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. 1990 Aug;172(8):4441–4447. doi: 10.1128/jb.172.8.4441-4447.1990

Location of a P1 plasmid replication inhibitor determinant within the initiator gene.

K Muraiso 1, G Mukhopadhyay 1, D K Chattoraj 1
PMCID: PMC213273  PMID: 2198259

Abstract

The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently. There are only about 20 RepA dimers present per repA gene. A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable. The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected. The activity is apparently not from RepA itself. Excess purified RepA did not inhibit replication in vitro. Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity. Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity. Further deletions affected both the activities. These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.

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Selected References

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  1. Abeles A. L. P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA. J Biol Chem. 1986 Mar 15;261(8):3548–3555. [PubMed] [Google Scholar]
  2. 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]
  3. Austin S. J., Mural R. J., Chattoraj D. K., Abeles A. L. Trans- and cis-acting elements for the replication of P1 miniplasmids. J Mol Biol. 1985 May 25;183(2):195–202. doi: 10.1016/0022-2836(85)90212-8. [DOI] [PubMed] [Google Scholar]
  4. Brosius J., Cate R. L., Perlmutter A. P. Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro. J Biol Chem. 1982 Aug 10;257(15):9205–9210. [PubMed] [Google Scholar]
  5. Chattoraj D. K., Snyder K. M., Abeles A. L. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc Natl Acad Sci U S A. 1985 May;82(9):2588–2592. doi: 10.1073/pnas.82.9.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crowl R. Expression of human interferon genes in E. coli with the lambda PL promoter. Methods Enzymol. 1986;119:376–383. doi: 10.1016/0076-6879(86)19057-4. [DOI] [PubMed] [Google Scholar]
  7. Das A. Overproduction of proteins in Escherichia coli: vectors, hosts, and strategies. Methods Enzymol. 1990;182:93–112. doi: 10.1016/0076-6879(90)82011-p. [DOI] [PubMed] [Google Scholar]
  8. Filutowicz M., McEachern M. J., Helinski D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9645–9649. doi: 10.1073/pnas.83.24.9645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fulford W., Model P. Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X. J Mol Biol. 1988 Sep 5;203(1):49–62. doi: 10.1016/0022-2836(88)90090-3. [DOI] [PubMed] [Google Scholar]
  10. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  11. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  12. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Manis J. J., Kline B. C. Restriction endonuclease mapping and mutagenesis of the F sex factor replication region. Mol Gen Genet. 1977 Apr 29;152(3):175–182. doi: 10.1007/BF00268815. [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. 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]
  16. Pal S. K., Mason R. J., Chattoraj D. K. P1 plasmid replication. Role of initiator titration in copy number control. J Mol Biol. 1986 Nov 20;192(2):275–285. doi: 10.1016/0022-2836(86)90364-5. [DOI] [PubMed] [Google Scholar]
  17. Patel I., Bastia D. A replication initiator protein enhances the rate of hybrid formation between a silencer RNA and an activator RNA. Cell. 1987 Nov 6;51(3):455–462. doi: 10.1016/0092-8674(87)90641-6. [DOI] [PubMed] [Google Scholar]
  18. Sternberg N., Austin S. Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L. J Bacteriol. 1983 Feb;153(2):800–812. doi: 10.1128/jb.153.2.800-812.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  20. Stormo G. D., Schneider T. D., Gold L., Ehrenfeucht A. Use of the 'Perceptron' algorithm to distinguish translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2997–3011. doi: 10.1093/nar/10.9.2997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Swack J. A., Pal S. K., Mason R. J., Abeles A. L., Chattoraj D. K. P1 plasmid replication: measurement of initiator protein concentration in vivo. J Bacteriol. 1987 Aug;169(8):3737–3742. doi: 10.1128/jb.169.8.3737-3742.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Terawaki Y., Nozue H., Zeng H., Hayashi T., Kamio Y., Itoh Y. Effects of mutations in the repA gene of plasmid Rts1 on plasmid replication and autorepressor function. J Bacteriol. 1990 Feb;172(2):786–792. doi: 10.1128/jb.172.2.786-792.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wickner S. H., Chattoraj D. K. Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3668–3672. doi: 10.1073/pnas.84.11.3668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yin J. C., Reznikoff W. S. p2 and inhibition of Tn5 transposition. J Bacteriol. 1988 Jul;170(7):3008–3015. doi: 10.1128/jb.170.7.3008-3015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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