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

Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2005; 280(6):4299-306. DOI: 10.1074/jbc.M411071200
Source: PubMed


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.

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    • "Fcp1 directly recognizes CTD repeats, in addition to binding to RNAPII at a functionally distinct site that may mediate its effect on elongation [16]. The dephosphorylation of Ser5 by Fcp1 after the termination of transcription, in vitro, can be explained by the lower specificity of Fcp1 for a ternary complex of DNA, RNAPII, TFIIH in comparison with that for free RNAPII [17]. Consequently, Fcp1 is modelled as acting on Ser2∼P during elongation and on both Ser2∼P and Ser5∼P during recycling. "
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