Mutagenesis of yeast TFIIIB70 reveals C-terminal residues critical for interaction with TBP and C34

The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B ", and TFIIIB70 or BRF. TFIIIB70 is a pivotal component sinc e it interacts with TBP, TFIIIC and RNA polymerase III (pol III). In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension . Conditional mutations lying in conserved regions II and III were obtained , some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations. Two conditional mutations in region I I impaired the interaction with TBP and were suppressed by its overexpressi on. The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D4 64 residues in vivo. As expected, this TFIIIB70 mutation impaired the assem bly of TFIIIB.TFIIIC.DNA complexes and affected in vitro transcription of t he SUP4 tRNA gene. Our results underscore the important role of region II o f TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding. (C) 1999 Academic Press.