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. 1987 May;7(5):1602–1611. doi: 10.1128/mcb.7.5.1602

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

M Nonet, C Scafe, J Sexton, R Young
PMCID: PMC365259  PMID: 3299050

Abstract

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.

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Selected References

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

  1. Allison L. A., Moyle M., Shales M., Ingles C. J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985 Sep;42(2):599–610. doi: 10.1016/0092-8674(85)90117-5. [DOI] [PubMed] [Google Scholar]
  2. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  4. 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. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Buchman A. R., Fromm M., Berg P. Complex regulation of simian virus 40 early-region transcription from different overlapping promoters. Mol Cell Biol. 1984 Sep;4(9):1900–1914. doi: 10.1128/mcb.4.9.1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gorenstein C., Warner J. R. Coordinate regulation of the synthesis of eukaryotic ribosomal proteins. Proc Natl Acad Sci U S A. 1976 May;73(5):1547–1551. doi: 10.1073/pnas.73.5.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greenleaf A. L. Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit. J Biol Chem. 1983 Nov 25;258(22):13403–13406. [PubMed] [Google Scholar]
  9. Greenleaf A. L., Borsett L. M., Jiamachello P. F., Coulter D. E. Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II. Cell. 1979 Nov;18(3):613–622. doi: 10.1016/0092-8674(79)90116-8. [DOI] [PubMed] [Google Scholar]
  10. Greenleaf A. L., Weeks J. R., Voelker R. A., Ohnishi S., Dickson B. Genetic and biochemical characterization of mutants at an RNA polymerase II locus in D. melanogaster. Cell. 1980 Oct;21(3):785–792. doi: 10.1016/0092-8674(80)90441-9. [DOI] [PubMed] [Google Scholar]
  11. Gross K. J., Pogo A. O. Control mechanism of ribonucleic acid synthesis in eukaryotes. The effect of amino acid and glucose starvation and cycloheximide on yeast deoxyribonucleic acid-dependent ribonucleic acid polymerases. J Biol Chem. 1974 Jan 25;249(2):568–576. [PubMed] [Google Scholar]
  12. Ingles C. J., Guialis A., Lam J., Siminovitch L. Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines. J Biol Chem. 1976 May 10;251(9):2729–2734. [PubMed] [Google Scholar]
  13. Ingles C. J., Himmelfarb H. J., Shales M., Greenleaf A. L., Friesen J. D. Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2157–2161. doi: 10.1073/pnas.81.7.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kirschbaum J. B., Claeys I. V., Nasi S., Molholt B., Miller J. H. Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2375–2379. doi: 10.1073/pnas.72.6.2375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindquist S. Regulation of protein synthesis during heat shock. Nature. 1981 Sep 24;293(5830):311–314. doi: 10.1038/293311a0. [DOI] [PubMed] [Google Scholar]
  16. Mann C., Davis R. W. Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jan;6(1):241–245. doi: 10.1128/mcb.6.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McAlister L., Finkelstein D. B. Alterations in translatable ribonucleic acid after heat shock of Saccharomyces cerevisiae. J Bacteriol. 1980 Aug;143(2):603–612. doi: 10.1128/jb.143.2.603-612.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meinkoth J., Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
  19. Miller M. J., Xuong N. H., Geiduschek E. P. A response of protein synthesis to temperature shift in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5222–5225. doi: 10.1073/pnas.76.10.5222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mortimer R. K., Hawthorne D. C. Genetic mapping in Saccharomyces. Genetics. 1966 Jan;53(1):165–173. doi: 10.1093/genetics/53.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reid G. A. Pulse labeling of yeast cells and spheroplasts. Methods Enzymol. 1983;97:324–329. doi: 10.1016/0076-6879(83)97144-6. [DOI] [PubMed] [Google Scholar]
  22. Riva M., Memet S., Micouin J. Y., Huet J., Treich I., Dassa J., Young R., Buhler J. M., Sentenac A., Fromageot P. Isolation of structural genes for yeast RNA polymerases by immunological screening. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1554–1558. doi: 10.1073/pnas.83.6.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosbash M., Harris P. K., Woolford J. L., Jr, Teem J. L. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell. 1981 Jun;24(3):679–686. doi: 10.1016/0092-8674(81)90094-5. [DOI] [PubMed] [Google Scholar]
  24. Rubin G. M. Three forms of the 5.8-S ribosomal RNA species in Saccharomyces cerevisiae. Eur J Biochem. 1974 Jan 3;41(1):197–202. doi: 10.1111/j.1432-1033.1974.tb03260.x. [DOI] [PubMed] [Google Scholar]
  25. Ruet A., Sentenac A., Fromageot P. A specific assay for yeast RNA polymerases in crude cell extracts. Eur J Biochem. 1978 Oct;90(2):325–330. doi: 10.1111/j.1432-1033.1978.tb12608.x. [DOI] [PubMed] [Google Scholar]
  26. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  27. Shortle D., Novick P., Botstein D. Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4889–4893. doi: 10.1073/pnas.81.15.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shulman R. W., Sripati C. E., Warner J. R. Noncoordinated transcription in the absence of protein synthesis in yeast. J Biol Chem. 1977 Feb 25;252(4):1344–1349. [PubMed] [Google Scholar]
  29. Shulman R. W., Warner J. R. Ribosomal RNA transcription in a mutant of Saccharomyces cerevisiae defective in ribosomal protein synthesis. Mol Gen Genet. 1978 May 3;161(2):221–223. doi: 10.1007/BF00274191. [DOI] [PubMed] [Google Scholar]
  30. Somers D. G., Pearson M. L., Ingles C. J. Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II. J Biol Chem. 1975 Jul 10;250(13):4825–4831. [PubMed] [Google Scholar]
  31. Struhl K. Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8419–8423. doi: 10.1073/pnas.82.24.8419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taketo M., Ishihama A. Biosynthesis of RNA polymerase in Escherichia coli. IV. Accumulation of intermediates in mutants defective in the subunit assembly. J Mol Biol. 1976 Apr 5;102(2):297–310. doi: 10.1016/s0022-2836(76)80055-1. [DOI] [PubMed] [Google Scholar]
  33. Warner J. R., Gorenstein C. Yeast has a true stringent response. Nature. 1978 Sep 28;275(5678):338–339. doi: 10.1038/275338a0. [DOI] [PubMed] [Google Scholar]
  34. Warner J. R., Udem S. A. Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae. J Mol Biol. 1972 Mar 28;65(2):243–257. doi: 10.1016/0022-2836(72)90280-x. [DOI] [PubMed] [Google Scholar]
  35. Yaffe M. P., Schatz G. Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4819–4823. doi: 10.1073/pnas.81.15.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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