Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Dec;10(12):6123–6131. doi: 10.1128/mcb.10.12.6123

A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

J Archambault 1, K T Schappert 1, J D Friesen 1
PMCID: PMC362887  PMID: 2247052

Abstract

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.

Full text

PDF
6123

Images in this article

6127Image
on p.6127
6127Image
on p.6127
6128Image
on p.6128
6128Image
on p.6128
6129Image
on p.6129

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. Allison L. A., Wong J. K., Fitzpatrick V. D., Moyle M., Ingles C. J. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol Cell Biol. 1988 Jan;8(1):321–329. doi: 10.1128/mcb.8.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arndt K. T., Styles C. A., Fink G. R. A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell. 1989 Feb 24;56(4):527–537. doi: 10.1016/0092-8674(89)90576-x. [DOI] [PubMed] [Google Scholar]
  4. Bell G. I., Valenzuela P., Rutter W. J. Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits. J Biol Chem. 1977 May 10;252(9):3082–3091. [PubMed] [Google Scholar]
  5. Bell G. I., Valenzuela P., Rutter W. J. Phosphorylation of yeast RNA polymerases. Nature. 1976 Jun 3;261(5559):429–431. doi: 10.1038/261429a0. [DOI] [PubMed] [Google Scholar]
  6. Biggs J., Searles L. L., Greenleaf A. L. Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II. Cell. 1985 Sep;42(2):611–621. doi: 10.1016/0092-8674(85)90118-7. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Broyles S. S., Moss B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A. 1986 May;83(10):3141–3145. doi: 10.1073/pnas.83.10.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bréant B., Buhler J. M., Sentenac A., Fromageot P. On the phosphorylation of yeast RNA polymerases A and B. Eur J Biochem. 1983 Feb 1;130(2):247–251. doi: 10.1111/j.1432-1033.1983.tb07143.x. [DOI] [PubMed] [Google Scholar]
  10. Bréant B., Huet J., Sentenac A., Fromageot P. Analysis of yeast RNA polymerases with subunit-specific antibodies. J Biol Chem. 1983 Oct 10;258(19):11968–11973. [PubMed] [Google Scholar]
  11. Budd M., Campbell J. L. Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci U S A. 1987 May;84(9):2838–2842. doi: 10.1073/pnas.84.9.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Buratowski S., Hahn S., Sharp P. A., Guarente L. Function of a yeast TATA element-binding protein in a mammalian transcription system. Nature. 1988 Jul 7;334(6177):37–42. doi: 10.1038/334037a0. [DOI] [PubMed] [Google Scholar]
  13. Cavallini B., Huet J., Plassat J. L., Sentenac A., Egly J. M., Chambon P. A yeast activity can substitute for the HeLa cell TATA box factor. Nature. 1988 Jul 7;334(6177):77–80. doi: 10.1038/334077a0. [DOI] [PubMed] [Google Scholar]
  14. Chodosh L. A., Olesen J., Hahn S., Baldwin A. S., Guarente L., Sharp P. A. A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable. Cell. 1988 Apr 8;53(1):25–35. doi: 10.1016/0092-8674(88)90484-9. [DOI] [PubMed] [Google Scholar]
  15. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  16. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  17. Clark-Adams C. D., Norris D., Osley M. A., Fassler J. S., Winston F. Changes in histone gene dosage alter transcription in yeast. Genes Dev. 1988 Feb;2(2):150–159. doi: 10.1101/gad.2.2.150. [DOI] [PubMed] [Google Scholar]
  18. Falkenburg D., Dworniczak B., Faust D. M., Bautz E. K. RNA polymerase II of Drosophila. Relation of its 140,000 Mr subunit to the beta subunit of Escherichia coli RNA polymerase. J Mol Biol. 1987 Jun 20;195(4):929–937. doi: 10.1016/0022-2836(87)90496-7. [DOI] [PubMed] [Google Scholar]
  19. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  20. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  21. Himmelfarb H. J., Simpson E. M., Friesen J. D. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2155–2164. doi: 10.1128/mcb.7.6.2155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huet J., Riva M., Sentenac A., Fromageot P. Yeast RNA polymerase C and its subunits. Specific antibodies as structural and functional probes. J Biol Chem. 1985 Dec 5;260(28):15304–15310. [PubMed] [Google Scholar]
  23. 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]
  24. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jarvik J., Botstein D. Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2738–2742. doi: 10.1073/pnas.72.7.2738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  29. Martin C., Young R. A. KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth. Mol Cell Biol. 1989 Jun;9(6):2341–2349. doi: 10.1128/mcb.9.6.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McKnight G. L., McConaughy B. L. Selection of functional cDNAs by complementation in yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4412–4416. doi: 10.1073/pnas.80.14.4412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mortimer R. K., Schild D., Contopoulou C. R., Kans J. A. Genetic map of Saccharomyces cerevisiae, edition 10. Yeast. 1989 Sep-Oct;5(5):321–403. doi: 10.1002/yea.320050503. [DOI] [PubMed] [Google Scholar]
  32. Nonet M., Scafe C., Sexton J., Young R. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis. Mol Cell Biol. 1987 May;7(5):1602–1611. doi: 10.1128/mcb.7.5.1602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nonet M., Sweetser D., Young R. A. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987 Sep 11;50(6):909–915. doi: 10.1016/0092-8674(87)90517-4. [DOI] [PubMed] [Google Scholar]
  34. Novick P., Osmond B. C., Botstein D. Suppressors of yeast actin mutations. Genetics. 1989 Apr;121(4):659–674. doi: 10.1093/genetics/121.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Percival-Smith A., Segall J. Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jul;6(7):2443–2451. doi: 10.1128/mcb.6.7.2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  38. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  39. Ruet A., Sentenac A., Fromageot P., Winsor B., Lacroute F. A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro. J Biol Chem. 1980 Jul 10;255(13):6450–6455. [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  42. Sturtevant A H. A Highly Specific Complementary Lethal System in Drosophila Melanogaster. Genetics. 1956 Jan;41(1):118–123. doi: 10.1093/genetics/41.1.118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sweetser D., Nonet M., Young R. A. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1192–1196. doi: 10.1073/pnas.84.5.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Woychik N. A., Liao S. M., Kolodziej P. A., Young R. A. Subunits shared by eukaryotic nuclear RNA polymerases. Genes Dev. 1990 Mar;4(3):313–323. doi: 10.1101/gad.4.3.313. [DOI] [PubMed] [Google Scholar]
  45. Woychik N. A., Young R. A. RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth. Mol Cell Biol. 1989 Jul;9(7):2854–2859. doi: 10.1128/mcb.9.7.2854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES