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,Jesper Q Svejstrup

doi:10.1074/jbc.m411071200

Stephanie E Kong, Michael S Kobor + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m411071200

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Abstract

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.

Highlights

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
Conditions that would be expected to disrupt ternary DNA-RNA-RNAPII0 complexes, such as DNase treatment and to an even larger extent DNase treatment plus addition of nucleotides, led to significant CTD de-phosphorylation (Fig. 1A, compare lanes 1 and 4). These experiments support the idea that the RNAPII CTD becomes susceptible to dephosphorylation upon disruption of the ternary complex and provides a possible explanation for our difficulties in isolating hyper-phosphorylated RNAPII after chromatin extraction
One of the hallmarks of Fcp1 is being unresponsive to such inhibitors [13], so in order to investigate the contribution of this protein to the observed CTD de-phosphorylation, chromatin was prepared from an fcp1 temperature-sensitive strain and its wild type counterpart

Summary

Introduction

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. 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.

Results

Conclusion