Central forkhead domain of human TFIIE beta plays a primary role in binding double-stranded DNA at transcription initiation.
The human general transcription factor, TFIIE, consists of two subunits, alpha and beta. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEbeta. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEbeta binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.
Tanaka
Aki
A
Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
Watanabe
Tomomichi
T
Iida
Yukari
Y
Hanaoka
Fumio
F
Ohkuma
Yoshiaki
Y
D004247:DNA / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D010766:Phosphorylation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051758:Transcription Factor TFIIH / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D051758:Transcription Factor TFIIH; D004247:DNA