Abstract
E protein, the 29 kd product of the F plasmid repE gene, plays both positive and negative roles in the autoregulation of F replication. We have cloned and expressed the repE gene in an inducible ATG-fusion vector and have detected specific binding of E protein to the repE operator and to four 19-base pair direct repeats (incB) within the F plasmid replication origin ori2. Binding of E protein at the repE operator occurs with higher affinity than at ori2(incB) and gives almost complete protection to at least 30 base pairs, whereas binding of E protein to the direct repeats in the ori2 region shows an alternating pattern of enhanced and reduced sensitivity to DNAase cleavage consistent with a protein-induced folding of the DNA. These results provide direct biochemical support for a model of F plasmid replication in which the E protein serves both as an initiator of replication and as an autorepressor of its own synthesis.
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Selected References
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- 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]
- 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]
- Carthew R. W., Chodosh L. A., Sharp P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell. 1985 Dec;43(2 Pt 1):439–448. doi: 10.1016/0092-8674(85)90174-6. [DOI] [PubMed] [Google Scholar]
- Collins J., Pritchard R. H. Relationship between chromosome replication and F'lac episome replication in Escherichia coli. J Mol Biol. 1973 Jun 25;78(1):143–155. doi: 10.1016/0022-2836(73)90434-8. [DOI] [PubMed] [Google Scholar]
- Drew H. R., Travers A. A. DNA bending and its relation to nucleosome positioning. J Mol Biol. 1985 Dec 20;186(4):773–790. doi: 10.1016/0022-2836(85)90396-1. [DOI] [PubMed] [Google Scholar]
- Filutowicz M., Davis G., Greener A., Helinski D. R. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. doi: 10.1093/nar/13.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
- Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [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]
- Hines J. C., Ray D. S. Construction and characterization of new coliphage M13 cloning vectors. Gene. 1980 Nov;11(3-4):207–218. doi: 10.1016/0378-1119(80)90061-x. [DOI] [PubMed] [Google Scholar]
- Hochschild A., Ptashne M. Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix. Cell. 1986 Mar 14;44(5):681–687. doi: 10.1016/0092-8674(86)90833-0. [DOI] [PubMed] [Google Scholar]
- Johnston S., Lee J. H., Ray D. S. High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA. Gene. 1985;34(2-3):137–145. doi: 10.1016/0378-1119(85)90121-0. [DOI] [PubMed] [Google Scholar]
- Kamio Y., Tabuchi A., Itoh Y., Katagiri H., Terawaki Y. Complete nucleotide sequence of mini-Rts1 and its copy mutant. J Bacteriol. 1984 Apr;158(1):307–312. doi: 10.1128/jb.158.1.307-312.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kelley W., Bastia D. Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A. 1985 May;82(9):2574–2578. doi: 10.1073/pnas.82.9.2574. [DOI] [PMC free article] [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]
- Komai N., Nishizawa T., Hayakawa Y., Murotsu T., Matsubara K. Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments. Mol Gen Genet. 1982;186(2):193–203. doi: 10.1007/BF00331850. [DOI] [PubMed] [Google Scholar]
- Lane H. E. Replication and incompatibility of F and plasmids in the IncFI Group. Plasmid. 1981 Jan;5(1):100–126. doi: 10.1016/0147-619x(81)90079-2. [DOI] [PubMed] [Google Scholar]
- Linder P., Churchward G., Xia G. X., Yu Y. Y., Caro L. An essential replication gene, repA, of plasmid pSC101 is autoregulated. J Mol Biol. 1985 Feb 5;181(3):383–393. doi: 10.1016/0022-2836(85)90227-x. [DOI] [PubMed] [Google Scholar]
- Maki S., Miki T., Horiuchi T. DNA sequence of an amber replication mutant indicates that a 29 kd protein is the product of the F plasmid replication gene. Mol Gen Genet. 1984;194(1-2):337–339. doi: 10.1007/BF00383537. [DOI] [PubMed] [Google Scholar]
- 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]
- Mukherjee S., Patel I., Bastia D. Conformational changes in a replication origin induced by an initiator protein. Cell. 1985 Nov;43(1):189–197. doi: 10.1016/0092-8674(85)90023-6. [DOI] [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]
- Ogura T., Hiraga S. Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell. 1983 Feb;32(2):351–360. doi: 10.1016/0092-8674(83)90454-3. [DOI] [PubMed] [Google Scholar]
- Rokeach L. A., Søgaard-Andersen L., Molin S. Two functions of the E protein are key elements in the plasmid F replication control system. J Bacteriol. 1985 Dec;164(3):1262–1270. doi: 10.1128/jb.164.3.1262-1270.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ryder K., Silver S., DeLucia A. L., Fanning E., Tegtmeyer P. An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell. 1986 Mar 14;44(5):719–725. doi: 10.1016/0092-8674(86)90838-x. [DOI] [PubMed] [Google Scholar]
- Stalker D. M., Kolter R., Helinski D. R. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol. 1982 Oct 15;161(1):33–43. doi: 10.1016/0022-2836(82)90276-5. [DOI] [PubMed] [Google Scholar]
- Søgaard-Andersen L., Rokeach L. A., Molin S. Regulated expression of a gene important for replication of plasmid F in E. coli. EMBO J. 1984 Feb;3(2):257–262. doi: 10.1002/j.1460-2075.1984.tb01794.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tolun A., Helinski D. R. Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment. Mol Gen Genet. 1982;186(3):372–377. doi: 10.1007/BF00729456. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Tsutsui H., Matsubara K. Replication control and switch-off function as observed with a mini-F factor plasmid. J Bacteriol. 1981 Aug;147(2):509–516. doi: 10.1128/jb.147.2.509-516.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson L. A., Phua S. H., Bergquist P. L., Lane H. E. An Mr 29000 protein is essential for mini-F maintenance in E. coli. Gene. 1982 Sep;19(2):173–178. doi: 10.1016/0378-1119(82)90003-8. [DOI] [PubMed] [Google Scholar]
- Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]