Open AccessFRET evidence for a conformational change in TFIIB upon TBP‐DNA binding
Author(s) -
Zheng Le,
Hoeflich Klaus P.,
Elsby Laura M.,
Ghosh Mahua,
Roberts Stefan G. E.,
Ikura Mitsuhiko
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.03983.x
Subject(s) - förster resonance energy transfer , conformational change , biophysics , transcription factor ii b , dna , chemistry , transcription factor ii a , computational biology , biology , polymerase , biochemistry , physics , fluorescence , gene , promoter , gene expression , rna dependent rna polymerase , quantum mechanics
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.