TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution

In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported for Srb8 and Srb9. Here, we identify a TRAP240/ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11. We find that spTrap240 and Sch. pombe Srb8 (spSrb8) regulate the same distinct subset of genes and have indistinguishable phenotypic characteristics. Importantly, Mediator containing the spSrb8/spTrap240/spSrb10/spSrb11 subunits is isolated only in free form, devoid of RNA polymerase II. In contrast, Mediator lacking this module associates with the polymerase. Our findings provide experimental evidence for recent suggestions that TRAP230/ARC240 and TRAP240/ARC250 may indeed be the Srb8 and Srb9 homologues of mammalian Mediator. Apparently Srb8/TRAP230/ARC240, Srb9/TRAP240/ARC250, Srb10, and Srb11 constitute a conserved Mediator submodule, which is involved in negative regulation of transcription in all eukaryotes.

[1]  K. Lund,et al.  Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12 , 2003, Nature.

[2]  Paul Tempst,et al.  A Complex of the Srb8, -9, -10, and -11 Transcriptional Regulatory Proteins from Yeast* , 2002, The Journal of Biological Chemistry.

[3]  M. Boube,et al.  Evidence for a Mediator of RNA Polymerase II Transcriptional Regulation Conserved from Yeast to Man , 2002, Cell.

[4]  R. Tjian,et al.  Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. , 2002, Genes & development.

[5]  Robert Tjian,et al.  Structure, Function, and Activator-Induced Conformations of the CRSP Coactivator , 2002, Science.

[6]  K. H. Byun,et al.  Negative regulation of filamentous growth and flocculation by Lkh1, a fission yeast LAMMER kinase homolog. , 2001, Biochemical and biophysical research communications.

[7]  R. Kornberg,et al.  Quantitation of the RNA Polymerase II Transcription Machinery in Yeast* , 2001, The Journal of Biological Chemistry.

[8]  T. Kieselbach,et al.  Analysis of Schizosaccharomyces pombe Mediator reveals a set of essential subunits conserved between yeast and metazoan cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Stillman,et al.  The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II. , 2001, Genes & development.

[10]  E. Kremmer,et al.  Novel critical role of a human Mediator complex for basal RNA polymerase II transcription , 2001, EMBO reports.

[11]  A. Berk,et al.  Characterization of Mediator Complexes from HeLa Cell Nuclear Extract , 2001, Molecular and Cellular Biology.

[12]  J. Lis,et al.  Mediator, not holoenzyme, is directly recruited to the heat shock promoter by HSF upon heat shock. , 2001, Molecular cell.

[13]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[14]  T. Samuelsson,et al.  Mediator – a universal complex in transcriptional regulation , 2001, Molecular microbiology.

[15]  K Nasmyth,et al.  Cdk1 triggers association of RNA polymerase to cell cycle promoters only after recruitment of the mediator by SBF. , 2001, Molecular cell.

[16]  K. Gould,et al.  Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe , 2001, Yeast.

[17]  H. Y. Liu,et al.  Characterization of CAF4 and CAF16 Reveals a Functional Connection between the CCR4-NOT Complex and a Subset of SRB Proteins of the RNA Polymerase II Holoenzyme* , 2001, The Journal of Biological Chemistry.

[18]  Y. Liu,et al.  Yeast Nuclear Extract Contains Two Major Forms of RNA Polymerase II Mediator Complexes* , 2001, The Journal of Biological Chemistry.

[19]  J. Qin,et al.  The USA-derived transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PCs. , 2000, Molecular cell.

[20]  C. Gustafsson,et al.  Purification and Characterization of RNA Polymerase II Holoenzyme from Schizosaccharomyces pombe * , 2000, The Journal of Biological Chemistry.

[21]  R. Tjian,et al.  Composite co-activator ARC mediates chromatin-directed transcriptional activation , 1999, Nature.

[22]  J. Qin,et al.  Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. , 1999, Molecules and Cells.

[23]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[24]  R. Young,et al.  RNA Polymerase II Holoenzymes and Subcomplexes* , 1998, The Journal of Biological Chemistry.

[25]  P. Philippsen,et al.  Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe , 1998, Yeast.

[26]  R. Young,et al.  Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. , 1998, Molecular cell.

[27]  J. Davey,et al.  Characterization of the Prk1 protein kinase from Schizosaccharomyces pombe , 1998, Yeast.

[28]  R. Young,et al.  General requirement for RNA polymerase II holoenzymes in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Young,et al.  A kinase–cyclin pair in the RNA polymerase II holoenzyme , 1995, Nature.

[30]  Yang Li,et al.  A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II , 1994, Cell.

[31]  R. Young,et al.  Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. , 1989, Genetics.

[32]  B. Miki,et al.  Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae , 1982, Journal of bacteriology.

[33]  R. Kornberg,et al.  Mediator of transcriptional regulation. , 2000, Annual review of biochemistry.

[34]  D. Bushnell,et al.  The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. , 1998, Genes & development.

[35]  S. Moreno,et al.  Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. , 1991, Methods in enzymology.