Summary
Assay conditions are described which permit detection of cryptic temperature sensitive RNA polymerases in vitro. RNA polymerase was prepared from fifteen different temperature sensitive mutants of Salmonella typhimurium chosen at random from a larger group isolated by localized mutagenesis and uridine suicide techniques. The dependence of enzyme activity on temperature, ionic strength and pH was studied in vitro. Assays at higher ionic strength (0.23 M) and temperature (50°C) distinguish three classes of mutants (Table 2). Activity of seven mutant RNA polymerases (called Class 1) under these conditions was 1% to 5% that of the parental RNA polymerase. Five mutant RNA polymerases (called Class 2) had 18% to 64% of the parental activity and three were not distinguishable from the parental enzyme under these conditions. Mixing experiments showed that the defect in Class 1 mutant enzymes is a property of the enzymes and not due to a diffusible inhibitor. In one case the lesion was shown to reside in the core enzyme. Class 1 mutant RNA polymerases were shown to be irreversibly inactivated during the assay at higher temperature and ionic strength. This suggests that the Class 1 enzymes may be more thermolabile than the wild type enzyme or may fail to be protected from thermal denaturation by formation of a ternary complex with template and product. We conclude that the method used to isolate these mutants (Young et al., 1976) and the assay described here (Table 2) are efficient ways to isolate and detect temperature sensitive RNA polymerase mutants of Salmonella typhimurium.
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References
Burgess, R., Jendrisak, J.J.: A procedure for the rapid, large scale purification of E. coli DNA dependent RNA polymerase involving polymicin P precipitation and DNA cellulose chromatography. Biochemistry 14, 4634–4638 (1975)
Burgess, R.R., Travers, A.A., Dunn, J.J., Bautz, E.K.F.: Factor stimulating transcription by RNA polymerase. Nature 221, 43–46 (1969)
Fuchs, E., Millette, R.L., Zillig, W., Walter, G.: Influence of salts on RNA synthesis by DNA dependent RNA polymerase from Escherichia coli. Eur. J. Biochem. 3, 183–193 (1967)
Gross, G., Fields, D.A., Bautz, E.: Characterization of a ts β′ mutant RNA polymerase of Escherichia coli. Mol. Gen. Genet. 147, 337–341 (1976)
Gross, G., Fields, D.A., Bautz, E.: Temperature sensitive mutants of Escherichia coli with defects in the assembly of RNA polymerase in vitro. Eur. J. Biochem. 81, 333–338 (1977)
Hong, J., Ames, B.: Localized mutagenesis of any specific small region of the bacterial chromosome. Proc. Natl. Acad. Sci. U.S.A. 68, 3158–3162 (1971)
Jendrisak, J.J., Burgess, R.: A new method for the large scale purification of wheat germ RNA polymerase. II. Biochemistry 14, 4639–4645 (1975)
Kawai, M., Ishihama, A., Yira, T.: RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant. Mol. Gen. Genet. 143, 233–241 (1976)
Khesin, R.B., Astaurova, R.B., Shemyakin, M., Kamzolova, S., Manyakov, V.: Changes in RNA polymerase properties during binding to DNA and initiation of RNA synthesis. Mol. Biol. 1, 617–636 (1967)
Krakow, J.S., Rhodes, G. Jovin, T.M.: In: RNA polymerase (R.C. Losick and M.J. Chamberlin, eds.), pp. 127–157. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratories 1976
Miller, J.H., Claeys, I.V., Kirschbaum, J.B., Nasi, S., Elsacker Van den, S., Mohold, B., Gross, G., Fields, D.A., Bautz, E.K.F.: Altered RNA polymerases resulting from temperature-sensitive mutations in the rif region of the E. coli chromosome. RNA polymerase (R. Losick and M. Chamberlin, eds.), pp. 519–538. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1976
Panny, S., Heil, A., Mazus, B., Palm, P., Zillig, W., Mindlin, S., Ilyina, T., Khesin, R.B.: A temperature sensitive mutation of the β′ subunit of DNA dependent RNA polymerase from E. coli T16. FEBS Lett. 48, 241–245 (1974)
Scaife, J.: Bacterial RNA polymerases: the genetics and control of their syntheses. RNA polymerase (R. Losick and M. Chamberlin, eds.), pp. 207–246. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1976
Stead, N., Jones, O.W.: The binding of RNA polymerase to DNA: stabilization by nucleoside triphosphates. Biochem. Biophys. Acta 145, 677–679 (1967)
Young, B., Guterman, S., Wright, A.: Temperature-sensitive ribonucleic acid polymerase mutant of Salmonella typhimurium with a defect in the β′ subunit. J. Bacteriol. 127, 1291–1297 (1976)
Young, B.S.: RNA polymerase in Salmonella typhimurium: effects of a temperature sensitive mutation on enzyme function. Ph.D. thesis. Tufts University (1977)
Young, B.S., Wright, A.: Multiple effects of an RNA polymerase β′ mutation on in vitro transcription. Mol. Gen. Genet. 155, 191–196 (1977)
Zillig, W., Zechel, I., Halbwachs, H.J.: A new method for large scale preparation of highly purified DNA-dependent RNA polymerase from E. coli. Hoppe-Seylers Z. Physiol. Chem. 351, 221–224 (1970)
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Boyd, D.H., Porter, L.M., Young, B.S. et al. The in vitro detection of defects in temperature sensitive RNA polymerases from mutants of Salmonella typhimurium . Molec. Gen. Genet. 173, 279–287 (1979). https://doi.org/10.1007/BF00268638
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DOI: https://doi.org/10.1007/BF00268638