Conserved structure-function relationships in the mediator complex

Abstract: The Mediator complex is an essential multiprotein coregulator of RNA polymerase II (pol II)-dependent transcription in fungi and metazoans. Mediator interacts directly with both pol II and sequence-specific transcriptional regulators and thus acting as a bridge between the two. We have performed a comparative biochemical and functional characterisation the Mediator in the two ascomycete yeasts S. cerevisiae and S. pombe to address to what degree subunit architecture and specific regulatory functions have been conserved between these two species. In Paper I we identified the Med31 subunit as a stable component of Mediator in S. cerevisiae and S. pombe. We defined a highly conserved central motif in the Med31 protein that appeared to be required but not sufficient for the association of Med31 with the rest of the Mediator complex. In Paper II we investigated the structural basis for the global shutdown of pol II-dependent transcription in the S. cerevisiae MED17 temperature sensitive allele srb4-138. We found that the srb4-138 allele disrupts the interaction between the head and middle domains both in vitro and in vivo. This suggested that the global shutdown of transcription in srb4-138 cells at the non-permissive temperature is caused by destabilisation of the core Mediator complex. In Paper III we characterised the architecture of the S. pombe Cdk8 module by biochemical means and addressed its relationship to the non-essential Med1 subunit. We found that the Med1 subunit is closely linked both structurally and functionally with the Cdk8 module. Electron microscopy of wildtype and med1 Mediator resolved the position of the Med1 subunit as proximal to the Cdk8 module within the pol II-binding cleft of the core Mediator complex. Expression profiling of med1 cells showed significant overlap with the changes seen in cells lacking either Med12 or Med13. In Paper IV we carried out a comprehensive functional characterisation of the S. pombe Mediator complex and identified two distinct morphological pathways that require different parts of the Mediator for their proper regulation. Head domain-linked mutants tended to grow as hyphae due to their inability to express genes required for proper cell separation after mitosis. Mutants of the Cdk8 module aggregated into large clumps due to impaired repression of genes that code for cell surface agglutinins. Comparison with similar studies done in S. cerevisiae revealed that both regulatory roles for these subcomplexes are conserved.