Molecular Genetics and Genomics

, Volume 276, Issue 2, pp 197–210 | Cite as

Functional and physical interactions within the middle domain of the yeast mediator

  • Magnus Hallberg
  • Guo-Zhen Hu
  • Susanna Tronnersjö
  • Zaki Shaikhibrahim
  • Darius Balciunas
  • Stefan Björklund
  • Hans Ronne
Original Paper

Abstract

Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserved in all eukaryotes. It is thought to play an important role in both transcriptional activation and repression. In the yeast Saccharomyces cerevisiae, Med21 is known to interact both with the Mediator subunit Med6 and the global co-repressor Tup1. We have made a temperature-sensitive med21-ts mutant, which we used in a high copy number suppressor screen. We found ten yeast genes that can suppress the med21-ts mutation in high copy number. The three strongest suppressors were MED7 and MED10 (NUT2), which encode other Mediator subunits, and ASH1, which encodes a repressor of the HO gene. 2-Hybrid experiments confirmed multiple interactions between Med21, Med10, Med7 and Med4, and also revealed a Med21 self-interaction. The interactions of Med21 with Med7 and Med10 were verified by co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, where both interactions were found to depend strongly on the amino acid residues 2–8 of Med21. These interactions, and the interactions of Med21 with Med6 and Tup1, suggest that Med21 may serve as a molecular switchboard that integrates different signals before they reach the core polymerase.

Keywords

Med7 Med10 Med21 Mediator Nut2 Srb7 

References

  1. Balciunas D, Gälman C, Ronne H, Björklund S (1999) The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression. Proc Natl Acad Sci USA 96:376–381PubMedCrossRefGoogle Scholar
  2. Balciunas D, Hallberg M, Björklund S, Ronne H (2002) Functional interactions within yeast mediator and evidence of differential subunit modifications. J Biol Chem 278:3831–3839PubMedCrossRefGoogle Scholar
  3. Baumli S, Hoeppner S, Cramer P (2005) A conserved Mediator hinge revealed in the structure of the MED7-MED21 (Med7-Srb7) heterodimer. J Biol Chem 280:18171–18178PubMedCrossRefGoogle Scholar
  4. Björklund S, Gustafsson CM (2004) The mediator complex. Adv Protein Chem 67:43–65PubMedCrossRefGoogle Scholar
  5. Boube M, Joulia L, Cribbs DL, Bourbon HM (2002) Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man. Cell 110:143–151PubMedCrossRefGoogle Scholar
  6. Bourbon HM, Aguilera A, Ansari AZ, Asturias FJ, Berk AJ, Bjorklund S, Blackwell TK, Borggrefe T, Carey M, Carlson M, Conaway JW, Conaway RC, Emmons SW, Fondell JD, Freedman LP, Fukasawa T, Gustafsson CM, Han M, He X, Herman PK, Hinnebusch AG, Holmberg S, Holstege FC, Jaehning JA, Kim YJ, Kuras L, Leutz A, Lis JT, Meisterernest M, Naar AM, Nasmyth K, Parvin JD, Ptashne M, Reinberg D, Ronne H, Sadowski I, Sakurai H, Sipiczki M, Sternberg PW, Stillman DJ, Strich R, Struhl K, Svejstrup JQ, Tuck S, Winston F, Roeder RG, Kornberg RD (2004) A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol Cell 14:553–557PubMedCrossRefGoogle Scholar
  7. Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292:1863–1876PubMedCrossRefGoogle Scholar
  8. Dotson MR, Yuan CX, Roeder RG, Myers LC, Gustafsson CM, Jiang YW, Li Y, Kornberg RD, Asturias FJ (2000) Structural organization of yeast and mammalian mediator complexes. Proc Natl Acad Sci USA 97:14307–14310PubMedCrossRefGoogle Scholar
  9. Estojak J, Brent R, Golemis ER (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15:5820–5829PubMedGoogle Scholar
  10. Farabaugh PJ (1996) Programmed translational frameshifting. Microbiol Rev 60:103–134PubMedGoogle Scholar
  11. Flanagan PM, Kelleher RJD, Sayre MH, Tschochner H, Kornberg RD (1991) A mediator required for activation of RNA polymerase II transcription in vitro. Nature 350:436–438PubMedCrossRefGoogle Scholar
  12. Fu J, Gnatt AL, Bushnell DA, Jensen GJ, Thompson NE, Burgess RR, David PR, Kornberg RD (1999) Yeast RNA polymerase II at 5 A resolution. Cell 98:799–810PubMedCrossRefGoogle Scholar
  13. Gromöller A, Lehming N (2000a) Srb7p is a physical and physiological target of Tup1p. EMBO J 19:6845–6852CrossRefGoogle Scholar
  14. Gromöller A, Lehming N (2000b). Srb7p is essential for the activation of a subset of genes. FEBS Lett 484:48–54CrossRefGoogle Scholar
  15. Guglielmi B, van Berkum NL, Klapholz B, Bijma T, Boube M, Boschiero C, Bourbon HM, Holstege FCP, Werner M (2004) A high resolution protein interaction map of the yeast Mediator complex. Nucl Acids Res 32:5379–5391PubMedCrossRefGoogle Scholar
  16. Gustafsson CM, Samuelsson T (2001) Mediator–a universal complex in transcriptional regulation. Mol Microbiol 41:1–8PubMedCrossRefGoogle Scholar
  17. Han SJ, Lee YC, Gim BS, Ryu GH, Park SJ, Lane WS, Kim YJ (1999) Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation. Mol Cell Biol 19:979–988PubMedGoogle Scholar
  18. Jäntti J, Aalto M, Öyen M, Sundqvist L, Keränen S, Ronne H (2002) Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation. J Cell Sci 115:409–420PubMedGoogle Scholar
  19. Kang JS, Kim SH, Hwang MS, Han SJ, Lee YC, Kim Y-J (2001) The structural and functional organization of the yeast mediator complex. J Biol Chem 276:42003–42010PubMedCrossRefGoogle Scholar
  20. Kelleher RJD, Flanagan PM, Kornberg RD (1990) A novel mediator between activator proteins and the RNA polymerase II transcription apparatus. Cell 61:1209–1215PubMedCrossRefGoogle Scholar
  21. Kim YJ, Björklund S, Li Y, Sayre MH, Kornberg RD (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77:599–608PubMedCrossRefGoogle Scholar
  22. Koleske AJ, Young RA (1994) An RNA polymerase II holoenzyme responsive to activators. Nature 368:466–469PubMedCrossRefGoogle Scholar
  23. Lee YC, Park JM, Min S, Han SJ, Kim YJ 1999. An activator binding module of yeast RNA polymerase II holoenzyme. Mol. Cell. Biol. 19:2967–2976PubMedGoogle Scholar
  24. Li Y, Björklund S, Jiang YW, Kim YJ, Lane WS, Stillman DJ, Kornberg RD (1995) Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc Natl Acad Sci USA 92:10864–10868PubMedCrossRefGoogle Scholar
  25. Liao SM, Zhang J, Jeffery DA, Koleske AJ, Thompson CM, Chao DM, Viljoen M, van Vuuren HJ, Young RA (1995) A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374:193–196PubMedCrossRefGoogle Scholar
  26. Linder T, Gustafsson CM (2004) The Soh1/MED31 protein is an ancient component of Schizosaccharomyces pombe and Saccharomyces cerevisiae Mediator. J Biol Chem 279:49455–49459PubMedCrossRefGoogle Scholar
  27. Liu Y, Ranish JA, Aebersold R, Hahn S (2001) Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes. J Biol Chem 276:7169–7175PubMedGoogle Scholar
  28. Maxon ME, Herskowitz I (2001) Ash1p is a site-specific DNA-binding protein that actively represses transcription. Proc Natl Acad Sci USA 98:1495–1500PubMedCrossRefGoogle Scholar
  29. Myers LC, Gustafsson CM, Bushnell DA, Lui M, Erdjument-Bromage H, Tempst P, Kornberg RD (1998) The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev 12:45–54PubMedCrossRefGoogle Scholar
  30. Myers LC, Gustafsson CM, Hayashibara KC, Brown PO, Kornberg RD (1999) Mediator protein mutations that selectively abolish activated transcription. Proc Natl Acad Sci USA 96:67–72PubMedCrossRefGoogle Scholar
  31. Nehlin JO, Carlberg M, Ronne H (1989) Yeast galactose permease is related to yeast and mammalian glucose transporters. Gene 85:313–319PubMedCrossRefGoogle Scholar
  32. Nonet ML, Young RA (1989) Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. Genetics 123:715–724PubMedGoogle Scholar
  33. Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Meth Enzymol 194:281–301PubMedCrossRefGoogle Scholar
  34. Sato S, Tomormori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CAS, Jin J, Cai Y, Washburn MP, Conaway JW, Conaway R (2004) A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol Cell 14:685–691PubMedCrossRefGoogle Scholar
  35. Tang H-L, Yeh L-S, Chen N-K, Ripmaster T, Schimmel P, Wang C-C (2004) Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J Biol Chem 279:49656–49663PubMedCrossRefGoogle Scholar
  36. Uetz PL, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockson D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang MJ, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623–627PubMedCrossRefGoogle Scholar
  37. Wilson CJ, Chao DM, Imbalzano AN, Schnitzler GR, Kingston RE, Young RA (1996) RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. Cell 84:235–244PubMedCrossRefGoogle Scholar
  38. Zervos AS, Gyuris J, Brent R (1993) Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell 72:223–232PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Magnus Hallberg
    • 1
    • 4
  • Guo-Zhen Hu
    • 2
    • 3
  • Susanna Tronnersjö
    • 2
    • 3
  • Zaki Shaikhibrahim
    • 1
  • Darius Balciunas
    • 2
    • 3
    • 5
  • Stefan Björklund
    • 1
  • Hans Ronne
    • 2
    • 3
  1. 1.Department of Medical Biochemistry and BiophysicsUmeå UniversityUmeåSweden
  2. 2.Department of Medical Biochemistry and MicrobiologyUppsala UniversityUppsalaSweden
  3. 3.Department of Plant Biology and Forest GeneticsSwedish University of Agricultural SciencesUppsalaSweden
  4. 4.Institute of Reproductive and Developmental Biology, Hammersmith Campus Imperial CollegeLondonUK
  5. 5.Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisUSA

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