Functional Relationships of Srb10-Srb11 Kinase, Carboxy-Terminal Domain Kinase CTDK-I, and Transcriptional Corepressor Ssn6-Tup1

Abstract

The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10⌬ and srb11⌬ mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10⌬ and ctk1⌬ suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.RNA polymerase II holoenzyme forms purified from the yeast Saccharomyces cerevisiae contain a mediator complex, which functions in transcriptional activation (3,12,18,22,23,53). Components of mediator/holoenzyme forms include Srb proteins, Gal11, Sin4, Rgr1, Rox3, and general transcription factors (16,18,22,23,29,30). Genetic evidence suggests that the mediator/holoenzyme plays a role not only in transcriptional activation but also in repression (for a review, see reference 4). Mutations in the genes encoding Srb8 to Srb11, Gal11, Sin4, Rgr1, and Rox3 appear to relieve negative regulation of diversely regulated genes (6,9,13,20,25,39,44,49,52,60,61).Two of these proteins, Srb10 and Srb11, constitute a cyclindependent kinase (cdk)-cyclin pair (25,30). The connection to RNA polymerase II was first established by the isolation of srb10 and srb11 alleles as suppressors of truncations in the carboxy-terminal repeat domain (CTD) of the largest subunit of polymerase (30). The Srb10-Srb11 kinase was found associated with an RNA polymerase II holoenzyme form and was shown to affect phosphorylation of the CTD in vitro (18,30). Mutations in SRB10 and SRB11 also reduced the activation of GAL promoters (25, 30). Mutations in both genes had previously been isolated in genetic selections for specific effects on gene regulation. Alleles called ssn3 and ssn8 were identified as suppressors of a defect in the Snf1 protein kinase and were shown to affect glucose repression of the SUC2 gene (5, 25, 60). A related selection for suppressors affecting the Snf1-dependent expression of gluconeogeni...

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

“…As a consequence, overexpression of cell cycle cyclins usually generates growth defects (9,60,68). The CTDK-I complex is implicated not in cell cycle regulation but rather in transcription regulation (26,31). Similarly to what has been observed for other C-type cyclins implicated in transcription control, CTK2 mRNA levels do not fluctuate during the cell cycle (data not shown).…”

“…Deletion of any of the CTK genes generates cryosensitive cells. CTDK-I is related to the Srb10/11 kinase complex, although it seems to display a distinct role in transcriptional control (26). In vitro studies carried out with HeLa nuclear extracts have shown that CTDK-I can modulate the elongation efficiency of RNA polymerase II (31).…”

Promoter-dependent Roles for the Srb10 Cyclin-dependent Kinase and the Hda1 Deacetylase in Tup1-mediated Repression inSaccharomyces cerevisiae

“…One component, Srb10, a nonessential cyclin-dependent kinase, is part of a distinct Mediator-associated complex (the Srb8-11 complex) that interacts with Tup1 (Myer and Young, 1998;Zaman et al, 2001;Borggrefe et al, 2002). Srb10 has been shown to negatively affect transcription, and its kinase function is necessary for full repression of a Tup1-controlled reporter construct (Holstege et al, 1998;Kuchin and Carlson, 1998;Song and Carlson, 1998;Lee et al, 2000 DNA-binding protein that recruits the Tup1-Ssn6 complex and is responsible for repressing hypoxia-induced genes (Holstege et al, 1998;Becerra et al, 2002). Previous work designed to dissect the mechanism of Tup1-mediated repression has concentrated mainly on the analysis of a few Tup1-repressed genes and reporter constructs.…”

Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

“…On the other hand, by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II, it can inactivate the holoenzyme, as assayed in vitro, and thus has been proposed to play a negative role in transcription (26). Deletion of Srb10͞11 or Srb7 has been reported to decrease Tup1-mediated repression and TUP1 has recently been reported to interact with Srb7 (23,27,28).…”