Mediator is a large, modular protein complex remotely conserved from yeast to man that conveys regulatory signals from DNA-binding transcription factors to RNA polymerase II. In Saccharomyces cerevisiae, Mediator is thought to be composed of 24 subunits organized in four sub-complexes, termed the head, middle, tail and Cdk8 (Srb8-11) modules. In this work, we have used screening and pair-wise two-hybrid approaches to investigate protein-protein contacts between budding yeast Mediator subunits. The derived interaction map includes the delineation of numerous interaction domains between Mediator subunits, frequently corresponding to segments that have been conserved in evolution, as well as novel connections between the Cdk8 (Srb8-11) and head modules, the head and middle modules, and the middle and tail modules. The two-hybrid analysis, together with co-immunoprecipitation studies and gel filtration experiments revealed that Med31 (Soh1) is associated with the yeast Mediator that therefore comprises 25 subunits. Finally, analysis of the protein interaction network within the Drosophila Mediator middle module indicated that the structural organization of the Mediator complex is conserved from yeast to metazoans. The resulting interaction map provides a framework for delineating Mediator structure-function and investigating how Mediator function is regulated.

Mediator is a general coactivator of RNA polymerase II (Pol II) transcription. Genomic location analyses of different Mediator subunits indicate a uniformly composed core complex upstream of active genes but unexpectedly also upstream of inactive genes and on the coding regions of some highly active genes. The repressive Cdk8 submodule is associated with core Mediator at all sites but with a lower degree of occupancy, indicating transient interaction, regardless of promoter activity. This suggests gene-specific regulation of Cdk8 activity, rather than regulated Cdk8 recruitment. Mediator presence is not necessarily linked to transcription. This goes beyond Cdk8-repressed genes, indicating that Mediator can mark some regulatory regions ahead of additional signals. Overlap with intergenic Pol II location in stationary phase points to a role as a binding platform for inactive Pol II during quiescence. These results shed light on Cdk8 repression, suggest additional roles for Mediator, and query models of recruitment-coupled regulation.

Assays capable of determining the properties of thousands of genes in parallel present challenges with regard to accurate data processing and functional annotation. Collections of microarray expression data are applied here to assess the quality of different high-throughput protein interaction data sets. Significant differences are found. Confidence in 973 out of 5342 putative two-hybrid interactions from S. cerevisiae is increased. Besides verification, integration of expression and interaction data is employed to provide functional annotation for over 300 previously uncharacterized genes. The robustness of these approaches is demonstrated by experiments that test the in silico predictions made. This study shows how integration improves the utility of different types of functional genomic data and how well this contributes to functional annotation.

Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.