The role of human TFIIB in transcription start site selection in vitro and in vivo.
The general transcription factor TFIIB plays a crucial role in selecting the transcription initiation site in yeast. We have analyzed the human homologs of TFIIB mutants that have previously been shown to affect transcription start site selection in the yeast Saccharomyces cerevisiae. Despite the distinct mechanisms of transcription start site selection observed in S. cerevisiae and humans, the role of TFIIB in this process is similar. However, unlike their yeast counterparts, the human mutants do not show a severe defect 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 vitro. Furthermore, although correct transcription start site selection is dependent upon an arginine residue at position 66 in human TFIIB, innate function in vivo is determined by the charge of the residue alone. Our observations raise questions as to the evolutionary conservation of TFIIB and uncover an additional function for TFIIB that is required in vivo but can be bypassed in vitro.
D017504:Adenovirus E4 Proteins / Q000235:genetics; D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic