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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Feb 15;88(4):1389–1393. doi: 10.1073/pnas.88.4.1389

Iteron inhibition of plasmid RK2 replication in vitro: evidence for intermolecular coupling of replication origins as a mechanism for RK2 replication control.

B L Kittell 1, D R Helinski 1
PMCID: PMC51023  PMID: 1996339

Abstract

The broad-host-range plasmid RK2 and its derivatives are maintained in Gram-negative bacteria at a specific copy number that appears to be determined by a series of direct repeats (iterons) located at the RK2 replication origin and by the RK2 replication initiation protein. TrfA. An in vitro replication system was developed from Escherichia coli that is active with either the intact eight-iteron RK2 origin or a minimal five-iteron RK2 origin when purified TrfA protein is provided. Using this in vitro replication system, we have examined the mechanism(s) of copy-number control. It was found that two or more RK2 iterons present on a supercoiled compatible plasmid molecule are capable of specifically inhibiting in vitro the replication of either functional RK2 origin plasmid and that this inhibition is not overcome by adding increasing amounts of TrfA protein. A mutant TrfA protein, TrfA-33(cop254D), that increases the copy number of an RK2 origin in vivo exhibits replication kinetics and activity levels in this in vitro system similar to that of the wild-type protein. However, RK2 in vitro replication initiated by TrfA-33(cop254D) has a much reduced sensitivity to iteron inhibition. These data support a model for RK2 copy-number control that involves intermolecular coupling between TrfA-bound iterons.

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Selected References

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

  1. Abeles A. L., Snyder K. M., Chattoraj D. K. P1 plasmid replication: replicon structure. J Mol Biol. 1984 Mar 5;173(3):307–324. doi: 10.1016/0022-2836(84)90123-2. [DOI] [PubMed] [Google Scholar]
  2. Chattoraj D., Cordes K., Abeles A. Plasmid P1 replication: negative control by repeated DNA sequences. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6456–6460. doi: 10.1073/pnas.81.20.6456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Durland R. H., Helinski D. R. Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number. J Bacteriol. 1990 Jul;172(7):3849–3858. doi: 10.1128/jb.172.7.3849-3858.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Durland R. H., Helinski D. R. The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa. Plasmid. 1987 Sep;18(2):164–169. doi: 10.1016/0147-619x(87)90044-8. [DOI] [PubMed] [Google Scholar]
  5. Durland R. H., Toukdarian A., Fang F., Helinski D. R. Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. J Bacteriol. 1990 Jul;172(7):3859–3867. doi: 10.1128/jb.172.7.3859-3867.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuller R. S., Kaguni J. M., Kornberg A. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7370–7374. doi: 10.1073/pnas.78.12.7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gaylo P. J., Turjman N., Bastia D. DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli. J Bacteriol. 1987 Oct;169(10):4703–4709. doi: 10.1128/jb.169.10.4703-4709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kamio Y., Terawaki Y. Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats. J Bacteriol. 1983 Sep;155(3):1185–1191. doi: 10.1128/jb.155.3.1185-1191.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kornacki J. A., West A. H., Firshein W. Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2. Plasmid. 1984 Jan;11(1):48–57. doi: 10.1016/0147-619x(84)90006-4. [DOI] [PubMed] [Google Scholar]
  11. Kües U., Stahl U. Replication of plasmids in gram-negative bacteria. Microbiol Rev. 1989 Dec;53(4):491–516. doi: 10.1128/mr.53.4.491-516.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lin L. S., Kim Y. J., Meyer R. J. The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro. Mol Gen Genet. 1987 Jul;208(3):390–397. doi: 10.1007/BF00328129. [DOI] [PubMed] [Google Scholar]
  13. Lin L. S., Meyer R. J. Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication, expression of incompatibility, and copy-number control. Plasmid. 1986 Jan;15(1):35–47. doi: 10.1016/0147-619x(86)90012-0. [DOI] [PubMed] [Google Scholar]
  14. McEachern M. J., Bott M. A., Tooker P. A., Helinski D. R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942–7946. doi: 10.1073/pnas.86.20.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mukherjee S., Erickson H., Bastia D. Detection of DNA looping due to simultaneous interaction of a DNA-binding protein with two spatially separated binding sites on DNA. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6287–6291. doi: 10.1073/pnas.85.17.6287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pal S. K., Chattoraj D. K. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol. 1988 Aug;170(8):3554–3560. doi: 10.1128/jb.170.8.3554-3560.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Persson C., Nordström K. Control of replication of the broad host range plasmid RSF1010: the incompatibility determinant consists of directly repeated DNA sequences. Mol Gen Genet. 1986 Apr;203(1):189–192. doi: 10.1007/BF00330402. [DOI] [PubMed] [Google Scholar]
  18. Pinkney M., Diaz R., Lanka E., Thomas C. M. Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III. J Mol Biol. 1988 Oct 20;203(4):927–938. doi: 10.1016/0022-2836(88)90118-0. [DOI] [PubMed] [Google Scholar]
  19. Roberts R. C., Burioni R., Helinski D. R. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J Bacteriol. 1990 Nov;172(11):6204–6216. doi: 10.1128/jb.172.11.6204-6216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schauzu M. A., Kücherer C., Kölling R., Messer W., Lother H. Transcripts within the replication origin, oriC, of Escherichia coli. Nucleic Acids Res. 1987 Mar 25;15(6):2479–2497. doi: 10.1093/nar/15.6.2479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schmidhauser T. J., Filutowicz M., Helinski D. R. Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid. 1983 May;9(3):325–330. doi: 10.1016/0147-619x(83)90010-0. [DOI] [PubMed] [Google Scholar]
  22. Schmidhauser T. J., Helinski D. R. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shingler V., Thomas C. M. Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. J Mol Biol. 1984 May 25;175(3):229–249. doi: 10.1016/0022-2836(84)90346-2. [DOI] [PubMed] [Google Scholar]
  24. Smith C. A., Thomas C. M. Nucleotide sequence of the trfA gene of broad host-range plasmid RK2. J Mol Biol. 1984 May 25;175(3):251–262. doi: 10.1016/0022-2836(84)90347-4. [DOI] [PubMed] [Google Scholar]
  25. Smith D. W., Garland A. M., Herman G., Enns R. E., Baker T. A., Zyskind J. W. Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. EMBO J. 1985 May;4(5):1319–1326. doi: 10.1002/j.1460-2075.1985.tb03779.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  27. Staudenbauer W. L. Replication of small plasmids in extracts of Escherichia coli. Mol Gen Genet. 1976 Jun 15;145(3):273–280. doi: 10.1007/BF00325823. [DOI] [PubMed] [Google Scholar]
  28. Thomas C. M. Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid. 1981 May;5(3):277–291. doi: 10.1016/0147-619x(81)90005-6. [DOI] [PubMed] [Google Scholar]
  29. Thomas C. M., Cross M. A., Hussain A. A., Smith C. A. Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2. EMBO J. 1984 Jan;3(1):57–63. doi: 10.1002/j.1460-2075.1984.tb01761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas C. M., Meyer R., Helinski D. R. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol. 1980 Jan;141(1):213–222. doi: 10.1128/jb.141.1.213-222.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thomas C. M., Stalker D. M., Helinski D. R. Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):1–7. doi: 10.1007/BF00338996. [DOI] [PubMed] [Google Scholar]
  32. Tolun A., Helinski D. R. Direct repeats of the F plasmid incC region express F incompatibility. Cell. 1981 Jun;24(3):687–694. doi: 10.1016/0092-8674(81)90095-7. [DOI] [PubMed] [Google Scholar]
  33. Trawick J. D., Kline B. C. A two-stage molecular model for control of mini-F replication. Plasmid. 1985 Jan;13(1):59–69. doi: 10.1016/0147-619x(85)90056-3. [DOI] [PubMed] [Google Scholar]
  34. Tsutsui H., Fujiyama A., Murotsu T., Matsubara K. Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control. J Bacteriol. 1983 Jul;155(1):337–344. doi: 10.1128/jb.155.1.337-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yamaguchi K., Yamaguchi M. The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region. Gene. 1984 Jul-Aug;29(1-2):211–219. doi: 10.1016/0378-1119(84)90181-1. [DOI] [PubMed] [Google Scholar]

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