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

Abstract

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 (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ 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 ceTFIIEβ 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, ceTFIIEβ 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.

ACKNOWLEDGMENTS

We thank Tetsuro Kokubo and Kiyoe Ura for critical reading of the manuscript, Takehiro Kobayashi and Kiyoji Tanaka for human XPB (ERCC3) and XPD (ERCC2) cDNA clones, Koji Hisatake for the human p52 cDNA clone, Jean-Marc Egly for human p52 and MAT1 cDNA clones, Charles J. Sherr for the mouse Cdk7 (MO15) cDNA clone, David O. Morgan for the human cyclin H cDNA clone, Yuji Kohara for the C. elegansembryonic and mixed-stage cDNA libraries, Hideyuki Okano for the C. elegans embryonic cDNA library, and Katsuyuki Tamai for raising antibodies. We also thank Masayuki Yokoi, Toshihiko Oka, and Tomoko Okamoto for technical assistance and Robert G. Roeder and our colleagues for helpful discussion.

This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (F.H. and Y.O.), the Core Research for Evolutional Science and Technology (CREST) (F.H. and Y.O.), the Biodesign Research Program of the Institute of Physical and Chemical Research (RIKEN) (F.H.), the Terumo Life Science Foundation (Y.O.), and the Yamanouchi Foundation for Research on Metabolic Disorders (Y.O.).