Multiple Roles of the τ131 Subunit of Yeast Transcription Factor IIIC (TFIIIC) in TFIIIB Assembly

Abstract

Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, τ131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (τ131-ΔTPR2) harboring a deletion in τ131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B”/TFIIIB90, but not Brf1 or TATA-binding protein. In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity. The in vitro transcription defect was abolished at high concentrations of B” but not Brf1. Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between τ131 and B”. τ131, therefore, appears to be involved in the recruitment of both Brf1 and B”.

We thank Emmanuel Favry for preparation of recombinant TBP, Brf1, B" fraction, and RNA polymerase III and Cécile Ducrot for technical assistance. We thank Micheline Wesolowski-Louvel (Université Claude Bernard Lyon 1) for the gift of the K. lactis genomic library. We thank Christine Conesa and Pierre Thuriaux for helpful discussions. Sequence data for C. albicans were obtained from the Stanford DNA Sequencing and Technology Center website at http://www-sequence.stanford.edu/group/candida . Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. The sequence data from S. pombe were produced by the S. pombe Sequencing Group at the Sanger Centre.

H.D.-O. acknowledges fellowships from the French Ministère de l’Éducation Nationale, de la Recherche et de la Technologie, and from the Association pour la Recherche contre le Cancer.