Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

Abstract

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions −9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions −5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

ACKNOWLEDGMENTS

We thank Diane Bourque and Vincent Trinh for the computer-generated models, and we thank Will Home and Diane Forget for critical reading of the manuscript. We are also grateful to our colleague Zachary Burton for helpful suggestions and for providing the TFIIF deletion mutants.

This work was supported by grants from the Medical Research Council of Canada and the Cancer Research Society, Inc. (to B.C.); the Human Frontier Science Program (grant RG0193/97M), INSERM, CNRS, and Association pour la Recherche sur le Cancer (to J.M.E.); and the Ministry of Education, Science and Culture of Japan and the Core Research for Evolutional Science and Technology (to Y.O.). B.C. is a junior research scholar of the Fonds de la Recherche en Santé du Québec. M.D. and F.C. hold studentships from the NSERC and the Association pour la Recherche sur le Cancer.