A SUPPRESSOR OF MUTATIONS IN THE CLASS-III TRANSCRIPTION SYSTEM ENCODES A COMPONENT OF YEAST TFIIIB

Class III genes depend on TFIIIB for recruitment of RNA polymerase III . Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 9 0 kDa polypeptide contained in the fraction B ''. We report the isolat ion of the yeast gene TFC7 which, based on genetic and biochemical evi dence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicop y suppressor of temperature-sensitive mutations in the two largest sub units of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of similar to 90 kDa. In gel shif t assays, recombinant TFC7 protein (rTFC7p) alone did not bind detecta bly to DNA, or to the TFIIIC-DNA complex even in the presence of TBP o r TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA comp lex. The two-hybrid assay pointed to an interaction between TFC7 prote in and tau(131), the second largest subunit of TFIIIC (that also inter acts with TFIIIB70). rTFC7p can replace the B '' component of TFIIIB f or synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surp risingly, specific transcription of the SUP4 tRNA(Tyr) gene promoted b y rTFC7p was much weaker than with B ''. An additional factor activity , provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.