Expression of human TFIIA subunits in Saccharomyces cerevisiae identifies regions with conserved and species-specific functions.
The transcription factor TFIIA stabilizes the interaction between the TATA-binding protein (TBP) and promoter DNA and facilitates activator function. In yeast, TFIIA is composed of large (TOA1) and small (TOA2) subunits that interact to form a beta-barrel domain and a helix bundle domain. Here we report plasmid shuffle experiments showing that the human subunits (TFIIAalpha/beta, ALF, and TFIIAgamma) are not able to support growth in yeast and that the failure is associated with morphological abnormalities related to cell division. To determine the regions responsible for species specificity, we examined a series of chimeric yeast-human subunits. The results showed that yeast-human hybrids that contained the N-termini of TFIIAgamma or TFIIAalpha/beta were viable, presumably because they could form a functional interspecies alpha-helical bundle. Likewise, a TOA1 hybrid that contained the nonconserved internal region from TFIIAalpha/beta also had no effect on TFIIA function. However, hybrids that contained the acidic region III or C-terminal region IV from TFIIAalpha/beta grew more slowly than the wild-type TOA1 subunit, and if both regions were exchanged, this effect was far more severe. Although these hybrids exchanged sequences which are involved in beta-barrel formation and interactions with TBP, they were all active in a TBP-dependent mobility shift assay. The results suggest that the growth phenotypes of these hybrids might be due to a failure to interact with components of the yeast transcription machinery other than TBP. Finally, we show that sequences from region III of TFIIA large subunits fall into classes that are either highly acidic or that are divergent and nonacidic, and provide the first evidence to suggest that, at least in yeast, this region is important for TFIIA function.
Upadhyaya
Ashok B
AB
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75080, USA.
DeJong
Jeff
J
D017124:Conserved Sequence / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D035561:Transcription Factor TFIIA / Q000096:biosynthesis*
D035561:Transcription Factor TFIIA