A conformational change in TFIIB is required for activator-mediated assembly of the preinitiation complex.
TFIIB plays a pivotal role during assembly of the RNA polymerase II transcription preinitiation complex. TFIIB is composed of two domains that engage in an intramolecular interaction that can be disrupted by the VP16 activation domain. In this study, we describe a novel human TFIIB derivative harbouring two point mutations in the highly conserved N-terminal charged cluster domain. This mutant, TFIIB R53E:R66E, exhibits an enhanced affinity in its intramolecular interaction when compared with wild-type TFIIB. Consistent with this, the mutant displays a significantly reduced affinity for VP16. However, its ability to complex with TATA-binding protein at a model promoter is equivalent to that of wild-type TFIIB. Furthermore, this TFIIB derivative is able to support high order preinitiation complex assembly in the absence of an activator. Strikingly though, an activator fails to recruit the TFIIB mutant to the promoter. Taken together, our results show that a TFIIB conformational change is critical for the formation of activator-dependent transcription complexes.
Glossop
James A
JA
School of Biological Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Dafforn
Tim R
TR
Roberts
Stefan G E
SG
D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D035581:Transcription Factor TFIIB / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D046911:Macromolecular Substances; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF