The Carboxy Terminus of the Small Subunit of TFIIE Regulates the Transition from Transcription Initiation to Elongation by RNA Polymerase II
AbstractThe Carboxy Terminus of the Small Subunit of TFIIE Regulates the Transition from Transcription Initiation to Elongation by RNA Polymerase II Tomomichi Watanabe 1 , 2 , † , Kazuhiro Hayashi 1 , 2 , Aki Tanaka 1 , 2 , Tadashi Furumoto 1 , 2 , Fumio Hanaoka 1 , 3 , 4 and Yoshiaki Ohkuma 1 , 3 , * 1 Graduate School of Frontier Biosciences 2 Graduate School of Pharmaceutical Sciences, Osaka University 3 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Suita, Osaka 565-0871 4 Cellular Physiology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan 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.