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. 2001 Oct;159(2):487–497. doi: 10.1093/genetics/159.2.487

Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae.

D L Lindstrom 1, G A Hartzog 1
PMCID: PMC1461841  PMID: 11606527

Abstract

Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

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Selected References

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  1. Akhtar A., Faye G., Bentley D. L. Distinct activated and non-activated RNA polymerase II complexes in yeast. EMBO J. 1996 Sep 2;15(17):4654–4664. [PMC free article] [PubMed] [Google Scholar]
  2. Andrulis E. D., Guzmán E., Döring P., Werner J., Lis J. T. High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 2000 Oct 15;14(20):2635–2649. doi: 10.1101/gad.844200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bensaude O., Bonnet F., Cassé C., Dubois M. F., Nguyen V. T., Palancade B. Regulated phosphorylation of the RNA polymerase II C-terminal domain (CTD). Biochem Cell Biol. 1999;77(4):249–255. [PubMed] [Google Scholar]
  4. Chiang P. W., Fogel E., Jackson C. L., Lieuallen K., Lennon G., Qu X., Wang S. Q., Kurnit D. M. Isolation, sequencing, and mapping of the human homologue of the yeast transcription factor, SPT5. Genomics. 1996 Dec 15;38(3):421–424. doi: 10.1006/geno.1996.0646. [DOI] [PubMed] [Google Scholar]
  5. Chiang P. W., Wang S. Q., Smithivas P., Song W. J., Crombez E., Akhtar A., Im R., Greenfield J., Ramamoorthy S., Van Keuren M. Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene. Genomics. 1996 Jun 15;34(3):368–375. doi: 10.1006/geno.1996.0299. [DOI] [PubMed] [Google Scholar]
  6. Cho H., Kim T. K., Mancebo H., Lane W. S., Flores O., Reinberg D. A protein phosphatase functions to recycle RNA polymerase II. Genes Dev. 1999 Jun 15;13(12):1540–1552. doi: 10.1101/gad.13.12.1540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahmus M. E. Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem. 1996 Aug 9;271(32):19009–19012. doi: 10.1074/jbc.271.32.19009. [DOI] [PubMed] [Google Scholar]
  8. Evans D. R., Brewster N. K., Xu Q., Rowley A., Altheim B. A., Johnston G. C., Singer R. A. The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain. Genetics. 1998 Dec;150(4):1393–1405. doi: 10.1093/genetics/150.4.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gu W., Malik S., Ito M., Yuan C. X., Fondell J. D., Zhang X., Martinez E., Qin J., Roeder R. G. A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol Cell. 1999 Jan;3(1):97–108. doi: 10.1016/s1097-2765(00)80178-1. [DOI] [PubMed] [Google Scholar]
  10. Guarente L. Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet. 1993 Oct;9(10):362–366. doi: 10.1016/0168-9525(93)90042-g. [DOI] [PubMed] [Google Scholar]
  11. Hartzog G. A., Basrai M. A., Ricupero-Hovasse S. L., Hieter P., Winston F. Identification and analysis of a functional human homolog of the SPT4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jun;16(6):2848–2856. doi: 10.1128/mcb.16.6.2848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hartzog G. A., Wada T., Handa H., Winston F. Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 1998 Feb 1;12(3):357–369. doi: 10.1101/gad.12.3.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hengartner C. J., Myer V. E., Liao S. M., Wilson C. J., Koh S. S., Young R. A. Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol Cell. 1998 Jul;2(1):43–53. doi: 10.1016/s1097-2765(00)80112-4. [DOI] [PubMed] [Google Scholar]
  14. Ivanov D., Kwak Y. T., Guo J., Gaynor R. B. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol Cell Biol. 2000 May;20(9):2970–2983. doi: 10.1128/mcb.20.9.2970-2983.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaplan C. D., Morris J. R., Wu C., Winston F. Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 2000 Oct 15;14(20):2623–2634. doi: 10.1101/gad.831900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim W. Y., Dahmus M. E. Purification of RNA polymerase IIO from calf thymus. J Biol Chem. 1988 Dec 15;263(35):18880–18885. [PubMed] [Google Scholar]
  17. Kobor M. S., Archambault J., Lester W., Holstege F. C., Gileadi O., Jansma D. B., Jennings E. G., Kouyoumdjian F., Davidson A. R., Young R. A. An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. Mol Cell. 1999 Jul;4(1):55–62. doi: 10.1016/s1097-2765(00)80187-2. [DOI] [PubMed] [Google Scholar]
  18. Komarnitsky P., Cho E. J., Buratowski S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 2000 Oct 1;14(19):2452–2460. doi: 10.1101/gad.824700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuchin S., Carlson M. Functional relationships of Srb10-Srb11 kinase, carboxy-terminal domain kinase CTDK-I, and transcriptional corepressor Ssn6-Tup1. Mol Cell Biol. 1998 Mar;18(3):1163–1171. doi: 10.1128/mcb.18.3.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LeRoy G., Orphanides G., Lane W. S., Reinberg D. Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science. 1998 Dec 4;282(5395):1900–1904. doi: 10.1126/science.282.5395.1900. [DOI] [PubMed] [Google Scholar]
  21. Malone E. A., Clark C. D., Chiang A., Winston F. Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Nov;11(11):5710–5717. doi: 10.1128/mcb.11.11.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mancebo H. S., Lee G., Flygare J., Tomassini J., Luu P., Zhu Y., Peng J., Blau C., Hazuda D., Price D. P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev. 1997 Oct 15;11(20):2633–2644. doi: 10.1101/gad.11.20.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marshall N. F., Price D. H. Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem. 1995 May 26;270(21):12335–12338. doi: 10.1074/jbc.270.21.12335. [DOI] [PubMed] [Google Scholar]
  24. Mortillaro M. J., Blencowe B. J., Wei X., Nakayasu H., Du L., Warren S. L., Sharp P. A., Berezney R. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8253–8257. doi: 10.1073/pnas.93.16.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murray S., Udupa R., Yao S., Hartzog G., Prelich G. Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Mol Cell Biol. 2001 Jul;21(13):4089–4096. doi: 10.1128/MCB.21.13.4089-4096.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Orphanides G., LeRoy G., Chang C. H., Luse D. S., Reinberg D. FACT, a factor that facilitates transcript elongation through nucleosomes. Cell. 1998 Jan 9;92(1):105–116. doi: 10.1016/s0092-8674(00)80903-4. [DOI] [PubMed] [Google Scholar]
  29. Patturajan M., Conrad N. K., Bregman D. B., Corden J. L. Yeast carboxyl-terminal domain kinase I positively and negatively regulates RNA polymerase II carboxyl-terminal domain phosphorylation. J Biol Chem. 1999 Sep 24;274(39):27823–27828. doi: 10.1074/jbc.274.39.27823. [DOI] [PubMed] [Google Scholar]
  30. Patturajan M., Schulte R. J., Sefton B. M., Berezney R., Vincent M., Bensaude O., Warren S. L., Corden J. L. Growth-related changes in phosphorylation of yeast RNA polymerase II. J Biol Chem. 1998 Feb 20;273(8):4689–4694. doi: 10.1074/jbc.273.8.4689. [DOI] [PubMed] [Google Scholar]
  31. Prelich G., Winston F. Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo. Genetics. 1993 Nov;135(3):665–676. doi: 10.1093/genetics/135.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Price D. H. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol. 2000 Apr;20(8):2629–2634. doi: 10.1128/mcb.20.8.2629-2634.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pérez Peña F., Aparicio Campillo G., López Asenjo J. A., Alonso-Castrillo C., Bru Amantegui S., Mata González P., Serrano Ríos M. Ascitis por acúmulo de líquido cefalorraquídeo. Rev Clin Esp. 1990 Jul-Aug;187(3):128–130. [PubMed] [Google Scholar]
  34. Rodriguez C. R., Cho E. J., Keogh M. C., Moore C. L., Greenleaf A. L., Buratowski S. Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. Mol Cell Biol. 2000 Jan;20(1):104–112. doi: 10.1128/mcb.20.1.104-112.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rowley A., Singer R. A., Johnston G. C. CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus. Mol Cell Biol. 1991 Nov;11(11):5718–5726. doi: 10.1128/mcb.11.11.5718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sterner D. E., Lee J. M., Hardin S. E., Greenleaf A. L. The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex. Mol Cell Biol. 1995 Oct;15(10):5716–5724. doi: 10.1128/mcb.15.10.5716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sun X., Zhang Y., Cho H., Rickert P., Lees E., Lane W., Reinberg D. NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. Mol Cell. 1998 Aug;2(2):213–222. doi: 10.1016/s1097-2765(00)80131-8. [DOI] [PubMed] [Google Scholar]
  38. Swanson M. S., Malone E. A., Winston F. SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat. Mol Cell Biol. 1991 Jun;11(6):3009–3019. doi: 10.1128/mcb.11.6.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swanson M. S., Winston F. SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. Genetics. 1992 Oct;132(2):325–336. doi: 10.1093/genetics/132.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uptain S. M., Kane C. M., Chamberlin M. J. Basic mechanisms of transcript elongation and its regulation. Annu Rev Biochem. 1997;66:117–172. doi: 10.1146/annurev.biochem.66.1.117. [DOI] [PubMed] [Google Scholar]
  41. Wada T., Takagi T., Yamaguchi Y., Ferdous A., Imai T., Hirose S., Sugimoto S., Yano K., Hartzog G. A., Winston F. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 1998 Feb 1;12(3):343–356. doi: 10.1101/gad.12.3.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Warren S. L., Landolfi A. S., Curtis C., Morrow J. S. Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle. J Cell Sci. 1992 Oct;103(Pt 2):381–388. doi: 10.1242/jcs.103.2.381. [DOI] [PubMed] [Google Scholar]
  43. West M. L., Corden J. L. Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations. Genetics. 1995 Aug;140(4):1223–1233. doi: 10.1093/genetics/140.4.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winston F., Chaleff D. T., Valent B., Fink G. R. Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics. 1984 Jun;107(2):179–197. doi: 10.1093/genetics/107.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Winston F., Dollard C., Ricupero-Hovasse S. L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. doi: 10.1002/yea.320110107. [DOI] [PubMed] [Google Scholar]
  46. Wittmeyer J., Formosa T. The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein. Mol Cell Biol. 1997 Jul;17(7):4178–4190. doi: 10.1128/mcb.17.7.4178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wu-Baer F., Lane W. S., Gaynor R. B. Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation. J Mol Biol. 1998 Mar 27;277(2):179–197. doi: 10.1006/jmbi.1997.1601. [DOI] [PubMed] [Google Scholar]
  48. Yamaguchi Y., Takagi T., Wada T., Yano K., Furuya A., Sugimoto S., Hasegawa J., Handa H. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell. 1999 Apr 2;97(1):41–51. doi: 10.1016/s0092-8674(00)80713-8. [DOI] [PubMed] [Google Scholar]
  49. Zhu Y., Pe'ery T., Peng J., Ramanathan Y., Marshall N., Marshall T., Amendt B., Mathews M. B., Price D. H. Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 1997 Oct 15;11(20):2622–2632. doi: 10.1101/gad.11.20.2622. [DOI] [PMC free article] [PubMed] [Google Scholar]

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