Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.
The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on the basis of their ability to compete with wild-type TFIIB in vivo. Moreover, analysis of the TFIIB mutant derivatives by chromatin immunoprecipitation showed that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II. Together, our results support a mode of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo.
Elsby
Laura M
LM
Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
O'Donnell
Amanda J M
AJ
Green
Laura M
LM
Sharrocks
Andrew D
AD
Roberts
Stefan G E
SG
D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics*; D035581:Transcription Factor TFIIB / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D035581:Transcription Factor TFIIB; D012319:RNA Polymerase II