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Molecular and Cellular Biology, April 2003, p. 2914-2926, Vol. 23, No. 8
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.8.2914-2926.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The 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*

Graduate School of Frontier Biosciences,¹ Graduate School of Pharmaceutical Sciences, Osaka University,² Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Suita, Osaka 565-0871,³ Cellular Physiology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan⁴

Received 9 October 2003/ Returned for modification 26 November 2002/ Accepted 28 January 2003

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 (hTFIIEß) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIEß 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 hTFIIEß 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 hTFIIEß 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.

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* Corresponding author. Mailing address: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7978. Fax: 81-6-6877-9382. E-mail: ohkumay{at}fbs.osaka-u.ac.jp.

Present address: Discovery Research Laboratories II, Pharmaceutical Research Division, Takeda Chemical Industries Ltd., Tsukuba, Ibaraki 300-4247, Japan.

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Molecular and Cellular Biology, April 2003, p. 2914-2926, Vol. 23, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.8.2914-2926.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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