An activator binding module of yeast RNA polymerase II holoenzyme

The Mediator complex of Saccharomyces cerevisiae is required for both gener al and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the f unction of each Mediator subcomplex and to delineate the functional relatio nship between the subcomplexes, we characterized the compositions and bioch emical activities of PolII-Mediator complexes (holoenzymes) prepared from s everal Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not ba sal transcription in a reconstituted in vitro system. This activation-speci fic defect was correlated with a crippled physical interaction to transcrip tional activator proteins, which could be bypassed by artificial recruitmen t of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene specific transcriptional activat or proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both ba sal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demo nstrate that the Gal11 module of the Rgr1 subcomplex is required for the ef ficient recruitment of PolII holoenzyme to a promoter via activator-specifi c interactions, while the Srb4 subcomplex functions in the modulation of ge neral polymerase activity.