Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Oct;173(20):6568–6577. doi: 10.1128/jb.173.20.6568-6577.1991

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

J L Vogel 1, Z J Li 1, M M Howe 1, A Toussaint 1, N P Higgins 1
PMCID: PMC208994  PMID: 1833382

Abstract

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.

Full text

PDF
6569

Images in this article

6572FIG. 3
on p.6572
6573FIG. 4
on p.6573
6574FIG. 6
on p.6574

Selected References

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

  1. Appleyard R K. Segregation of Lambda Lysogenicity during Bacterial Recombination in Escherichia Coli K12. Genetics. 1954 Jul;39(4):429–439. doi: 10.1093/genetics/39.4.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourret R. B., Fox M. S. Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts. J Bacteriol. 1988 Apr;170(4):1683–1690. doi: 10.1128/jb.170.4.1683-1690.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bourret R. B., Fox M. S. Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu. J Bacteriol. 1988 Apr;170(4):1672–1682. doi: 10.1128/jb.170.4.1672-1682.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  6. Falconi M., McGovern V., Gualerzi C., Hillyard D., Higgins N. P. Mutations altering chromosomal protein H-NS induce mini-Mu transposition. New Biol. 1991 Jun;3(6):615–625. [PubMed] [Google Scholar]
  7. Germino J., Gray J. G., Charbonneau H., Vanaman T., Bastia D. Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6848–6852. doi: 10.1073/pnas.80.22.6848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geuskens V., Vogel J. L., Grimaud R., Desmet L., Higgins N. P., Toussaint A. Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage. J Bacteriol. 1991 Oct;173(20):6578–6585. doi: 10.1128/jb.173.20.6578-6585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goosen N., van Heuvel M., Moolenaar G. F., van de Putte P. Regulation of Mu transposition. II. The escherichia coli HimD protein positively controls two repressor promoters and the early promoter of bacteriophage Mu. Gene. 1984 Dec;32(3):419–426. doi: 10.1016/0378-1119(84)90017-9. [DOI] [PubMed] [Google Scholar]
  10. Gray T. M., Matthews B. W. Structural analysis of the temperature-sensitive mutant of bacteriophage T4 lysozyme, glycine 156----aspartic acid. J Biol Chem. 1987 Dec 15;262(35):16858–16864. doi: 10.2210/pdb1l16/pdb. [DOI] [PubMed] [Google Scholar]
  11. Harshey R. M., Getzoff E. D., Baldwin D. L., Miller J. L., Chaconas G. Primary structure of phage mu transposase: homology to mu repressor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7676–7680. doi: 10.1073/pnas.82.22.7676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hecht M. H., Nelson H. C., Sauer R. T. Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding. Proc Natl Acad Sci U S A. 1983 May;80(9):2676–2680. doi: 10.1073/pnas.80.9.2676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Higgins N. P., Collier D. A., Kilpatrick M. W., Krause H. M. Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu. J Biol Chem. 1989 Feb 15;264(5):3035–3042. [PubMed] [Google Scholar]
  14. Hu J. C., O'Shea E. K., Kim P. S., Sauer R. T. Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. Science. 1990 Dec 7;250(4986):1400–1403. doi: 10.1126/science.2147779. [DOI] [PubMed] [Google Scholar]
  15. Krause H. M., Higgins N. P. On the mu repressor and early DNA intermediates of transposition. Cold Spring Harb Symp Quant Biol. 1984;49:827–834. doi: 10.1101/sqb.1984.049.01.093. [DOI] [PubMed] [Google Scholar]
  16. Krause H. M., Higgins N. P. Positive and negative regulation of the Mu operator by Mu repressor and Escherichia coli integration host factor. J Biol Chem. 1986 Mar 15;261(8):3744–3752. [PubMed] [Google Scholar]
  17. Krause H. M., Rothwell M. R., Higgins N. P. The early promoter of bacteriophage Mu: definition of the site of transcript initiation. Nucleic Acids Res. 1983 Aug 25;11(16):5483–5495. doi: 10.1093/nar/11.16.5483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mizuuchi M., Weisberg R. A., Mizuuchi K. DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu. Nucleic Acids Res. 1986 May 12;14(9):3813–3825. doi: 10.1093/nar/14.9.3813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moskaluk C., Bastia D. DNA bending is induced in an enhancer by the DNA-binding domain of the bovine papillomavirus E2 protein. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1826–1830. doi: 10.1073/pnas.85.6.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nelson H. C., Hecht M. H., Sauer R. T. Mutations defining the operator-binding sites of bacteriophage lambda repressor. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):441–449. doi: 10.1101/sqb.1983.047.01.052. [DOI] [PubMed] [Google Scholar]
  21. Nishi K., Müller M., Schnier J. Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli. J Bacteriol. 1987 Oct;169(10):4854–4856. doi: 10.1128/jb.169.10.4854-4856.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pato M. L., Howe M. M., Higgins N. P. A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8716–8720. doi: 10.1073/pnas.87.22.8716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Priess H., Kamp D., Kahmann R., Bräuer B., Delius H. Nucleotide sequence of the immunity region of bacteriophage Mu. Mol Gen Genet. 1982;186(3):315–321. doi: 10.1007/BF00729448. [DOI] [PubMed] [Google Scholar]
  24. Ross W., Shore S. H., Howe M. M. Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu. J Bacteriol. 1986 Sep;167(3):905–919. doi: 10.1128/jb.167.3.905-919.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Signer E. R., Weil J. Recombination in bacteriophage lambda. I. Mutants deficient in general recombination. J Mol Biol. 1968 Jul 14;34(2):261–271. doi: 10.1016/0022-2836(68)90251-9. [DOI] [PubMed] [Google Scholar]
  27. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  28. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  29. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]
  30. Yu M. H., King J. Single amino acid substitutions influencing the folding pathway of the phage P22 tail spike endorhamnosidase. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6584–6588. doi: 10.1073/pnas.81.21.6584. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES