LETHAL MUTATIONS IN A YEAST U6 RNA GENE-B BLOCK PROMOTER ELEMENT IDENTIFY ESSENTIAL CONTACTS WITH TRANSCRIPTION FACTOR-IIIC

The B block promoter element is the primary binding site for the RNA p olymerase III transcription initiation factor TFIIIC. It is always loc ated within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pair s downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interac tion with TFIIIC. The in vitro and in vivo effects of all possible sin gle base pair substitutions in the 9-base pair core of the B block wer e determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TF IIIC and the B block promoter element. Transcript analysis reveals tha t lethal B block substitutions reduce U6 RNA synthesis at least 10-fol d in vivo and 20-fold in vitro. One viable B block mutant strain has o ne-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The location s of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact over-lapping sites on the B block eleme nt.