Abstract
The RepE initiator protein (251 residues) is essential for mini-F replication in Escherichia coli and exhibits two major functions: initiation of DNA replication from ori2 and autogenous repression of repE transcription. Whereas the initiation is mediated by RepE monomers that bind to the ori2 iterons (direct repeats), the autogenous repression is mediated by dimers that bind to the repE operator, which contains an inverted repeat sequence related to the iterons. We now report that the binding of RepE to these DNA sites is primarily determined by the C-terminal region of this protein. The mutant RepE proteins lacking either the N-terminal 33 (or more) residues or the C-terminal 7 (or more) residues were first shown to be defective in binding to both the ori2 and the operator DNAs. However, direct screening and analysis of mutant RepEs which are specifically affected in binding to the ori2 iterons revealed that the mutations (mostly amino acid substitutions) occur exclusively in the C-terminal region (residues 168 to 242). These mutant proteins exhibited reduced binding to ori2 and no detectable binding to the operator. Thus, whereas truncation of either end of RepE can destroy the DNA-binding activities, the C-terminal region appears to represent a primary DNA-binding domain of RepE for both ori2 and the operator. Analogous DNA-binding domains seem to be conserved among the initiator proteins of certain related plasmids.
Full Text
The Full Text of this article is available as a PDF (361.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Armstrong K. A., Acosta R., Ledner E., Machida Y., Pancotto M., McCormick M., Ohtsubo H., Ohtsubo E. A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J Mol Biol. 1984 May 25;175(3):331–348. doi: 10.1016/0022-2836(84)90352-8. [DOI] [PubMed] [Google Scholar]
- BECKWITH J. RESTORATION OF OPERON ACTIVITY BY SUPPRESSORS. Biochim Biophys Acta. 1963 Sep 17;76:162–164. [PubMed] [Google Scholar]
- Bex F., Piérard P., Desmyter A., Drèze P., Colet M., Couturier M. Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control. J Mol Biol. 1986 May 20;189(2):293–303. doi: 10.1016/0022-2836(86)90511-5. [DOI] [PubMed] [Google Scholar]
- Dodd I. B., Egan J. B. Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res. 1990 Sep 11;18(17):5019–5026. doi: 10.1093/nar/18.17.5019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ezaki B., Ogura T., Mori H., Niki H., Hiraga S. Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene. Mol Gen Genet. 1989 Aug;218(2):183–189. doi: 10.1007/BF00331267. [DOI] [PubMed] [Google Scholar]
- Filutowicz M., Dellis S., Levchenko I., Urh M., Wu F., York D. Regulation of replication of an iteron-containing DNA molecule. Prog Nucleic Acid Res Mol Biol. 1994;48:239–273. doi: 10.1016/s0079-6603(08)60857-0. [DOI] [PubMed] [Google Scholar]
- Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
- Germino J., Bastia D. Primary structure of the replication initiation protein of plasmid R6K. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5475–5479. doi: 10.1073/pnas.79.18.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Germino J., Bastia D. Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4692–4696. doi: 10.1073/pnas.81.15.4692. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilbride K. A., Brunton J. L. Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. J Bacteriol. 1990 May;172(5):2439–2446. doi: 10.1128/jb.172.5.2439-2446.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giraldo R., Nieto C., Fernandez-Tresguerres M. E., Diaz R. Bacterial zipper. Nature. 1989 Dec 21;342(6252):866–866. doi: 10.1038/342866a0. [DOI] [PubMed] [Google Scholar]
- Greener A., Filutowicz M. S., McEachern M. J., Helinski D. R. N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. Mol Gen Genet. 1990 Oct;224(1):24–32. doi: 10.1007/BF00259447. [DOI] [PubMed] [Google Scholar]
- Hansen E. B., Yarmolinsky M. B. Host participation in plasmid maintenance: dependence upon dnaA of replicons derived from P1 and F. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4423–4427. doi: 10.1073/pnas.83.12.4423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirano M., Shigesada K., Imai M. Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli. Gene. 1987;57(1):89–99. doi: 10.1016/0378-1119(87)90180-6. [DOI] [PubMed] [Google Scholar]
- Ishiai M., Wada C., Kawasaki Y., Yura T. Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein. J Bacteriol. 1992 Sep;174(17):5597–5603. doi: 10.1128/jb.174.17.5597-5603.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ishiai M., Wada C., Kawasaki Y., Yura T. Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor). Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3839–3843. doi: 10.1073/pnas.91.9.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kawasaki Y., Wada C., Yura T. Binding of RepE initiator protein to mini-F DNA origin (ori2). Enhancing effects of repE mutations and DnaJ heat shock protein. J Biol Chem. 1992 Jun 5;267(16):11520–11524. [PubMed] [Google Scholar]
- Kawasaki Y., Wada C., Yura T. Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein. J Bacteriol. 1991 Feb;173(3):1064–1072. doi: 10.1128/jb.173.3.1064-1072.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kawasaki Y., Wada C., Yura T. Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication. Mol Gen Genet. 1990 Jan;220(2):277–282. doi: 10.1007/BF00260494. [DOI] [PubMed] [Google Scholar]
- Kline B. C. A review of mini-F plasmid maintenance. Plasmid. 1985 Jul;14(1):1–16. doi: 10.1016/0147-619x(85)90027-7. [DOI] [PubMed] [Google Scholar]
- Kline B. C., Kogoma T., Tam J. E., Shields M. S. Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance. J Bacteriol. 1986 Oct;168(1):440–443. doi: 10.1128/jb.168.1.440-443.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kline B. C., Sandhu G. S., Eckloff B. W., Aleff R. A. Site-specific proteolysis of mini-F plasmid replication protein RepE destroys initiator function and generates an incompatibility substance. J Bacteriol. 1992 May;174(9):3004–3010. doi: 10.1128/jb.174.9.3004-3010.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krishnan B. R., Fobert P. R., Seitzer U., Iyer V. N. Mutations within the replicon of the IncN plasmid pCU1 that affect its Escherichia coli polA-independence but not its autonomous replication ability. Gene. 1990 Jul 2;91(1):1–7. doi: 10.1016/0378-1119(90)90155-k. [DOI] [PubMed] [Google Scholar]
- Manen D., Upegui-Gonzalez L. C., Caro L. Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8923–8927. doi: 10.1073/pnas.89.19.8923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masson L., Ray D. S. Mechanism of autonomous control of the Escherichia coli F plasmid: different complexes of the initiator/repressor protein are bound to its operator and to an F plasmid replication origin. Nucleic Acids Res. 1986 Jul 25;14(14):5693–5711. doi: 10.1093/nar/14.14.5693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masson L., Ray D. S. Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product. Nucleic Acids Res. 1988 Jan 25;16(2):413–424. doi: 10.1093/nar/16.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McEachern M. J., Filutowicz M., Helinski D. R. Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1480–1484. doi: 10.1073/pnas.82.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muraiso K., Tokino T., Murotsu T., Matsubara K. Replication of mini-F plasmid in vitro promoted by purified E protein. Mol Gen Genet. 1987 Mar;206(3):519–521. doi: 10.1007/BF00428895. [DOI] [PubMed] [Google Scholar]
- Murakami Y., Ohmori H., Yura T., Nagata T. Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication. J Bacteriol. 1987 Apr;169(4):1724–1730. doi: 10.1128/jb.169.4.1724-1730.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murotsu T., Matsubara K., Sugisaki H., Takanami M. Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene. 1981 Nov;15(2-3):257–271. doi: 10.1016/0378-1119(81)90135-9. [DOI] [PubMed] [Google Scholar]
- Nieto C., Giraldo R., Fernández-Tresguerres E., Díaz R. Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi. J Mol Biol. 1992 Jan 20;223(2):415–426. doi: 10.1016/0022-2836(92)90661-3. [DOI] [PubMed] [Google Scholar]
- Ogura T., Niki H., Kano Y., Imamoto F., Hiraga S. Maintenance of plasmids in HU and IHF mutants of Escherichia coli. Mol Gen Genet. 1990 Jan;220(2):197–203. doi: 10.1007/BF00260482. [DOI] [PubMed] [Google Scholar]
- Tokino T., Murotsu T., Matsubara K. Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4109–4113. doi: 10.1073/pnas.83.12.4109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wada C., Imai M., Yura T. Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8849–8853. doi: 10.1073/pnas.84.24.8849. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wada M., Kohno K., Imamoto F., Kano Y. Participation of hup gene product in ori2-dependent replication of fertility plasmid F. Gene. 1988 Oct 30;70(2):393–397. doi: 10.1016/0378-1119(88)90211-9. [DOI] [PubMed] [Google Scholar]
- Zerbib D., Jakowec M., Prentki P., Galas D. J., Chandler M. Expression of proteins essential for IS1 transposition: specific binding of InsA to the ends of IS1. EMBO J. 1987 Oct;6(10):3163–3169. doi: 10.1002/j.1460-2075.1987.tb02627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- da Silva-Tatley F. M., Steyn L. M. Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini-replicon of pCU1 and the ori-2 of F. Mol Microbiol. 1993 Mar;7(5):805–823. doi: 10.1111/j.1365-2958.1993.tb01171.x. [DOI] [PubMed] [Google Scholar]