CHARACTERIZATION OF SUA7 MUTATIONS DEFINES A DOMAIN OF TFIIB INVOLVEDIN TRANSCRIPTION START SITE SELECTION IN YEAST

The SUA7 gene of Saccharomyces cerevisiae encodes the general transcri ption factor TFIIB. SUA7 was identified based on the ability of mutati ons at this locus to shift transcription start site selection at the c yc1 gene downstream of normal. Here, we report the nature of these mut ations; the sua7-1 and sua7-2 alleles encode identical E62K replacemen ts, and sua7-3 encodes an R78C replacement. Both Glu-62 and Arg-78 are phylogenetically invariant and occur within the most highly conserved region of TFIIB, immediately distal to a zinc finger motif. A double E62K,R78C mutant was constructed and exhibited the same phenotypes ass ociated with the single mutants, including cold sensitivity and altere d start site selection, suggesting that Glu-62 and Arg-78 are function ally related. This observation, and the opposite charge of the 2 resid ues, suggested that Glu-62 and Arg-78 might interact to form a salt br idge. This was tested by constructing reciprocal E62R and R78E replace ments. The E62R mutant is phenotypically identical to the E62K mutant, whereas the R78E mutant is inviable. However, an E62R,R78E double mut ant was not only viable but is phenotypically similar to the single mu tants. These results define the highly conserved sequence adjacent to the zinc finger of TFIIB as a critical determinant of start site selec tion and suggest that an Glu-62 Arg-78 salt bridge is an important str uctural element of that domain.