The role of human TFIIB in transcription start site selection in vitro andin vivo

The general transcription factor TFIIB plays a crucial role in selecting th e transcription initiation site in yeast. We have analyzed the human homolo gs of TFIIB mutants that have previously been shown to affect transcription start site selection in the yeast Saccharomyces cerevisiae. Despite the di stinct mechanisms of transcription start site selection observed in S. cere visiae and humans, the role of TFIIB in this process is similar. However, u nlike their yeast counterparts, the human mutants do not show a severe defe ct in supporting either basal transcription or transcription stimulated by an acidic activator in vitro. Transient transfection analysis revealed that , in addition to a role in transcription start site selection, human TFIIB residue Arg-66 performs a critical function in vivo that is bypassed in vit ro. Furthermore, although correct transcription start site selection is dep endent upon an arginine residue at position 66 in human TFIIB, innate funct ion in vivo is determined by the charge of the residue alone. Our observati ons raise questions as to the evolutionary conservation of TFIIB and uncove r an additional function for TFIIB that is required in vivo but can be bypa ssed in vitro.