Summary
Promoter sequences recognized by Escherichia coli RNA polymerase have been isolated from bacteriophage T7 DNA using the plasmid pBRH4. T7 DNA was digested with the restriction endonucleases Hae III, Alu I, and Eco RI* and the products of these digestions were ligated into the Eco RI site of pBRH4. Cloning of Hae III and Alu I-digested T7 DNA was achieved by blunt-end ligation of these fragments to the polymerized ends of Eco RI-cleaved pBRH4. This converts blunt-end fragments of T7 DNA into cohesive-end Eco RI fragments. Promoter-containing T7 restriction fragments were selected by activation of the tetracycline-resistance gene located on the plasmid vector. The genomic location of each T7 insert was determined using the “Southern-blotting” technique and Hpa I-cleaved T7 DNA. Two promoter-active restriction fragments are thought to contain the C and E promoters of T7. However, the majority of the promoter-active fragments cloned map within the late gene region of T7. In vitro binding studies indicate that E. coli RNA polymerase can form heparin resistant complexes with the cloned T7 DNA promoter fragments. These results suggest that while E. coli RNA polymerase may not participate directly in the transcription of late T7 genes, promoters for this enzyme are present in this region of the DNA.
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References
Backman K, Ptashne M, Gilbert W (1976) Construction of plasmids carrying the cI gene of bacteriophage λ. Proc Natl Acad Sci USA 73:4174–4178
Bolivar F, Rodriguez RL, Betlach MC, Boyer HW (1977) Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene 2:75–93
Burgess RR, Jendrisak JJ (1975) A procedure for the rapid, largescale purification of Escherichia coli DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14:4634–4638
Campbell JL, Richardson CC, Studier FW (1978) Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc Natl Acad Sci USA 75:2276–2280
Chamberlin M, Ring J (1973) Characterization of T7-specific ribonucleic acid polymerase. I. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem 248:2235–2244
Delius H, Westphal H, Axelrod N (1973) Length measurements of RNA synthesized in vitro by Escherichia coli RNA polymerase. J Mol Biol 74:677–687
DeWyngaert MA, Hinkle DC (1979) Bacterial mutants affecting phage T7 DNA replication produce RNA polymerase resistant to inhibition by the T7 gene 2 protein. J Biol Chem 254:11247–11253
Dunn JJ, Studier FW (1973) T7 early RNAs are generated by site-specific cleavages. Proc Natl Acad Sci USA 70:1559–1563
Golomb M, Chamberlin M (1974) A preliminary map of the major transcription units read by T7 RNA polymerase on the T7 and T3 bacteriophage chromosomes. Proc Natl Acad Sci USA 71:760–764
Gordon RL, Humphries P, McConnell DJ (1978) Restriction enzyme cleavage mapping of T7 virus early region. Mol Gen Genet 162:329–339
Greene PJ, Heyneker HL, Bolivar F, Rodriguez RL, Betlach MC, Covarrubias AA, Backman K, Russel DJ, Tait R, Boyer HW (1978) A general method for the purification of restriction enzymes. Nucleic Acids Res 5:2373–2380
Hsieh T, Wang J (1976) Physicochemical studies on interactions between DNA and RNA polymerase. Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase. Biochemistry 15:5776–5783
Issinger OG, Hausmann R (1973) Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1. J Virol 11:465–472
Jones BB, Chan H, Rothstein S, Wells RD, Reznikoff WS (1977) RNA polymerase binding sites in λ plac DNA. Proc Natl Acad Sci USA 74:4914–1918
Kassavetis GA, Kaya KM, Chamberlin MJ (1978) Escherichia coli RNA polymerase-rifampicin complexes bound at promoter sites block RNA chain eleongation by Escherichia coli RNA polymerase and T7-specific RNA polymerase. Biochemistry 17:5798–5804
Kadesch TR, Williams RC, Chamberlin MJ (1980) Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA. II. Formation of multiple promoter-like complexes at non-promoter sites. J Mol Biol 136:79–93
Koller T, Kübler O, Portmann R, Sogo JM (1978) High resolution physical mapping of specific binding sites of Escherichia coli RNA polymerase on the DNA of bacteriophage T7. J Mol Biol 120:121–131
LeClerc JE, Richardson CC (1979) Gene 2 protein of bacteriophage T7: Purification and requirement for packaging of T7 DNA in vitro. Proc Natl Acad. Sci USA 76:4852–4856
Maniatis T, Jeffrey A, Kleid DG (1975) Nucleotide sequence of the rightward operator of phage λ. Proc Natl Acad Sci USA 72:1184–1188
McConnell DJ (1979a) The DNA sequence at the T7 C promoter. Nucleic Acids Res 6:525–544
McConnell DJ (1979b) Control sites in the sequence at the beginning of T7 gene 1. Nucleic Acids Res 6:3491–3504
McDonnell MW, Simon MN, Studier FW (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–146
Minkley EG, Pribnow D (1973) Transcription of the early region of bacteriophage T7: Selective initiation with dinucleotides. J Mol Biol 77:255–277
Neve RL, West RW, Rodriguez RL (1979) Bukaryotic DNA fragments which act as promoters for a plasmid gene. Nature 277:324–325
Oakley JL, Coleman JE (1977) Structure of a promoter for T7 RNA polymerase. Proc Natl Acad Sci USA 74:4266–4270
Oakley JL, Strothkamp RE, Sarris AE, Coleman JE (1979) T7 RNA polymerase: Promoter structure and polymerase binding. Biochemistry 18:528–537
Oen H, Wu CW, Haas R, Cole PE (1979) T7 deoxyribonucleic acid directed, rapid-turnover, single-step addition reactions catalyzed by Escherichia coli ribonucleic acid polymerase. Biochemistry 18:4148–4154
Panayotatos N, Wells RW (1979) Recognition and initiation site for four late promoters of phage T7 is a 22-base pair DNA sequence. Nature 280:35–39
Pfeffer SR, Stahl SJ, Chamberlin MJ (1977) Binding of Escherichia coli RNA polymerase to T7 DNA-displacement of holoenzyme from promoter complexes by heparin. J Biol Chem 252:5403–5407
Polisky B, Greene P, Garfin DE, McCarthy BJ, Goodman HM, Boyer HW (1975) Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc Natl Acad Sci USA 72:3310–3314
Portmann R, Sogo JM, Koller T, Zillig W (1974) Binding sites of E. coli RNA polymerase on T7 DNA as determined by electron microscopy. FEBS Letters 45:64–67
Pribnow D (1975) Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci USA 72:784–788
Rodriguez RL, West RW, Heyneker HL, Bolivar F, Boyer HW (1979) Characterizing wild-type and mutant promoters of the tetracycline-resistance gene in pBR313. Nucleic Acids Res 6:3267–3287
Rosa MD (1979) Four T7 RNA polymerase promoters contain and identical 23 bp sequence. Cell 16:815–824
Rosenberg AH, Simon MN, Studier FW, Roberts RJ (1980) Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA. J Mol Biol 135:907–916
Siebenlist U (1979) Nucleotide sequence of the three major early promoters of bacteriophage T7. Nucleic Acids Res 6:1895–1907
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stahl SJ, Chamberlin MJ (1977) An expanded transcription map of T7 bacteriophage. Reading of minor T7 promoter sites in vitro by Escherichia coli RNA polymerase. J Mol Biol 112:577–601
Struhl K, Davis RW (1977) Production of a functional eukaryotic enzyme in Escherichia coli: Cloning and expression of the yeast structual gene for for imidazole-glycerolphosphate dehydratase (his3). Proc Natl Acad Sci USA 74:5255–5259
Studier FW (1972) Bacteriophage T7. Science 176:367–376
Studier FW, Rosenberg AH, Simon MN, Dunn JJ (1980) Genetic and physical mapping in the early region of bacteriophage T7 DNA. J Mol Biol 135:917–938
Tait RC, Rodriguez RL, West RW Jr (1980) The rapid purification of T4 DNA ligase from a λT4 lig lysogen. J Biol Chem, 255:813–815
Vapnek D, Hautala JA, Jacobson JW, Giles NH, Kushner SR (1977) Expression in Escherichia coli K-12 of the structural gene for catabolic dihydroquinase of Neurospora crassa. Proc Natl Acad Sci USA 74:3508–4512
West RW Jr, Neve RL, Rodriguez RL (1979) Construction and characterization of E. coli promoter-probe plasmid vectors. I. Cloning of promoter-containing DNA fragments. Gene 7:271–288
West RW Jr, Rodriguez RL (1980) Construction and characterization of E. coli promoter-probe plasmid vectors. II. RNA polymerase binding studies on antibiotic resistance promoters. Gene 9:175–193
Zollinger M, Guertin M, Mamet-Bratley MD (1977) A new electron microscopic method for studying protein-nucleic acid interactions. An Biochem 82:196–203
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Communicated by H. W. Boyer
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West, R.W., McConnell, D. & Rodriguez, R.L. Isolation of E. coli promoters from the late region of bacteriophage T7 DNA. Molec. Gen. Genet. 180, 439–447 (1980). https://doi.org/10.1007/BF00425860
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DOI: https://doi.org/10.1007/BF00425860