Functional studies in yeast of cyclin C and the RNA polymerase II Mediator complex

Abstract: Cyclin C belongs to a group of cyclins that are not cell cycle-regulated. It was first cloned from Drosophila and rat, but its role was not understood until the yeast cyclin C homologue Srb 11 was identified in several genetic screens for transcriptional repressors and subsequently was shown to be associated with the RNA polymerase II Mediator complex. The Mediator is a multisubunit complex that enables RNA polymerase II to respond to activators in vitro.In the work presented here, the yeast genes encoding cyclin C (Srb11/Gig3), its cyclin-dependent kinase (Srb10/Gig2), and a third associated protein (Srb8/Gig1) were identified in a genetic screen for negative regulators of the gluconeogenic genes. A further analysis of the cloned genes suggested that the encoded proteins function closely together.The Med1 subunit of the yeast Mediator complex was characterized. Evidence was found of a functional connection between Med1 and the cyclin C-dependent kinase. The expression of the GAL1 promoter is partly deregulated in cells lacking cyclin C, Med1, or another mediator subunit, Med2. This deregulated expression is seen also under derepressed non-inducing conditions, and is therefore not due to a failure of glucose repression.An analysis of the ability of different Mediator subunits to activate transcription when fused to a DNA binding domain indicated that Med1 and Srb7 are negatively regulated both by cyclin C and by the Sin4 subunit of the Mediator, but not by the Med2 or Gal11 subunits, even though Sin4, Med2 and Gal11 are a part of the same module within the Mediator.A screen was made for multicopy suppressors of disruptions in the SRB8, SRB10 and SRB11 genes. Since these disruptions lack selectable phenotypes in a wild type background, the failure of snf1 mig1 srb8/10/11 cells to grow on galactose was used to select suppressors. Four new genes were identified and named GISI-4. Evidence was obtained of a functional interaction between these genes and the RAS/cAMP pathway.