Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1996 Mar;142(3):737–747. doi: 10.1093/genetics/142.3.737

Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces Cerevisiae

J Archambault 1, D B Jansma 1, J D Friesen 1
PMCID: PMC1207015  PMID: 8849884

Abstract

In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steadystate amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

Full Text

The Full Text of this article is available as a PDF (5.0 MB).

Selected References

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

  1. Allison L. A., Ingles C. J. Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2794–2798. doi: 10.1073/pnas.86.8.2794. [DOI] [PMC free article] [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. Ambroziak J., Henry S. A. INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter. J Biol Chem. 1994 May 27;269(21):15344–15349. [PubMed] [Google Scholar]
  4. Andrews B. J., Herskowitz I. Identification of a DNA binding factor involved in cell-cycle control of the yeast HO gene. Cell. 1989 Apr 7;57(1):21–29. doi: 10.1016/0092-8674(89)90168-2. [DOI] [PubMed] [Google Scholar]
  5. Archambault J., Friesen J. D. Genetics of eukaryotic RNA polymerases I, II, and III. Microbiol Rev. 1993 Sep;57(3):703–724. doi: 10.1128/mr.57.3.703-724.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Archambault J., Schappert K. T., Friesen J. D. A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III. Mol Cell Biol. 1990 Dec;10(12):6123–6131. doi: 10.1128/mcb.10.12.6123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arndt K. M., Ricupero-Hovasse S., Winston F. TBP mutants defective in activated transcription in vivo. EMBO J. 1995 Apr 3;14(7):1490–1497. doi: 10.1002/j.1460-2075.1995.tb07135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Ashburner B. P., Lopes J. M. Autoregulated expression of the yeast INO2 and INO4 helix-loop-helix activator genes effects cooperative regulation on their target genes. Mol Cell Biol. 1995 Mar;15(3):1709–1715. doi: 10.1128/mcb.15.3.1709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Berroteran R. W., Ware D. E., Hampsey M. The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations. Mol Cell Biol. 1994 Jan;14(1):226–237. doi: 10.1128/mcb.14.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bonneaud N., Ozier-Kalogeropoulos O., Li G. Y., Labouesse M., Minvielle-Sebastia L., Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. doi: 10.1002/yea.320070609. [DOI] [PubMed] [Google Scholar]
  12. Carman G. M., Henry S. A. Phospholipid biosynthesis in yeast. Annu Rev Biochem. 1989;58:635–669. doi: 10.1146/annurev.bi.58.070189.003223. [DOI] [PubMed] [Google Scholar]
  13. Chiannilkulchai N., Stalder R., Riva M., Carles C., Werner M., Sentenac A. RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4433–4440. doi: 10.1128/mcb.12.10.4433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Côté J., Quinn J., Workman J. L., Peterson C. L. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science. 1994 Jul 1;265(5168):53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]
  15. Dalley B. K., Rogalski T. M., Tullis G. E., Riddle D. L., Golomb M. Post-transcriptional regulation of RNA polymerase II levels in Caenorhabditis elegans. Genetics. 1993 Feb;133(2):237–245. doi: 10.1093/genetics/133.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol. 1988 Jul;8(7):2690–2697. doi: 10.1128/mcb.8.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Furter-Graves E. M., Hall B. D., Furter R. Role of a small RNA pol II subunit in TATA to transcription start site spacing. Nucleic Acids Res. 1994 Nov 25;22(23):4932–4936. doi: 10.1093/nar/22.23.4932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gudenus R., Mariotte S., Moenne A., Ruet A., Memet S., Buhler J. M., Sentenac A., Thuriaux P. Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae. Genetics. 1988 Jul;119(3):517–526. doi: 10.1093/genetics/119.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guialis A., Beatty B. G., Ingles C. J., Crerar M. M. Regulation of RNA polymerase II activity in alpha-amanitin-resistant CHO hybrid cells. Cell. 1977 Jan;10(1):53–60. doi: 10.1016/0092-8674(77)90139-8. [DOI] [PubMed] [Google Scholar]
  20. Guialis A., Morrison K. E., Ingles C. J. Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines. J Biol Chem. 1979 May 25;254(10):4171–4176. [PubMed] [Google Scholar]
  21. Hekmatpanah D. S., Young R. A. Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection. Mol Cell Biol. 1991 Nov;11(11):5781–5791. doi: 10.1128/mcb.11.11.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hengartner C. J., Thompson C. M., Zhang J., Chao D. M., Liao S. M., Koleske A. J., Okamura S., Young R. A. Association of an activator with an RNA polymerase II holoenzyme. Genes Dev. 1995 Apr 15;9(8):897–910. doi: 10.1101/gad.9.8.897. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hirsch J. P., Henry S. A. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis. Mol Cell Biol. 1986 Oct;6(10):3320–3328. doi: 10.1128/mcb.6.10.3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hudak K. A., Lopes J. M., Henry S. A. A pleiotropic phospholipid biosynthetic regulatory mutation in Saccharomyces cerevisiae is allelic to sin3 (sdi1, ume4, rpd1). Genetics. 1994 Feb;136(2):475–483. doi: 10.1093/genetics/136.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hull M. W., McKune K., Woychik N. A. RNA polymerase II subunit RPB9 is required for accurate start site selection. Genes Dev. 1995 Feb 15;9(4):481–490. doi: 10.1101/gad.9.4.481. [DOI] [PubMed] [Google Scholar]
  27. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  28. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  29. Koleske A. J., Buratowski S., Nonet M., Young R. A. A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID. Cell. 1992 May 29;69(5):883–894. doi: 10.1016/0092-8674(92)90298-q. [DOI] [PubMed] [Google Scholar]
  30. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  31. Koleske A. J., Young R. A. The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem Sci. 1995 Mar;20(3):113–116. doi: 10.1016/s0968-0004(00)88977-x. [DOI] [PubMed] [Google Scholar]
  32. Kolodziej P. A., Young R. A. Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. Mol Cell Biol. 1991 Sep;11(9):4669–4678. doi: 10.1128/mcb.11.9.4669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kruger W., Herskowitz I. A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMG1. Mol Cell Biol. 1991 Aug;11(8):4135–4146. doi: 10.1128/mcb.11.8.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kuchin S., Yeghiayan P., Carlson M. Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):4006–4010. doi: 10.1073/pnas.92.9.4006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lopes J. M., Hirsch J. P., Chorgo P. A., Schulze K. L., Henry S. A. Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors. Nucleic Acids Res. 1991 Apr 11;19(7):1687–1693. doi: 10.1093/nar/19.7.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mosrin C., Riva M., Beltrame M., Cassar E., Sentenac A., Thuriaux P. The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail. Mol Cell Biol. 1990 Sep;10(9):4737–4743. doi: 10.1128/mcb.10.9.4737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Myers A. M., Tzagoloff A., Kinney D. M., Lusty C. J. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene. 1986;45(3):299–310. doi: 10.1016/0378-1119(86)90028-4. [DOI] [PubMed] [Google Scholar]
  38. Nicholson R. C., Williams D. B., Moran L. A. An essential member of the HSP70 gene family of Saccharomyces cerevisiae is homologous to immunoglobulin heavy chain binding protein. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1159–1163. doi: 10.1073/pnas.87.3.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nonet M. L., Young R. A. Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. Genetics. 1989 Dec;123(4):715–724. doi: 10.1093/genetics/123.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Peterson C. L., Dingwall A., Scott M. P. Five SWI/SNF gene products are components of a large multisubunit complex required for transcriptional enhancement. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2905–2908. doi: 10.1073/pnas.91.8.2905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Peterson C. L., Herskowitz I. Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription. Cell. 1992 Feb 7;68(3):573–583. doi: 10.1016/0092-8674(92)90192-f. [DOI] [PubMed] [Google Scholar]
  43. Peterson C. L., Tamkun J. W. The SWI-SNF complex: a chromatin remodeling machine? Trends Biochem Sci. 1995 Apr;20(4):143–146. doi: 10.1016/s0968-0004(00)88990-2. [DOI] [PubMed] [Google Scholar]
  44. Sawadogo M., Sentenac A. RNA polymerase B (II) and general transcription factors. Annu Rev Biochem. 1990;59:711–754. doi: 10.1146/annurev.bi.59.070190.003431. [DOI] [PubMed] [Google Scholar]
  45. Scafe C., Nonet M., Young R. A. RNA polymerase II mutants defective in transcription of a subset of genes. Mol Cell Biol. 1990 Mar;10(3):1010–1016. doi: 10.1128/mcb.10.3.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Stettler S., Mariotte S., Riva M., Sentenac A., Thuriaux P. An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae. J Biol Chem. 1992 Oct 25;267(30):21390–21395. [PubMed] [Google Scholar]
  48. Thompson C. M., Koleske A. J., Chao D. M., Young R. A. A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell. 1993 Jul 2;73(7):1361–1375. doi: 10.1016/0092-8674(93)90362-t. [DOI] [PubMed] [Google Scholar]
  49. Thompson N. E., Steinberg T. H., Aronson D. B., Burgess R. R. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem. 1989 Jul 5;264(19):11511–11520. [PubMed] [Google Scholar]
  50. Tyers M., Tokiwa G., Nash R., Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. doi: 10.1002/j.1460-2075.1992.tb05229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wahi M., Johnson A. D. Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae. Genetics. 1995 May;140(1):79–90. doi: 10.1093/genetics/140.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Winston F., Carlson M. Yeast SNF/SWI transcriptional activators and the SPT/SIN chromatin connection. Trends Genet. 1992 Nov;8(11):387–391. doi: 10.1016/0168-9525(92)90300-s. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Young R. A. RNA polymerase II. Annu Rev Biochem. 1991;60:689–715. doi: 10.1146/annurev.bi.60.070191.003353. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES