Abstract
The anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.
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- Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
- Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
- Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43–46. doi: 10.1038/221043a0. [DOI] [PubMed] [Google Scholar]
- Creighton T. E. Intermediates in the refolding of reduced pancreatic trypsin inhibitor. J Mol Biol. 1974 Aug 15;87(3):579–602. doi: 10.1016/0022-2836(74)90105-3. [DOI] [PubMed] [Google Scholar]
- Daegelen P., Brody E. The rIIA gene of bacteriophage T4. I. Its DNA sequence and discovery of a new open reading frame between genes 60 and rIIA. Genetics. 1990 Jun;125(2):237–248. doi: 10.1093/genetics/125.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gansz A., Kruse U., Rüger W. Gene product dsbA of bacteriophage T4 binds to late promoters and enhances late transcription. Mol Gen Genet. 1991 Mar;225(3):427–434. doi: 10.1007/BF00261683. [DOI] [PubMed] [Google Scholar]
- Geiduschek E. P. Regulation of expression of the late genes of bacteriophage T4. Annu Rev Genet. 1991;25:437–460. doi: 10.1146/annurev.ge.25.120191.002253. [DOI] [PubMed] [Google Scholar]
- Hahn S., Kruse U., Rüger W. The region of phage T4 genes 34, 33 and 59: primary structures and organization on the genome. Nucleic Acids Res. 1986 Dec 9;14(23):9311–9327. doi: 10.1093/nar/14.23.9311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Kassavetis G. A., Geiduschek E. P. Defining a bacteriophage T4 late promoter: bacteriophage T4 gene 55 protein suffices for directing late promoter recognition. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5101–5105. doi: 10.1073/pnas.81.16.5101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Liebig H. D., Rüger W. Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J Mol Biol. 1989 Aug 20;208(4):517–536. doi: 10.1016/0022-2836(89)90145-9. [DOI] [PubMed] [Google Scholar]
- Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
- Matthias P. D., Bernard H. U., Scott A., Brady G., Hashimoto-Gotoh T., Schütz G. A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells. EMBO J. 1983;2(9):1487–1492. doi: 10.1002/j.1460-2075.1983.tb01612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Montag D., Degen M., Henning U. Nucleotide sequence of gene t (lysis gene) of the E. coli phage T4. Nucleic Acids Res. 1987 Aug 25;15(16):6736–6736. doi: 10.1093/nar/15.16.6736. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orsini G., Brody E. N. Phage T4 DNA codes for two distinct 10-kDa proteins which strongly bind to RNA polymerase. Virology. 1988 Feb;162(2):397–405. doi: 10.1016/0042-6822(88)90480-1. [DOI] [PubMed] [Google Scholar]
- Ratner D. Letter to the editor: Bacteriophage T4 transcriptional control gene 55 codes for a protein bound to Escherichia coli RNA polymerase. J Mol Biol. 1974 Nov 15;89(4):803–807. doi: 10.1016/0022-2836(74)90054-0. [DOI] [PubMed] [Google Scholar]
- Ratner D. The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase. J Mol Biol. 1974 Sep 15;88(2):373–383. doi: 10.1016/0022-2836(74)90488-4. [DOI] [PubMed] [Google Scholar]
- Ruckman J., Parma D., Tuerk C., Hall D. H., Gold L. Identification of a T4 gene required for bacteriophage mRNA processing. New Biol. 1989 Oct;1(1):54–65. [PubMed] [Google Scholar]
- 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]
- Saunders S. E., Burke J. F. Rapid isolation of miniprep DNA for double strand sequencing. Nucleic Acids Res. 1990 Aug 25;18(16):4948–4948. doi: 10.1093/nar/18.16.4948. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- Stevens A. Deoxyribonucleic acid dependent ribonucleic acid polymerases from two T4 phage-infected systems. Biochemistry. 1974 Jan 29;13(3):493–503. doi: 10.1021/bi00700a015. [DOI] [PubMed] [Google Scholar]
- Stevens A. Inhibition of DNA-enzyme binding by an RNA polymerase inhibitor from T4 phage-infected Escherichia coli. Biochim Biophys Acta. 1977 Mar 2;475(1):193–196. doi: 10.1016/0005-2787(77)90355-0. [DOI] [PubMed] [Google Scholar]
- Stevens A. New small polypeptides associated with DNA-dependent RNA polymerase of Escherichia coli after infection with bacteriophage T4. Proc Natl Acad Sci U S A. 1972 Mar;69(3):603–607. doi: 10.1073/pnas.69.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stevens A., Rhoton J. C. Characterization of an inhibitor causing potassium chloride sensitivity of an RNA polymerase from T4 phage-infected Escherichia coli. Biochemistry. 1975 Nov 18;14(23):5074–5079. doi: 10.1021/bi00694a007. [DOI] [PubMed] [Google Scholar]
- Stormo G. D., Schneider T. D., Gold L., Ehrenfeucht A. Use of the 'Perceptron' algorithm to distinguish translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2997–3011. doi: 10.1093/nar/10.9.2997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Uzan M., Brody E., Favre R. Nucleotide sequence and control of transcription of the bacteriophage T4 motA regulatory gene. Mol Microbiol. 1990 Sep;4(9):1487–1496. doi: 10.1111/j.1365-2958.1990.tb02059.x. [DOI] [PubMed] [Google Scholar]
- Uzan M., Favre R., Brody E. A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8895–8899. doi: 10.1073/pnas.85.23.8895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Williams J. G., Gratzer W. B. Limitations of the detergent-polyacrylamide gel electrophoresis method for molecular weight determination of proteins. J Chromatogr. 1971 Apr 22;57(1):121–125. doi: 10.1016/0021-9673(71)80013-4. [DOI] [PubMed] [Google Scholar]
- Williams K. P., Kassavetis G. A., Esch F. S., Geiduschek E. P. Identification of the gene encoding an RNA polymerase-binding protein of bacteriophage T4. J Virol. 1987 Feb;61(2):597–599. doi: 10.1128/jvi.61.2.597-599.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Williams K. P., Müller R., Rüger W., Geiduschek E. P. Overproduced bacteriophage T4 gene 33 protein binds RNA polymerase. J Bacteriol. 1989 Jun;171(6):3579–3582. doi: 10.1128/jb.171.6.3579-3582.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- de Franciscis V., Brody E. In vitro system for middle T4 RNA. I. Studies with Escherichia coli RNA polymerase. J Biol Chem. 1982 Apr 25;257(8):4087–4096. [PubMed] [Google Scholar]