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. 1990 Dec;172(12):6641–6650. doi: 10.1128/jb.172.12.6641-6650.1990

Identification of a positive regulator of the Mu middle operon.

K Mathee 1, M M Howe 1
PMCID: PMC210775  PMID: 2147678

Abstract

Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late. The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription. A plasmid containing a Pm-lacZ operon fusion was constructed. beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction. Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17. Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator. Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insertion of a linker into mor substantially reduced the ability of Mu to form plaques. When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu. The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression. Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology.

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Selected References

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

  1. Bedwell D. M., Nomura M. Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli. Mol Gen Genet. 1986 Jul;204(1):17–23. doi: 10.1007/BF00330181. [DOI] [PubMed] [Google Scholar]
  2. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Bowie J. U., Sauer R. T. Identifying determinants of folding and activity for a protein of unknown structure. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2152–2156. doi: 10.1073/pnas.86.7.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bowie J. U., Sauer R. T. TraY proteins of F and related episomes are members of the Arc and Mnt repressor family. J Mol Biol. 1990 Jan 5;211(1):5–6. doi: 10.1016/0022-2836(90)90004-6. [DOI] [PubMed] [Google Scholar]
  8. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  9. Bölker M., Wulczyn F. G., Kahmann R. Role of bacteriophage Mu C protein in activation of the mom gene promoter. J Bacteriol. 1989 Apr;171(4):2019–2027. doi: 10.1128/jb.171.4.2019-2027.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  12. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dodd I. B., Egan J. B. Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins. J Mol Biol. 1987 Apr 5;194(3):557–564. doi: 10.1016/0022-2836(87)90681-4. [DOI] [PubMed] [Google Scholar]
  15. Evans R. M., Hollenberg S. M. Zinc fingers: gilt by association. Cell. 1988 Jan 15;52(1):1–3. doi: 10.1016/0092-8674(88)90522-3. [DOI] [PubMed] [Google Scholar]
  16. Ferguson B., Jones N., Richter J., Rosenberg M. Adenovirus E1a gene product expressed at high levels in Escherichia coli is functional. Science. 1984 Jun 22;224(4655):1343–1346. doi: 10.1126/science.6374895. [DOI] [PubMed] [Google Scholar]
  17. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  18. Grundy F. J., Howe M. M. Involvement of the invertible G segment in bacteriophage mu tail fiber biosynthesis. Virology. 1984 Apr 30;134(2):296–317. doi: 10.1016/0042-6822(84)90299-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Howe M. M., O'Day K. J., Schultz D. W. Isolation of mutations defining five new cistrons essential for development of bacteriophage Mu. Virology. 1979 Mar;93(2):303–319. doi: 10.1016/0042-6822(79)90235-6. [DOI] [PubMed] [Google Scholar]
  22. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  23. Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984 Jul;12(1):19–36. doi: 10.1016/0147-619x(84)90063-5. [DOI] [PubMed] [Google Scholar]
  24. Knight K. L., Bowie J. U., Vershon A. K., Kelley R. D., Sauer R. T. The Arc and Mnt repressors. A new class of sequence-specific DNA-binding protein. J Biol Chem. 1989 Mar 5;264(7):3639–3642. [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  28. Margolin W., Howe M. M. Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase. J Bacteriol. 1990 Mar;172(3):1424–1429. doi: 10.1128/jb.172.3.1424-1429.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Margolin W., Rao G., Howe M. M. Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence. J Bacteriol. 1989 Apr;171(4):2003–2018. doi: 10.1128/jb.171.4.2003-2018.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marrs C. F., Howe M. M. Kinetics and regulation of transcription of bacteriophage Mu. Virology. 1990 Jan;174(1):192–203. doi: 10.1016/0042-6822(90)90068-3. [DOI] [PubMed] [Google Scholar]
  32. Merrick M. J., Gibbins J. R. The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 1985 Nov 11;13(21):7607–7620. doi: 10.1093/nar/13.21.7607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Day K., Schultz D., Ericsen W., Rawluk L., Howe M. Correction and refinement of the genetic map of bacteriophage Mu. Virology. 1979 Mar;93(2):320–328. doi: 10.1016/0042-6822(79)90236-8. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Raibaud O., Schwartz M. Positive control of transcription initiation in bacteria. Annu Rev Genet. 1984;18:173–206. doi: 10.1146/annurev.ge.18.120184.001133. [DOI] [PubMed] [Google Scholar]
  37. Reznikoff W. S., Siegele D. A., Cowing D. W., Gross C. A. The regulation of transcription initiation in bacteria. Annu Rev Genet. 1985;19:355–387. doi: 10.1146/annurev.ge.19.120185.002035. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Stark M. J. Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene. 1987;51(2-3):255–267. doi: 10.1016/0378-1119(87)90314-3. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Stoddard S. F., Howe M. M. DNA sequence within the Mu C operon. Nucleic Acids Res. 1987 Sep 11;15(17):7198–7198. doi: 10.1093/nar/15.17.7198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Van Leerdam E., Karreman C., van de Putte P. Ner, a cro-like function of bacteriophage Mu. Virology. 1982 Nov;123(1):19–28. doi: 10.1016/0042-6822(82)90291-4. [DOI] [PubMed] [Google Scholar]
  48. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  49. Wijffelman C., Gassler M., Stevens W. F., van de Putte P. On the control of transcription of bacteriophage Mu. Mol Gen Genet. 1974;131(2):85–96. doi: 10.1007/BF00266145. [DOI] [PubMed] [Google Scholar]
  50. Wijffelman C., Lotterman B. Kinetics of Mu DNA synthesis. Mol Gen Genet. 1977 Mar 7;151(2):169–174. doi: 10.1007/BF00338691. [DOI] [PubMed] [Google Scholar]
  51. Wijffelman C., van de Putte P. Transcription of bacteriophage mu. An analysis of the transcription pattern in the early phase of phage development. Mol Gen Genet. 1974;135(4):327–337. doi: 10.1007/BF00271147. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. de Jong W. W., Zweers A., Cohen L. H. Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem Biophys Res Commun. 1978 May 30;82(2):532–539. doi: 10.1016/0006-291x(78)90907-5. [DOI] [PubMed] [Google Scholar]
  54. van de Putte P., Giphart-Gassler M., Goosen N., Goosen T., van Leerdam E. Regulation of integration and replication functions of bacteriophage Mu. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):347–353. doi: 10.1101/sqb.1981.045.01.048. [DOI] [PubMed] [Google Scholar]

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