The τ95 Subunit of Yeast TFIIIC Influences Upstream and Downstream Functions of TFIIIC·DNA Complexes

The yeast transcription factor IIIC (TFIIIC) is organized in two distinct multisubunit domains, τA and τB, that are respectively responsible for TFIIIB assembly and stable anchoring of TFIIIC on the B block of tRNA genes. Surprisingly, we found that the removal of τA by mild proteolysis stabilizes the residual τB·DNA complexes at high temperatures. Focusing on the well conserved τ95 subunit that belongs to the τA domain, we found that the τ95-E447K mutation has long distance effects on the stability of TFIIIC·DNA complexes and start site selection. Mutant TFIIIC·DNA complexes presented a shift in their 5′ border, generated slow-migrating TFIIIB·DNA complexes upon stripping TFIIIC by heparin or heat treatment, and allowed initiation at downstream sites. In addition, mutant TFIIIC·DNA complexes were highly unstable at high temperatures. Coimmunoprecipitation experiments indicated that τ95 participates in the interconnection of τA with τB via its contacts with τ138 and τ91 polypeptides. The results suggest that τ95 serves as a scaffold critical for τA·DNA spatial configuration and τB·DNA stability.