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Molecular and Cellular Biology, January 2001, p. 1-15, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.1-15.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Seiji Yamamoto,^1,2 Yoshinori Watanabe,¹ Peter J. van der Spek,^1, Tomomichi Watanabe,^1,2 Hiroyuki Fujimoto,^1,2 Fumio Hanaoka,^1,3 and Yoshiaki Ohkuma^1,3,*

Institute for Molecular and Cellular Biology¹ and The Graduate School of Pharmaceutical Sciences,² Osaka University, and CREST, Japan Science and Technology Corporation,³ Suita, Osaka 565-0871, Japan

Received 30 May 2000/Returned for modification 9 August 2000/Accepted 13 October 2000

The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIE and ceTFIIE). ceTFIIE could partially replace hTFIIE, whereas ceTFIIE could not replace hTFIIE. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIE did not bind tightly to hTFIIE, and ceTFIIE showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIE induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIE was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

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^* Corresponding author. Mailing address: Institute for Molecular and Cellular Biology, 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}imcb.osaka-u.ac.jp.

Present address: N. V. Organon, Akzo-Nobel, 5340 BH Oss, The Netherlands.

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Molecular and Cellular Biology, January 2001, p. 1-15, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.1-15.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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