ACCESSIBILITY OF EPITOPES ON HUMAN TRANSCRIPTION FACTOR IIB IN THE NATIVE PROTEIN AND IN A COMPLEX WITH DNA

Transcription factor IIB (TFIIB) plays a central role in the assembly of the RNA polymerase If initiation complex. Monoclonal antibodies (mA bs) that react with human TFIIB were prepared and used as probes to id entify portions of TFIIB that are accessible when the factor is in sol ution and when it is contained in a complex with DNA. Seven mAbs were examined and were mapped to three regions of the TFIIB molecule. Only the mAbs that mapped to residues 52-105 inhibited transcription, immun oprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extra ct (NE), and supershifted a complex containing TFIIB, the TATA-binding protein and DNA. The mAbs that mapped to residues 1-51 and the mAb th at mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 151), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa ce ll NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation com plex and that some far N-terminal epitopes are accessible on the purif ied protein, but become blocked when TFIIB is in HeLa cell NE or in th e preinitiation complex.