Functional analysis of the Ume3p/ Srb11p-RNA polymerase II holoenzyme interaction.
The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis. This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery. This report describes two domains required for Ume3p-RNA Pol II holoenzyme association. One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding. The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box. In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo. Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity. In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo. Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.
Cooper
K F
KF
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Strich
R
R
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D020035:Holoenzymes; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D016213:Cyclins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D020035:Holoenzymes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; C093345:SSN8 protein, S cerevisiae; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae