Abstract: The Mediator complex is an essential co-activator for RNA polymerase II-dependent transcription in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae core Mediator complex consists of three larger domains that are termed head, middle, and tail. The Med17 subunit is located within the head domain and is essential for cell viability. A temperature-sensitive allele of the MED17 gene known as srb4-138 causes all RNA polymerase II-dependent transcription to cease at the non-permissive temperature. The phenotype of srb4-138 allele has served as the main in vivo proof of the importance of Mediator, but the molecular basis for the effect of this mutant has not been determined. We here characterize Mediator from cells carrying the srb4-138 allele and find that the Mediator complex consistently breaks apart at the head/middle domain boundary even at lower temperatures. We find that both the head and middle domains are able to associate with the RNA polymerase independently of each other. Interestingly, both sub-complexes are able to associate with an active promoter at the permissive temperature but at the non-permissive temperature the head domain is lost from the promoter.