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J. Biol. Chem., Vol. 280, Issue 6, 4299-4306, February 11, 2005

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Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator^*

Stephanie E. Kong, Michael S. Kobor¶||, Nevan J. Krogan¶, Baggavalli P. Somesh, T. Max M. Søgaard, Jack F. Greenblatt¶, and Jesper Q. Svejstrup**

From the Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, EN6 3LD, United Kingdom and ^¶Banting and Best Department of Medical Research, Toronto Yeast Proteomics Organization, University of Toronto, Toronto, Ontario M5G 1L6, Canada

Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNAPII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex. Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1. Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo. Genetic interactions between Elongator and a number of other transcription factors are also reported. Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes.

Received for publication, September 27, 2004

^* This work was supported by an Overseas Research Grant and Bath Fellowship (to S. E. K.) and a grant from Cancer Research UK (to J. Q. S.). Work in the Greenblatt lab was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Stowers Institute for Medical Research, Kansas City, MO 64110.

^|| Present address: Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

^** To whom correspondence should be addressed. Tel.: 44-1707-62-5960; Fax: 44-207-269-3801; E-mail: J.Svejstrup{at}Cancer.org.UK.

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