The SUA7 gene of Saccharomyces cerevisiae encodes the general transcription factor TFIIB. SUA7 was identified based on the ability of mutations at this locus to shift transcription start site selection at the cyc1 gene downstream of normal. Here, we report the nature of these mutations; the sua7-1 and sua7-2 alleles encode identical E62K replacements, 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 associated with the single mutants, including cold sensitivity and altered start site selection, suggesting that Glu-62 and Arg-78 are functionally related. This observation, and the opposite charge of the 2 residues, suggested that Glu-62 and Arg-78 might interact to form a salt bridge. This was tested by constructing reciprocal E62R and R78E replacements. The E62R mutant is phenotypically identical to the E62K mutant, whereas the R78E mutant is inviable. However, an E62R,R78E double mutant was not only viable but is phenotypically similar to the single mutants. These results define the highly conserved sequence adjacent to the zinc finger of TFIIB as a critical determinant of start site selection and suggest that an Glu-62-Arg-78 salt bridge is an important structural element of that domain.