Reconstitution of the transcription factor TFIIH: Assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7

To understand the initiation of the transcription of protein-coding genes, we have dissected the role of the basal transcription/DNA repair factor TFI IH. Having succeeded in reconstituting a functionally active TFIIH from bac ulovirus recombinant polypeptides, we were able to analyze the role of XPB, XPD, and cdk7 subunits in the transcription reaction. Designing mutated re combinant subunits, we show that the XPB helicase is absolutely required fo r transcription to open the promoter around the start site whereas the XPD helicase, which is dispensable, stimulates transcription and allows the CAK complex to be anchored to TFIIH. In addition, we also show that cdk7 may p hosphorylate the carboxy-terminal domain (CTD) of RNA pol II in the absence of promoter opening.