Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

Abstract

Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1-CTD interaction has remained ambiguous because two different modes have been proposed: the ''dockingsite'' model versus the ''distributive'' mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.C-terminal domain dephosphorylation ͉ C-terminal domain hyperphosphorylation R NA polymerase II (Pol II) is distinguished from RNA polymerases I and III by the unique C-terminal domain (CTD) of its largest subunit, Rpb1 (1-3). The CTD, through its interaction with the Mediator complex, plays a central role in transcription activation (4-6), and it also plays critical roles in coupling mRNA synthesis to mRNA processing events, such as 5Ј capping, splicing, and 3Ј cleavage and polyadenylation (7,8). Composed of a tandemly repeated heptapeptide of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , the CTD undergoes reversible phosphorylation (9-11). Whereas the hypophosphorylated Pol II isoform (Pol IIA) assembles with general transcription factors into the transcription preinitiation complex (12-14), the hyperphosphorylated isoform (Pol IIO) is the dominant species found in transcription elongation complexes in vivo (15)(16)(17). The phosphorylation state of the CTD is controlled by the activities of CTD-specific kinases and phosphatases. Several transcription factor-associated CTD kinases have been identified, including CDK7͞Kin28, CDK9͞Bur1͞Ctk1 and CDK8͞Srb10 (11,(18)(19)(20). Other kinases (e.g., Cdc2 kinase and mitogen-activated protein kinase 2) can phosphorylate the CTD in vitro, but their contributions to CTD phosphorylation in vivo are uncertain (18,(21)(22)(23).Pol IIO is believed to be dephosphorylated before it can recycle for another round of transcription initiation (13,14,21,24). Fcp1 was the first CTD phosphatase discovered to fulfill such a function (25, 26). Fcp1 was reported to remove phosphates specifically from the Pol II CTD, but not from the other phosphoproteins tested (25). The human and...

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

“…In the S. pombe Fcp1 crystal structure (Ghosh et al 2008), the equivalent peptide makes extensive intramolecular hydrophobic contacts that would preclude its binding to TFIIF in the mode reported for a synthetic peptide derived from human Fcp1. Moreover, the relevance of TFIIF binding to S. pombe Fcp1 is unclear, because available evidence points to a direct interaction of SpFcp1 with Pol2 as a likely mode of SpFcp1 binding to the elongation complex (Kimura et al 2002;Suh et al 2005).…”

The Rpb4/7 Module of RNA Polymerase II Is Required for Carbon Catabolite Repressor Protein 4-Negative on TATA (Ccr4-Not) Complex to Promote Elongation

“…Ccr4-Not complex was purified from yeast from a Caf40-or Not4-TAP strain containing a deletion of DST1 by tandem affinity purification (TAP) as described in a previous publication (13). RNAPII and its mutant derivatives were purified through Rpb4-TAP or Rpb8-TAP (for 4/7⌬ RNAPII) (18). The amount of RNAPII was estimated by Bradford assay using bovine serum albumin as a standard.…”

The multifunctional Ccr4–Not complex directly promotes transcription elongation

“…3A). Rbp1 is predominantly hypophosphorylated (Suh et al 2005; data not shown). All of the known core subunits of the Ccr4-Not complex were detected in the TAP purification, and its composition matched that published by others (Mulder et al 2007b;Azzouz et al 2009).…”

“…TFIIS was expressed in Escherichia coli and purified as a histidinetagged protein (Kim et al 2007). Yeast RNAPII was purified as described in a previous study (Suh et al 2005). …”

“…However, these assays cannot determine if Ccr4-Not directly interacts with RNAPII or if it can bind elongating RNAPII. Yeast RNAPII (yRNAPII) was isolated to high purity, as described previously (Suh et al 2005), and the Ccr4-Not complex was purified by tandem affinity purification (TAP) from a TAP-Not4 strain. Silver-staining reveals that all 12 subunits of RNAPII are present (Fig.…”