Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Feb 1;24(3):450–457. doi: 10.1093/nar/24.3.450

Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function.

I Artsimovitch 1, M M Howe 1
PMCID: PMC145667  PMID: 8602357

Abstract

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region. Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR. The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production. Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them. Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding. In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer. These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint. These mutations alter promoter activity without affecting Mor binding. A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence.

Full Text

The Full Text of this article is available as a PDF (128.9 KB).

Selected References

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

  1. Ansari A. Z., Chael M. L., O'Halloran T. V. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature. 1992 Jan 2;355(6355):87–89. doi: 10.1038/355087a0. [DOI] [PubMed] [Google Scholar]
  2. Balzer D., Ziegelin G., Pansegrau W., Kruft V., Lanka E. KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucleic Acids Res. 1992 Apr 25;20(8):1851–1858. doi: 10.1093/nar/20.8.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baxa C. A., Chiang L., Howe M. M. DNA sequence characterization of the G gene region of bacteriophage Mu. DNA Seq. 1992;2(5):329–333. doi: 10.3109/10425179209030967. [DOI] [PubMed] [Google Scholar]
  4. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bukhari A. I. Reversal of mutator phage Mu integration. J Mol Biol. 1975 Jul 25;96(1):87–99. doi: 10.1016/0022-2836(75)90183-7. [DOI] [PubMed] [Google Scholar]
  6. Carey J. Gel retardation. Methods Enzymol. 1991;208:103–117. doi: 10.1016/0076-6879(91)08010-f. [DOI] [PubMed] [Google Scholar]
  7. Chiang L. W., Howe M. M. Mutational analysis of a C-dependent late promoter of bacteriophage Mu. Genetics. 1993 Nov;135(3):619–629. doi: 10.1093/genetics/135.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chiang L. W., Kovari I., Howe M. M. Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element. PCR Methods Appl. 1993 Feb;2(3):210–217. doi: 10.1101/gr.2.3.210. [DOI] [PubMed] [Google Scholar]
  9. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crothers D. M., Haran T. E., Nadeau J. G. Intrinsically bent DNA. J Biol Chem. 1990 May 5;265(13):7093–7096. [PubMed] [Google Scholar]
  11. Gralla J. D. Promoter recognition and mRNA initiation by Escherichia coli E sigma 70. Methods Enzymol. 1990;185:37–54. doi: 10.1016/0076-6879(90)85006-a. [DOI] [PubMed] [Google Scholar]
  12. Hattman S., Ives J., Margolin W., Howe M. M. Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region. Gene. 1985;39(1):71–76. doi: 10.1016/0378-1119(85)90109-x. [DOI] [PubMed] [Google Scholar]
  13. Heisig P., Kahmann R. The sequence and mom-transactivation function of the C gene of bacteriophage Mu. Gene. 1986;43(1-2):59–67. doi: 10.1016/0378-1119(86)90008-9. [DOI] [PubMed] [Google Scholar]
  14. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Livrelli V., Lee I. W., Summers A. O. In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants. J Biol Chem. 1993 Feb 5;268(4):2623–2631. [PubMed] [Google Scholar]
  17. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  18. Margolin W., Howe M. M. Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription. Nucleic Acids Res. 1986 Jun 25;14(12):4881–4897. doi: 10.1093/nar/14.12.4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mathee K., Howe M. M. Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter. J Bacteriol. 1993 Sep;175(17):5314–5323. doi: 10.1128/jb.175.17.5314-5323.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mathee K., Howe M. M. Identification of a positive regulator of the Mu middle operon. J Bacteriol. 1990 Dec;172(12):6641–6650. doi: 10.1128/jb.172.12.6641-6650.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  22. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  23. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  24. Ross W., Gosink K. K., Salomon J., Igarashi K., Zou C., Ishihama A., Severinov K., Gourse R. L. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. doi: 10.1126/science.8248780. [DOI] [PubMed] [Google Scholar]
  25. Sasse-Dwight S., Gralla J. D. Footprinting protein-DNA complexes in vivo. Methods Enzymol. 1991;208:146–168. doi: 10.1016/0076-6879(91)08012-7. [DOI] [PubMed] [Google Scholar]
  26. Stoddard S. F., Howe M. M. Characterization of the C operon transcript of bacteriophage Mu. J Bacteriol. 1990 Jan;172(1):361–371. doi: 10.1128/jb.172.1.361-371.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stoddard S. F., Howe M. M. Localization and regulation of bacteriophage Mu promoters. J Bacteriol. 1989 Jun;171(6):3440–3448. doi: 10.1128/jb.171.6.3440-3448.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  29. Toussaint A., Lecocq J. P. Sensitivity of bacteriophage Mu-1 development to rifampicin and streptolydigin. Mol Gen Genet. 1974 Mar 14;129(2):185–188. doi: 10.1007/BF00268631. [DOI] [PubMed] [Google Scholar]
  30. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  31. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES