A MUTATION IN THE LARGEST SUBUNIT OF YEAST TFIIIC AFFECTS TRANSFER-RNA AND 5-S RNA-SYNTHESIS - IDENTIFICATION OF 2 CLASSES OF SUPPRESSORS

We report the characterization of a mutation affecting tau(138), the l argest subunit of yeast transcription factor IIIC (TFIIIC). A previous ly described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S. D., and Pringle, J. R. (1991) G enetics 127, 279-285) is shown to lie in the TFC3 gene which encodes t au(138). The tau(138) subunit carrying this mutation bears a single su bstitution of Glu for Gly at position 349 (G349E). In extracts from mu tant cells, both the level of TFIIIC and its affinity for tDNA were fo und to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel sh ift assays. In addition, the tsv115 mutation affected 5 S RNA synthesi s in vitro, suggesting that the tau(138) subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of th e TFIIIC defect were sought to reveal components participating in TFII IC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau(95) and tau(131), th e 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymeras e (pol) I, II, and III, ABC10 alpha; it also includes genes potentiall y related to pol III function, such as SRP40 which also suppresses a m utation in a subunit shared by RNA polymerases I and III. A second cla ss of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.