DELINEATION OF 2 FUNCTIONAL REGIONS OF TRANSCRIPTION FACTOR-TFIIB

Human transcription factor TFIIB, a protein of 316 amino acids, was su bjected to limited proteolysis in order to define stable structural do mains. We find that the C-terminal region of TFIIB, residues 106-316, is relatively stable, while the N-terminal region is very sensitive to proteases. Like full-length TFIIB, the stable domain, which we refer to as TFIIBc, interacts with the TATA-binding protein (TBP) on DNA. Ho wever, TFIIBc is unable to substitute for TFIIB in an in vitro transcr iption assay. We show by gel mobility-shift experiments that TFIIBc ar rests formation of the transcription complex after binding to TBP, and we conclude that the N-terminal region of TFIIB, which is missing fro m TFIIBc, is responsible for the recruitment of RNA polymerase II to t he promoter. We also show that TFIIBc inhibits transcription by compet ing with full-length TFIIB for the interaction with TBP, either in the presence or in the absence of the TBP-associated factors. The acidic transcriptional activator GAL4-VP16 does not favor the assembly of the functional transcription complex over the nonfunctional complex conta ining TFIIBc. Thus, if the function of GAL4-VP16 is enhancement of the interaction between TFIIB and the TFIID-DNA complex, then this functi on can also be exerted on the protease-resistant domain TFIIBc.