The tau95 subunit of yeast TFIIIC influences upstream and downstream functions of TFIIIC.DNA complexes.

ABSTRACT

The yeast transcription factor IIIC (TFIIIC) is organized in two distinct multisubunit domains, tauA and tauB, 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 tauA by mild proteolysis stabilizes the residual tauB.DNA complexes at high temperatures. Focusing on the well conserved tau95 subunit that belongs to the tauA domain, we found that the tau95-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 tau95 participates in the interconnection of tauA with tauB via its contacts with tau138 and tau91 polypeptides. The results suggest that tau95 serves as a scaffold critical for tauA.DNA spatial configuration and tauB.DNA stability.