Identifying a species-specific region of yeast TF11B in vivo.
The general transcription factor IIB (TFIIB) is required for RNA polymerase II transcription in eukaryotes. It provides a physical link between the TATA-binding protein (TBP) and the RNA polymerase and is a component previously suggested to respond to transcriptional activators in vitro. In this report, we compare the yeast (Saccharomyces cerevisiae) and human forms of the protein in yeast cells to study their functional differences. We demonstrate that human TFIIB fails to functionally replace yeast TFIIB in yeast cells. By analyzing various human-yeast hybrid TFIIB molecules, we show that a 14-amino-acid region at the amino terminus of the first repeat of yeast TFIIB plays an important role in determining species specificity in vivo. In addition, we identify four amino acids in this region that are critical for an amphipathic helix unique to yeast TFIIB. By site-directed mutagenesis analyses we demonstrate that these four amino acids are important for yeast TFIIB's activity in vivo. Finally, we show that mutations in the species-specific region of yeast TFIIB can differentially affect the expression of genes activated by different activators in vivo. These results provide strong evidence suggesting that yeast TFIIB is involved in the process of transcriptional activation in living cells.
Shaw
S P
SP
Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
Wingfield
J
J
Dorsey
M J
MJ
Ma
J
J
D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014981:Xenopus
D035581:Transcription Factor TFIIB; D014157:Transcription Factors