Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Jul;169(7):3379–3384. doi: 10.1128/jb.169.7.3379-3384.1987

Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

A Heltzel, D Gambill, W J Jackson, P A Totis, A O Summers
PMCID: PMC212397  PMID: 3036786

Abstract

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.

Full text

PDF
3379

Images in this article

Selected References

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

  1. Barrineau P., Gilbert P., Jackson W. J., Jones C. S., Summers A. O., Wisdom S. The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1. J Mol Appl Genet. 1984;2(6):601–619. [PubMed] [Google Scholar]
  2. Barrineau P., Gilbert P., Jackson W. J., Jones C. S., Summers A. O., Wisdom S. The structure of the mer operon. Basic Life Sci. 1985;30:707–718. doi: 10.1007/978-1-4613-2447-8_49. [DOI] [PubMed] [Google Scholar]
  3. Barrineau P., Summers A. O. A second positive regulatory function in the mer (mercury resistance) operon. Gene. 1983 Nov;25(2-3):209–221. doi: 10.1016/0378-1119(83)90225-1. [DOI] [PubMed] [Google Scholar]
  4. Chamberlin M., Ring J. Characterization of T7-specific ribonucleic acid polymerase. II. Inhibitors of the enzyme and their application to the study of the enzymatic reaction. J Biol Chem. 1973 Mar 25;248(6):2245–2250. [PubMed] [Google Scholar]
  5. Coleman J., Inouye M., Nakamura K. Mutations upstream of the ribosome-binding site affect translational efficiency. J Mol Biol. 1985 Jan 5;181(1):139–143. doi: 10.1016/0022-2836(85)90332-8. [DOI] [PubMed] [Google Scholar]
  6. Dempsey W. B., McIntire S. A., Willetts N., Schottel J., Kinscherf T. G., Silver S., Shannon W. A., Jr Properties of lambda transducing bacteriophages carrying R100 plasmid DNA: mercury resistance genes. J Bacteriol. 1978 Dec;136(3):1084–1093. doi: 10.1128/jb.136.3.1084-1093.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Foster T. J., Brown N. L. Identification of the merR gene of R100 by using mer-lac gene and operon fusions. J Bacteriol. 1985 Sep;163(3):1153–1157. doi: 10.1128/jb.163.3.1153-1157.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Foster T. J., Ginnity F. Some mercurial resistance plasmids from different incompatibility groups specify merR regulatory functions that both repress and induce the mer operon of plasmid R100. J Bacteriol. 1985 May;162(2):773–776. doi: 10.1128/jb.162.2.773-776.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foster T. J., Nakahara H., Weiss A. A., Silver S. Transposon A-generated mutations in the mercuric resistance genes of plasmid R100-1. J Bacteriol. 1979 Oct;140(1):167–181. doi: 10.1128/jb.140.1.167-181.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gambill B. D., Summers A. O. Versatile mercury-resistant cloning and expression vectors. Gene. 1985;39(2-3):293–297. doi: 10.1016/0378-1119(85)90326-9. [DOI] [PubMed] [Google Scholar]
  12. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gheysen D., Iserentant D., Derom C., Fiers W. Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene. Gene. 1982 Jan;17(1):55–63. doi: 10.1016/0378-1119(82)90100-7. [DOI] [PubMed] [Google Scholar]
  14. Jackson W. J., Summers A. O. Biochemical characterization of HgCl2-inducible polypeptides encoded by the mer operon of plasmid R100. J Bacteriol. 1982 Aug;151(2):962–970. doi: 10.1128/jb.151.2.962-970.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson W. J., Summers A. O. Polypeptides encoded by the mer operon. J Bacteriol. 1982 Feb;149(2):479–487. doi: 10.1128/jb.149.2.479-487.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelley R. L., Yanofsky C. Trp aporepressor production is controlled by autogenous regulation and inefficient translation. Proc Natl Acad Sci U S A. 1982 May;79(10):3120–3124. doi: 10.1073/pnas.79.10.3120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lathe R., Kieny M. P., Skory S., Lecocq J. P. Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA. 1984;3(2):173–182. doi: 10.1089/dna.1984.3.173. [DOI] [PubMed] [Google Scholar]
  19. Lund P. A., Ford S. J., Brown N. L. Transcriptional regulation of the mercury-resistance genes of transposon Tn501. J Gen Microbiol. 1986 Feb;132(2):465–480. doi: 10.1099/00221287-132-2-465. [DOI] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Misra T. K., Brown N. L., Fritzinger D. C., Pridmore R. D., Barnes W. M., Haberstroh L., Silver S. Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5975–5979. doi: 10.1073/pnas.81.19.5975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Halloran T., Walsh C. Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization. Science. 1987 Jan 9;235(4785):211–214. doi: 10.1126/science.3798107. [DOI] [PubMed] [Google Scholar]
  23. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  24. Schottel J. L., Sninsky J. J., Cohen S. N. Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system. Gene. 1984 May;28(2):177–193. doi: 10.1016/0378-1119(84)90255-5. [DOI] [PubMed] [Google Scholar]
  25. Sherratt D., Arthur A., Burke M. Transposon-specified, site-specific recombination systems. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):275–281. doi: 10.1101/sqb.1981.045.01.040. [DOI] [PubMed] [Google Scholar]
  26. Summers A. O. Organization, expression, and evolution of genes for mercury resistance. Annu Rev Microbiol. 1986;40:607–634. doi: 10.1146/annurev.mi.40.100186.003135. [DOI] [PubMed] [Google Scholar]
  27. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  28. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Thomas J. O., Kornberg R. D. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2626–2630. doi: 10.1073/pnas.72.7.2626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. de Boer H. A., Hui A., Comstock L. J., Wong E., Vasser M. Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites. DNA. 1983;2(3):231–235. doi: 10.1089/dna.1983.2.231. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES