A., van Knippenberg P. H. Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli. Gene. 1985;37(1-3):145–154. doi: 10.1016/0378-1119(85)90267-7. [DOI] [PubMed] [Google Scholar]
  29. Masai H., Kaziro Y., Arai K. Definition of oriR, the minimum DNA segment essential for initiation of R1 plasmid replication in vitro. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6814–6818. doi: 10.1073/pnas.80.22.6814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Molin S., Stougaard P., Light J., Nordström M., Nordström K. Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control. Mol Gen Genet. 1981;181(1):123–130. doi: 10.1007/BF00339015. [DOI] [PubMed] [Google Scholar]
  33. Molin S., Stougaard P., Uhlin B. E., Gustafsson P., Nordström K. Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19. J Bacteriol. 1979 Apr;138(1):70–79. doi: 10.1128/jb.138.1.70-79.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nordström K., Molin S., Aagaard-Hansen H. Partitioning of plasmid R1 in Escherichia coli. II. Incompatibility properties of the partitioning system. Plasmid. 1980 Nov;4(3):332–339. doi: 10.1016/0147-619x(80)90071-2. [DOI] [PubMed] [Google Scholar]
  35. Nordström K., Molin S., Light J. Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. Plasmid. 1984 Sep;12(2):71–90. doi: 10.1016/0147-619x(84)90054-4. [DOI] [PubMed] [Google Scholar]
  36. Ofverstedt L. G., Hammarström K., Balgobin N., Hjertén S., Pettersson U., Chattopadhyaya J. Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis). Biochim Biophys Acta. 1984 Jun 16;782(2):120–126. doi: 10.1016/0167-4781(84)90014-9. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Poulsen P., Bonekamp F., Jensen K. F. Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attentuation. EMBO J. 1984 Aug;3(8):1783–1790. doi: 10.1002/j.1460-2075.1984.tb02046.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Riise E., Molin S. Purification and characterization of the CopB replication control protein, and precise mapping of its target site in the R1 plasmid. Plasmid. 1986 May;15(3):163–171. doi: 10.1016/0147-619x(86)90034-x. [DOI] [PubMed] [Google Scholar]
  40. Riise E., Stougaard P., Bindslev B., Nordström K., Molin S. Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1. J Bacteriol. 1982 Sep;151(3):1136–1145. doi: 10.1128/jb.151.3.1136-1145.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roberts T. M., Kacich R., Ptashne M. A general method for maximizing the expression of a cloned gene. Proc Natl Acad Sci U S A. 1979 Feb;76(2):760–764. doi: 10.1073/pnas.76.2.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosen J., Ryder T., Inokuchi H., Ohtsubo H., Ohtsubo E. Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet. 1980;179(3):527–537. doi: 10.1007/BF00271742. [DOI] [PubMed] [Google Scholar]
  43. Ryder T. B., Davidson D. B., Rosen J. I., Ohtsubo E., Ohtsubo H. Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli. Gene. 1982 Mar;17(3):299–310. doi: 10.1016/0378-1119(82)90146-9. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stewart V., Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):383–386. doi: 10.1128/jb.167.1.383-386.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Vogt V. M. Purification and properties of an aminopeptidase from Escherichia coli. J Biol Chem. 1970 Sep 25;245(18):4760–4769. [PubMed] [Google Scholar]
  49. 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]
  50. Womble D. D., Sampathkumar P., Easton A. M., Luckow V. A., Rownd R. H. Transcription of the replication control region of the IncFII R-plasmid NR1 in vitro and in vivo. J Mol Biol. 1985 Feb 5;181(3):395–410. doi: 10.1016/0022-2836(85)90228-1. [DOI] [PubMed] [Google Scholar]
  51. Zubay G. In vitro synthesis of protein in microbial systems. Annu Rev Genet. 1973;7:267–287. doi: 10.1146/annurev.ge.07.120173.001411. [DOI] [PubMed] [Google Scholar]

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