Mutational analysis of yeast TFIIB: A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB

TFIIB is an essential component of the RNA polymerase II core transcription al machinery. Previous studies have defined TFIIB domains required for inte raction with other transcription factors and for basal transcription in vit ro. In the study reported here we investigated the TFIIB structural require ments for transcription initiation in vivo. A library of sua7 mutations enc oding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two sing le amino acid replacements in TFIIB were defined and characterized. These r eplacements are distributed throughout the protein and occur primarily at p hylogenetically conserved positions. Most replacements have little or no ef fect on the steady-state protein levels, implying that each affects TFIIB f unction rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start sit e selection, indicating that specific TFIIB structural defects affect trans criptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactio ns between ssu72 and sub1, both of which were initially identified as eithe r suppressors (SUB1 2 mu) or enhancers (sub1 Delta, ssu72-1) of sua7 mutati ons. We show that the interactions of ssu72-1 and sub1 Delta with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; a nd that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstr ate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that t hese interactions play a role in the mechanism of start site selection by R NA polymerase II.