Photo-Cross-Linking of a Purified Preinitiation Complex Reveals Central Roles for the RNA Polymerase II Mobile Clamp and TFIIE in Initiation Mechanisms

Abstract

The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIEα closely approaches promoter DNA at nucleotide −10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIEα is obtained when the DNA template is artificially premelted in the −11/−1 region, suggesting that TFIIEα facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

We are grateful to the members of our laboratory for helpful discussions. We thank Bryan Wilkes for his invaluable help in producing the model in Fig. 1B, Diane Bourque for artwork, and Julie Edwards for critical reading of the manuscript. We also thank members of the laboratory of Yvan Guindon for help with chemical procedures.

This work is supported by a grant from the Canadian Institutes for Health Research. M.-F.L. and V.T. hold studentships from the Natural Sciences and Engineering Research Council of Canada and the Fonds de la recherche en santé du Québec. B.C. is a senior scholar from the Fonds de la Recherche en Santé du Québec.