FRET evidence for a conformational change in TFIIB upon TBP-DNA binding.
As a critical step of the preinitiation complex assembly in transcription, the general transcription factor TFIIB forms a complex with the TATA-box binding protein (TBP) bound to a promoter element. Transcriptional activators such as the herpes simplex virus VP16 facilitate this complex formation through conformational activation of TFIIB, a focal molecule of transcriptional initiation and activation. Here, we used fluorescence resonance energy transfer to investigate conformational states of human TFIIB fused to enhanced cyan fluorescent protein and enhanced yellow fluorescent protein at its N- and C-terminus, respectively. A significant reduction in fluorescence resonance energy transfer ratio was observed when this fusion protein, hereafter named CYIIB, was mixed with promoter-loaded TBP. The rate for the TFIIB-TBP-DNA complex formation is accelerated drastically by GAL4-VP16 and is also dependent on the type of promoter sequences. These results provide compelling evidence for a 'closed-to-open' conformational change of TFIIB upon binding to the TBP-DNA complex, which probably involves alternation of the spatial orientation between the N-terminal zinc ribbon domain and the C-terminal conserved core domain responsible for direct interactions with TBP and a DNA element.
Zheng
Le
L
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Ontario, Canada.
Hoeflich
Klaus P
KP
Elsby
Laura M
LM
Ghosh
Mahua
M
Roberts
Stefan G E
SG
Ikura
Mitsuhiko
M
D019943:Amino Acid Substitution; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000378:metabolism*; D035581:Transcription Factor TFIIB / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D011189:Potassium Chloride; D004247:DNA