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RNA polymerase from Rhizobium japonicum

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Abstract

DNA-dependend RNA polymerase (EC 2.7.7.6) from Rhizobium japonicum was purified. The subunit structure was found to be ββ′α2σ, with the following apparent molecular weights determined by electrophoresis: M r (β and β') 150,000 each, M r (σ) 96,000, M r (α) 40,000, M r (holoenzyme) 490,000, M r (core enzyme) 380,000. The recovery of σ was 28%. RNA polymerase from aerobically grown R. japonicum cells and from nitrogen-fixing cells have the same electrophoretic properties suggesting that no chemical modification of the enzyme occurs when cells undergo this metabolic differentiation.

The enzyme is Mg2+-dependent, rifampicin-sensitive, and has optimal activity at alkaline pH (8–10) and at 35–40° C. It binds strongly to bacteriophage T7 promoters, weakly to antibiotic resistance genes, and not at all to cloned R. japonicum nif DNA. Preliminary in vitro transcription experiments, including nif DNA as template, revealed that additional factors may be required for selective transcription from promoters.

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References

  • Allen RC, Maurer HR (1974) Electrophoresis and isoelectric focusing in polyacrylamide gels. Walter de Gruyter, Berlin New York

    Google Scholar 

  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker ML, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multiple cloning system. Gene 2:95–113

    Article  Google Scholar 

  • Burgess RR (1976) Purification and physical properties of E. coli RNA polymerase. In: Losick R, Chamberlin M (eds) RNA polymerase. Cold Spring Harbor Laboratory, Cold Spring Harbor New York, pp 69–100

    Google Scholar 

  • Burgess RR (1969a) A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem 244:6160–6167

    PubMed  Google Scholar 

  • Burgess RR (1969b) Separation and characterization of the subunits of ribonucleic acid polymerase. J Biol Chem 244:6168–6176

    PubMed  Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor New York

    Google Scholar 

  • Dunn JJ, Studier FW (1981) Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4. J Mol Biol 148:303–330

    PubMed  Google Scholar 

  • Ernst H, Hartmann GR, Domdey H (1982) Species specifity of promoter recognition by RNA polymerase and its transfer by the sigma factor. Eur J Biochem 124:427–433

    PubMed  Google Scholar 

  • Fuhrmann M, Hennecke H (1982) Coding properties of cloned nitrogenase structural genes from Rhizobium japonicum. Mol Gen Genet 187:419–425

    Google Scholar 

  • Hennecke H (1981a) Regulation of nitrogenase biosynthesis in free-living and symbiotic N2-fixing bacteria: a comparison. In: Bothe H, Trebst A (eds) Biology of inorganic nitrogen and sulfur. Springer, Berlin Heidelberg New York, pp 309–316

    Google Scholar 

  • Hennecke H (1981b) recombinant plasmids carrying nitrogen fixation genes from Rhizobium japonicum. Nature 291:354–355

    Google Scholar 

  • Ishihama A, Taketo M, Saitoh T, Fukuda R (1976) Control of formation of RNA polymerase in Escherichia coli. In: Losick R, Chamberlin M (eds) RNA polymerase. Cold Spring Harbor Laboratory, Cold Spring Harbor New York, pp 485–502

    Google Scholar 

  • Jovin T, Chrambach A, Naughton MA (1964) An apparatus for preparative temperature-regulated polyacrylamide gel electrophoresis. Anal Biochem 9:351–369

    Google Scholar 

  • Knopf UC (1974) The nucleoside triphosphate-ribonucleic acid nucleotidyltransferase (EC 2.7.7.6.) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Biochem J 143:511–520

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Locker J (1979) Analytical and preparative electrophoresis of RNA in agarose-urea. Anal Biochem 98:358–367

    PubMed  Google Scholar 

  • Lotz W, Fees H, Wohlleben W, Burkhardt HJ (1981) Isolation and characterization of the DNA-dependent RNA polymerase of Rhizobium leguminosarum 300. J Gen Microbiol 125:301–309

    Google Scholar 

  • Lowe PA, Hager DA, Burgess RR (1979) Purification and properties of the σ-subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry 18:1344–1352

    PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  Google Scholar 

  • Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. In: Grossmann L, Moldave K (eds) Methods in enzymology, vol 65. Academic Press, London New York, pp 499–560

    Google Scholar 

  • Melançon P, Burgess RR, Record Jr MT (1982) Nitrocellulose filter binding studies of the interaction of Escherichia coli RNA polymerase holoenzyme with deoxyribonucleic acid restriction fragments: evidence for multiple classes of nonpromoter interactions, some of which display promoter-like properties. Biochemistry 21:4318–4331

    PubMed  Google Scholar 

  • O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 230:4007–4021

    Google Scholar 

  • Ovchinnikov YA, Monasturskaya GS, Gubanov VV, Guryev VV, Salomatina IS, Shuvaeva TM, Lipkin VM, Sverdlov ED (1981a) The primary structure of E. coli RNA polymerase. Nucleotide sequence of the rpoC gene and amino acid sequence of the β'-subunit. Dokl Akad Nauk USSR 261:763–768

    Google Scholar 

  • Ovchinnikov YA, Monasturskaya GS, Gubanov VV, Guryev SO, Chertov OY, Modyanov NN, Grinkevich VA, Makarova IA, Marchenko TV, Polovnikova IN, Lipkin VM, Sverdlov ED (1981b) The primary structure of E. coli RNA polymerase. Nucleotide sequence of the rpoB gene and amino acid sequence of the β-subunit. Eur J Biochem 116:621–629

    PubMed  Google Scholar 

  • Ovchinnikov YA, Lipkin VM, Modyanov NN, Chertov OY, Smirnov YV (1977) Primary structure of α-subunit of DNA-dependent RNA polymerase from E. coli. FEBS Lett 76:108–111

    Article  PubMed  Google Scholar 

  • Pain AN (1979) Symbiotic properties of antibiotic-resistant and auxotrophic mutants of Rhizobium leguminosarum. J Appl Bacteriol 47:53–64

    Google Scholar 

  • Pankhurst CE (1977) Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species. Can J Microbiol 23:1026–1033

    PubMed  Google Scholar 

  • Pankhurst CE, Scott DB, Ronson CW, White DWR (1981) Effect of rifampicin and ethidium bromide on nitrogenase biosynthesis and expression in free living cultures of Rhizobium strain 32H1. In: Gibson AH, Newton WE (eds) Current perspectives in nitrogen fixation. Australian Academy of Science, Canberra, p 447

    Google Scholar 

  • Pankhurst CE, Scott DB, Ronson CW (1982) Correlation between rifampicin resistance of slow-growing Rhizobium strains and their ability to express nitrogenase activity in culture. FEMS Microbiol Lett 15:137–139

    Google Scholar 

  • Scott DB, Hennecke H, Lim ST (1979) The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum. Biochem Biophys Acta 565:365–378

    PubMed  Google Scholar 

  • Seeburg PH, Schaller H (1975) Mapping and characterization of promoters in bacteriophages fd, f1 and M13. J Mol Biol 92:261–277

    PubMed  Google Scholar 

  • Stetter KO, Zillig W (1974) Transcription in Lactobacillaceae. DNA-dependent RNA polymerase from Lactobacillus curvatus. Eur J Biochem 48:527–540

    PubMed  Google Scholar 

  • Strauss CS, Boston RS, Record Jr MT, Burgess RR (1981) Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose-filter-binding assay. Gene 13:75–87

    PubMed  Google Scholar 

  • Taylor WE, Burgess RR (1979) Escherichia coli RNA polymerase binding and initiation of transcription on fragments of rifd 118 DNA containing promoters for λ genes and for rrnB, tufB, rplK, A, rplJ, L and rpoB,C genes. Gene 6:331–365

    PubMed  Google Scholar 

  • Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific Publications. Oxford Edinburgh

    Google Scholar 

  • Werner D (1978) Differentiation of Rhizobium japonicum. III. Inhibition of nitrogenase derepression by chloramphenicol and rifampicin concentrations, not inhibiting growth. Z Naturforsch 33:859–862

    Google Scholar 

  • Wiggs JL, Bush JW, Chamberlin MJ (1979) Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA Polymerases from a variety of bacterial orders. Cell 16:97–109

    Article  PubMed  Google Scholar 

  • Zillig W, Stetter KO, Janekovic D (1979) DNA-dependent RNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Eur J Biochem 96:597–604

    PubMed  Google Scholar 

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  1. Brigitte Regensburger & Hauke Hennecke

    Present address: Mikrobiologisches Institut, ETH Zürich, Universitätsstrasse 2, CH-8092, Zürich, Switzerland

Authors and Affiliations

  1. Lehrstuhl für Mikrobiologie der Universität München, Maria-Ward-Strasse 1 a, D-8000, München 19, Federal Republic of Germany

    Brigitte Regensburger & Hauke Hennecke

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  1. Brigitte Regensburger
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  2. Hauke Hennecke
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Regensburger, B., Hennecke, H. RNA polymerase from Rhizobium japonicum . Arch. Microbiol. 135, 103–109 (1983). https://doi.org/10.1007/BF00408017

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  • DOI: https://doi.org/10.1007/BF00408017

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