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 II initiation complex. Monoclonal antibodies (mAbs) that react with human TFIIB were prepared and used as probes to identify portions of TFIIB that are accessible when the factor is in solution 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, immunoprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extract (NE), and supershifted a complex containing TFIIB, the TATA-binding protein, and DNA. The mAbs that mapped to residues 1-51 and the mAb that mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 1-51), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa cell NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation complex and that some far N-terminal epitopes are accessible on the purified protein, but become blocked when TFIIB is in HeLa cell NE or in the preinitiation complex.
Thompson
N E
NE
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Strasheim
L A
LA
Nolan
K M
KM
Burgess
R R
RR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012996:Solutions; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000737:chemistry* / Q000276:immunology; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D012996:Solutions; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D004247:DNA