Abstract
Partially purified DNA-dependent RNA polymerase ofStreptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits, represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage ϕ29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases fromEscherichia coli andBacillus subtilis.
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Barthelmy I., Salas M., Mellado R.P.:In vivo transcription of bacteriophage ϕ29 DNA: transcription initiation sites.J. Virol. 60, 874–879 (1986).
Bradford M.M.: A rapid and sensitive method for the quantitation of, microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–254 (1976).
Burgess R.R., Jendrisak J.J.: A procedure for the rapid large-scale purification ofEscherichia coli DNA dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography.Biochemistry 14, 4634–4638 (1975).
Davis R.W., Simon M., Davidson N.: Electron microscopic heteroduplex methods for mapping regions of base sequence homology in nucleic acid, pp. 413–428 inMethods in Enzymology (L. Grossman, K. Moldave, Eds.), Vol. 210. Academic Press, New York 1971.
Gonzales N., Wiggs J., Chamberlin M.J.: A simple procedure for resolution ofEscherichia coli RNA polymerase holoenzyme from core enzyme polymerase.Arch. Biochem. Biophys. 182, 404–408 (1977).
Inciarte M.R., Lázaro J.M., Salas M., Vinuela E.: Physical map of bacteriophage ϕ29 DNA.Virology 74, 314–323 (1976).
Jones G.H.: Purification of RNA polymerase from actinomycin producing and nonproducing cells ofStreptomyces antibioticus.Arch. Biochem. Biophys. 198, 195–204 (1979).
Laemmli U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685 (1970).
Lowe P.A., Hager D.A., Burgess R.R.: Purification and properties of the σ subunit ofEscherichia coli DNA-dependent RNA polymerase.Biochemistry 18, 1344–1352 (1979).
Mellado R.P., Barthelmy I., Salas M.:In vivo transcription of bacteriophage ϕ29. Early and late promoter sequences.J. Mol. Biol. 191, 191–197 (1986a).
Mellado R.P., Barthelmy I., Salas M.:In vitro transcription of bacteriophage ϕ29. Correlation betweenin vitro andin vivo promoters.Nucl. Acids Res. 14, 4731–4741 (1986b).
Mukai R., Iida Y.: Separation of RNA polymerase from core enzyme on DNA-cellulose column.Biochem. Biophys. Res. Commun. 54, 134–139 (1973).
Nüsslein C., Heyden B.: Chromatography of RNA polymerase fromEscherichia coli on single stranded DNA-agarose columns.Biochem. Biophys. Res. Commun. 47, 282–289 (1972).
Portmann R., Schaffner W., Birnstiel M.: Partial denaturation mapping of cloned histone DNA from the sea urchinPsammechinus milliaris.Nature 264, 31–34 (1976).
Pulido D., Jiménez A., Salas M., Mellado R.P.:Bacillus subtilis phage ϕ29 main promoters are efficiently recognizedin vitro by theStreptomyces lividans RNA polymerase.Gene 49, 377–382 (1986).
Saneyoshi M., Tohyama J., Nakayama C., Takiya S., Iwabuchi X.: Inhibitory effects of 3′-deoxycytidine 5′-triphosphate on DNA-dependent RNA polymerase I and II purified fromDictyostelium discoideum cells.Nucl Acids Res. 9, 3129–3138 (1981).
Sogo J.M., Inciarte M.R., Corral J., Vinuela E., Solas M.: RNA polymerase binding sites and transcription map of the DNA ofBacillus subtilis phage ϕ29.J. Mol. Biol. 127, 411–436 (1979).
Sogo J.M., Lozano M., Salas M.:In vitro transcription of theBacillus subtilis phage ϕ29 DNA byBacillus subtilis andEscherichia coli RNA polymerase.Nucl. Acids Res. 12, 1943–1960 (1984).
Vollenweider H.J., Szybalski W.: Electron microscopic mapping of RNA polymerase binding to coliphage λ DNA.J. Mol. Biol. 123, 485–498 (1978).
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Ŝmardová, J., Felsberg, J., Ŝmarda, J. et al. Isolation of DNA-dependent RNA polymerase fromStreptomyces granaticolor and its binding to phage ϕ29 DNA. Folia Microbiol 36, 120–126 (1991). https://doi.org/10.1007/BF02814489
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DOI: https://doi.org/10.1007/BF02814489