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. 1990 Mar;172(3):1385–1391. doi: 10.1128/jb.172.3.1385-1391.1990

Purified Escherichia coli F-factor TraY protein binds oriT.

E E Lahue 1, S W Matson 1
PMCID: PMC208610  PMID: 2407722

Abstract

The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.

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Selected References

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

  1. Abdel-Monem M., Taucher-Scholz G., Klinkert M. Q. Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4659–4663. doi: 10.1073/pnas.80.15.4659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Cohen A., Fisher W. D., Curtiss R., 3rd, Adler H. I. The properties of DNA transferred to minicells during conjugation. Cold Spring Harb Symp Quant Biol. 1968;33:635–641. doi: 10.1101/sqb.1968.033.01.071. [DOI] [PubMed] [Google Scholar]
  4. Everett R., Willetts N. Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F. J Mol Biol. 1980 Jan 15;136(2):129–150. doi: 10.1016/0022-2836(80)90309-5. [DOI] [PubMed] [Google Scholar]
  5. Finlay B. B., Frost L. S., Paranchych W. Nucleotide sequences of the R1-19 plasmid transfer genes traM, finP, traJ, and traY and the traYZ promoter. J Bacteriol. 1986 May;166(2):368–374. doi: 10.1128/jb.166.2.368-374.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Finlay B. B., Frost L. S., Paranchych W. Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1. J Bacteriol. 1986 Oct;168(1):132–139. doi: 10.1128/jb.168.1.132-139.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fowler T., Taylor L., Thompson R. The control region of the F plasmid transfer operon: DNA sequence of the traJ and traY genes and characterisation of the traY leads to Z promoter. Gene. 1983 Dec;26(1):79–89. doi: 10.1016/0378-1119(83)90038-0. [DOI] [PubMed] [Google Scholar]
  8. Fürste J. P., Pansegrau W., Ziegelin G., Kröger M., Lanka E. Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1771–1775. doi: 10.1073/pnas.86.6.1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  10. Inamoto S., Yoshioka Y., Ohtsubo E. Identification and characterization of the products from the traJ and traY genes of plasmid R100. J Bacteriol. 1988 Jun;170(6):2749–2757. doi: 10.1128/jb.170.6.2749-2757.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ippen-Ihler K. A., Minkley E. G., Jr The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet. 1986;20:593–624. doi: 10.1146/annurev.ge.20.120186.003113. [DOI] [PubMed] [Google Scholar]
  12. Johnson D., Everett R., Willetts N. Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP. J Mol Biol. 1981 Dec 5;153(2):187–202. doi: 10.1016/0022-2836(81)90273-4. [DOI] [PubMed] [Google Scholar]
  13. Kennedy N., Beutin L., Achtman M., Skurray R., Rahmsdorf U., Herrlich P. Conjugation proteins encoded by the F sex factor. Nature. 1977 Dec 15;270(5638):580–585. doi: 10.1038/270580a0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lahue E. E., Matson S. W. Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction. J Biol Chem. 1988 Mar 5;263(7):3208–3215. [PubMed] [Google Scholar]
  16. 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]
  17. Ohki M., Tomizawa J. Asymmetric transfer of DNA strands in bacterial conjugation. Cold Spring Harb Symp Quant Biol. 1968;33:651–658. doi: 10.1101/sqb.1968.033.01.074. [DOI] [PubMed] [Google Scholar]
  18. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sarathy P. V., Siddiqi O. DNA synthesis during bacterial conjugation. II. Is DNA replication in the Hfr obligatory for chromosome transfer? J Mol Biol. 1973 Aug 15;78(3):443–451. doi: 10.1016/0022-2836(73)90467-1. [DOI] [PubMed] [Google Scholar]
  20. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thompson R., Taylor L., Kelly K., Everett R., Willetts N. The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks. EMBO J. 1984 May;3(5):1175–1180. doi: 10.1002/j.1460-2075.1984.tb01947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thompson R., Taylor L. Promoter mapping and DNA sequencing of the F plasmid transfer genes traM and traJ. Mol Gen Genet. 1982;188(3):513–518. doi: 10.1007/BF00330058. [DOI] [PubMed] [Google Scholar]
  23. Thompson T. L., Centola M. B., Deonier R. C. Location of the nick at oriT of the F plasmid. J Mol Biol. 1989 Jun 5;207(3):505–512. doi: 10.1016/0022-2836(89)90460-9. [DOI] [PubMed] [Google Scholar]
  24. Traxler B. A., Minkley E. G., Jr Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein. J Mol Biol. 1988 Nov 5;204(1):205–209. doi: 10.1016/0022-2836(88)90609-2. [DOI] [PubMed] [Google Scholar]
  25. Traxler B. A., Minkley E. G., Jr Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport. J Bacteriol. 1987 Jul;169(7):3251–3259. doi: 10.1128/jb.169.7.3251-3259.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vapnek D., Rupp W. D. Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli. J Mol Biol. 1971 Sep 28;60(3):413–424. doi: 10.1016/0022-2836(71)90178-1. [DOI] [PubMed] [Google Scholar]
  27. Ziegelin G., Fürste J. P., Lanka E. TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin. J Biol Chem. 1989 Jul 15;264(20):11989–11994. [PubMed] [Google Scholar]

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