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. 1987 Jul 10;15(13):5413–5432. doi: 10.1093/nar/15.13.5413

Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties.

K A Chapman, R R Burgess
PMCID: PMC305969  PMID: 3299271

Abstract

This paper describes the construction of 18 cloned bacteriophage T7 late promoters with single point mutations. In vitro transcription experiments were used to characterize the properties of these promoters. Since the mutated promoters are cloned into identical backgrounds, differences seen in the transcription assays are directly attributable to the point mutations. All of the mutated promoters are less active than wildtype, but they can be divided into two types. Type A mutations map from -4 to +1 and reduce promoter activity when the template is linearized or when 60mM NaCl is added to the reaction buffer. Type B mutations map from -9 to -7 and reduce promoter activity under all conditions tested. At several sites all three possible point mutations are available. At these sites we observed hierarchies of base pair preference, as determined by promoter activity, that may indicate that T7 RNA polymerase interacts with groups in the major groove.

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Selected References

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

  1. Basu S., Maitra U. Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleoside triphosphate (GTP). J Mol Biol. 1986 Aug 5;190(3):425–437. doi: 10.1016/0022-2836(86)90013-6. [DOI] [PubMed] [Google Scholar]
  2. Blake R. D., Haydock P. V. Effect of sodium ion on the high-resolution melting of lambda DNA. Biopolymers. 1979 Dec;18(12):3089–3109. doi: 10.1002/bip.1979.360181214. [DOI] [PubMed] [Google Scholar]
  3. Chamberlin M., Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem. 1973 Mar 25;248(6):2235–2244. [PubMed] [Google Scholar]
  4. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dickerson R. E. Base sequence and helix structure variation in B and A DNA. J Mol Biol. 1983 May 25;166(3):419–441. doi: 10.1016/s0022-2836(83)80093-x. [DOI] [PubMed] [Google Scholar]
  6. Gross C. A., Grossman A. D., Liebke H., Walter W., Burgess R. R. Effects of the mutant sigma allele rpoD800 on the synthesis of specific macromolecular components of the Escherichia coli K12 cell. J Mol Biol. 1984 Jan 25;172(3):283–300. doi: 10.1016/s0022-2836(84)80027-3. [DOI] [PubMed] [Google Scholar]
  7. Gunderson S. I., Chapman K. A., Burgess R. R. Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II). Biochemistry. 1987 Mar 24;26(6):1539–1546. doi: 10.1021/bi00380a007. [DOI] [PubMed] [Google Scholar]
  8. Ikeda R. A., Richardson C. C. Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3614–3618. doi: 10.1073/pnas.83.11.3614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lohman T. M., deHaseth P. L., Record M. T., Jr Pentalysine-deoxyribonucleic acid interactions: a model for the general effects of ion concentrations on the interactions of proteins with nucleic acids. Biochemistry. 1980 Jul 22;19(15):3522–3530. doi: 10.1021/bi00556a017. [DOI] [PubMed] [Google Scholar]
  10. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  11. McAllister W. T., Carter A. D. Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro. Nucleic Acids Res. 1980 Oct 24;8(20):4821–4837. doi: 10.1093/nar/8.20.4821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  14. Nakanishi S., Adhya S., Gottesman M., Pastan I. Activation of transcription at specific promoters by glycerol. J Biol Chem. 1974 Jul 10;249(13):4050–4056. [PubMed] [Google Scholar]
  15. Nakanishi S., Adhya S., Gottesman M., Pastan I. Selective effects of MgCl2 and temperature on the initiation of transcription at lac, gal, and lambda promoters. J Biol Chem. 1975 Oct 25;250(20):8202–8208. [PubMed] [Google Scholar]
  16. Oakley J. L., Coleman J. E. Structure of a promoter for T7 RNA polymerase. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4266–4270. doi: 10.1073/pnas.74.10.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Panayotatos N., Wells R. D. Identification, cloning and characterization of three late promoters at 14.6, 14.8 and 15.9% of T7 DNA. J Mol Biol. 1979 Nov 25;135(1):91–109. doi: 10.1016/0022-2836(79)90342-5. [DOI] [PubMed] [Google Scholar]
  19. Rosa M. D. Four T7 RNA polymerase promoters contain an identical 23 bp sequence. Cell. 1979 Apr;16(4):815–825. doi: 10.1016/0092-8674(79)90097-7. [DOI] [PubMed] [Google Scholar]
  20. Rosenberg J. M., Greene P. Eco RI* specificity and hydrogen bonding. DNA. 1982;1(2):117–124. doi: 10.1089/dna.1.1982.1.117. [DOI] [PubMed] [Google Scholar]
  21. Ryan M. J., Wells R. D. Coumerimycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. II. In vivo characterization. Biochemistry. 1976 Aug 24;15(17):3778–3782. doi: 10.1021/bi00662a021. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stahl S. J., Chamberlin M. J. Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase. J Biol Chem. 1978 Jul 25;253(14):4951–4959. [PubMed] [Google Scholar]
  25. Strothkamp R. E., Oakley J. L., Coleman J. E. Promoter melting by T7 ribonucleic acid polymerase as detected by single-stranded endonuclease digestion. Biochemistry. 1980 Mar 18;19(6):1074–1080. doi: 10.1021/bi00547a005. [DOI] [PubMed] [Google Scholar]
  26. Vollenweider H. J., Fiandt M., Szybalski W. A relationship between DNA helix stability and recognition sites for RNA polymerase. Science. 1979 Aug 3;205(4405):508–511. doi: 10.1126/science.377494. [DOI] [PubMed] [Google Scholar]

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