The Carboxy Terminus of the Small Subunit of TFIIE Regulates the Transition from Transcription Initiation to Elongation by RNA Polymerase II

Abstract

The general transcription factor TFIIE plays essential roles in both transcription initiation and the transition from initiation to elongation. Previously, we systematically deleted the structural motifs and characteristic sequences of the small subunit of human TFIIE (hTFIIEβ) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIEβ and identified the functionally essential amino acid residues that bind to RNA polymerase II (Pol II), the general transcription factors, and single-stranded DNA. Although most residues identified were essential for transcription initiation, use of an in vitro transcription assay with a linearized template revealed that several residues in the carboxy-terminal helix-loop region are crucially involved in the transition stage. Mutations in these residues also affected the ability of hTFIIEβ to stimulate TFIIH-mediated phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of Pol II. Furthermore, these mutations conspicuously augmented the binding of hTFIIEβ to the p44 subunit of TFIIH. The antibody study indicated that they thus altered the conformation of one side of TFIIH, consisting of p44, XPD, and Cdk-activating kinase subunits, that is essential for the transition stage. This is an important clue for elucidating the molecular mechanisms involved in the transition stage.

ACKNOWLEDGMENTS

We thank Shunji Natori for providing the mouse SII cDNA clone pSII-3, Thomas Oelgeschläger, Sohail Malik, and Tetsuro Kokubo for critically reading the manuscript, Yoshinori Watanabe and Mitsuo Iwasaki for technical assistance, and Yoshifumi Nishimura, Masahiko Okuda, Katsuhiko Kamada, and our present and former laboratory members for helpful discussions.

This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (F.H. and Y.O.), the Japan Society for the Promotion of Science (Y.O.), the Biodesign Research Program of the Institute of Physical and Chemical Research (RIKEN) (F.H.), and the Core Research for Evolutional Science and Technology (F.H. and Y.O.).