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. 1989 May;86(10):3624–3628. doi: 10.1073/pnas.86.10.3624

A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

J M Lee 1, A L Greenleaf 1
PMCID: PMC287190  PMID: 2657724

Abstract

The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts.

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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. 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. Bartholomew B., Dahmus M. E., Meares C. F. RNA contacts subunits IIo and IIc in HeLa RNA polymerase II transcription complexes. J Biol Chem. 1986 Oct 25;261(30):14226–14231. [PubMed] [Google Scholar]
  4. Bartolomei M. S., Halden N. F., Cullen C. R., Corden J. L. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II. Mol Cell Biol. 1988 Jan;8(1):330–339. doi: 10.1128/mcb.8.1.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  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. Buhler J. M., Iborra F., Sentenac A., Fromageot P. The presence of phosphorylated subunits in yeast RNA polymerases A and B. FEBS Lett. 1976 Nov 15;72(1):37–41. doi: 10.1016/0014-5793(76)80893-9. [DOI] [PubMed] [Google Scholar]
  8. Cadena D. L., Dahmus M. E. Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II. J Biol Chem. 1987 Sep 15;262(26):12468–12474. [PubMed] [Google Scholar]
  9. Christmann J. L., Dahmus M. E. Monoclonal antibody specific for calf thymus RNA polymerases IIO and IIA. J Biol Chem. 1981 Nov 25;256(22):11798–11803. [PubMed] [Google Scholar]
  10. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  11. Corden J. L., Cadena D. L., Ahearn J. M., Jr, Dahmus M. E. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7934–7938. doi: 10.1073/pnas.82.23.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dahmus M. E., Kedinger C. Transcription of adenovirus-2 major late promoter inhibited by monoclonal antibody directed against RNA polymerases IIO and IIA. J Biol Chem. 1983 Feb 25;258(4):2303–2307. [PubMed] [Google Scholar]
  13. Dahmus M. E. Phosphorylation of eukaryotic DNA-dependent RNA polymerase. Identification of calf thymus RNA polymerase subunits phosphorylated by two purified protein kinases, correlation with in vivo sites of phosphorylation in HeLa cell RNA polymerase II. J Biol Chem. 1981 Apr 10;256(7):3332–3339. [PubMed] [Google Scholar]
  14. Hunter T. A thousand and one protein kinases. Cell. 1987 Sep 11;50(6):823–829. doi: 10.1016/0092-8674(87)90509-5. [DOI] [PubMed] [Google Scholar]
  15. Jokerst R. S., Weeks J. R., Zehring W. A., Greenleaf A. L. Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila. Mol Gen Genet. 1989 Jan;215(2):266–275. doi: 10.1007/BF00339727. [DOI] [PubMed] [Google Scholar]
  16. Kim W. Y., Dahmus M. E. Immunochemical analysis of mammalian RNA polymerase II subspecies. Stability and relative in vivo concentration. J Biol Chem. 1986 Oct 25;261(30):14219–14225. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Padmanabha R., Glover C. V. Casein kinase II of yeast contains two distinct alpha polypeptides and an unusually large beta subunit. J Biol Chem. 1987 Feb 5;262(4):1829–1835. [PubMed] [Google Scholar]
  21. Price D. H., Sluder A. E., Greenleaf A. L. Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts. J Biol Chem. 1987 Mar 5;262(7):3244–3255. [PubMed] [Google Scholar]
  22. Rio D. C., Laski F. A., Rubin G. M. Identification and immunochemical analysis of biologically active Drosophila P element transposase. Cell. 1986 Jan 17;44(1):21–32. doi: 10.1016/0092-8674(86)90481-2. [DOI] [PubMed] [Google Scholar]
  23. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  25. Sigler P. B. Transcriptional activation. Acid blobs and negative noodles. Nature. 1988 May 19;333(6170):210–212. doi: 10.1038/333210a0. [DOI] [PubMed] [Google Scholar]
  26. Wang S. Y., Williams D. L. Biosynthesis of the vitellogenins. Identification and characterization of nonphosphorylated precursors to avian vitellogenin I and vitellogenin II. J Biol Chem. 1982 Apr 10;257(7):3837–3846. [PubMed] [Google Scholar]
  27. Zehring W. A., Lee J. M., Weeks J. R., Jokerst R. S., Greenleaf A. L. The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3698–3702. doi: 10.1073/pnas.85.11.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]

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