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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    ABSTRACT: The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3' end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics.
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    • "region [168] [169] [229] [230] "
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    ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the "CTD code" hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a "code." Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.
    02/2012; 2012(9):347214. DOI:10.1155/2012/347214
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    • "Elp3 is a subunit of Elongator, a HAT complex originally purified with elongating RNAPII (Otero et al., 1999). Importantly, Elongator disruptions (including ELP3) exacerbate deletions in ASF1 (Li et al., 2009) and FACT (Formosa et al., 2002), or are lethal with NAP1 deletions (Kong et al., 2005). Gcn5 activity is also linked to chaperone functions since mutations in ASF1 (Adkins et al., 2007), and FACT (Biswas et al., 2005; VanDemark et al., 2006), are lethal or a deletion in VPS75 (Fillingham et al., 2008) grows poorly when combined with a GCN5 deletion. "
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