Abstract
We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the termination of transcription for a subset of genes in yeast. Srb5/Med18 could affect termination either indirectly by modulating CTD-Ser(2) phosphorylation near the 3' end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser(2) phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3' end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5(-) cells. Furthermore, Srb5-dependent termination of transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated termination of transcription, and the looped gene architecture may have a general role in termination of transcription in budding yeast.