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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 "docking-site" 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. [ABSTRACT FROM AUTHOR]

The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of (¹YSPTSPS7)n heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser5 in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure. Whereas the CPLs rely on Tyr¹ and Pro3 located on the upstream side of the Ser5-PO4 target site, Ssu72 recognizes Thr4 and Pro6 flanking the target Ser5-PO4 plus the downstream Tyr¹ residue of the adjacent heptad. We surmise that the reading of the CTD code does not obey uniform rules with respect to the location and phasing of specificity determinants. Thus, CTD code, like the CTD structure, is plastic. [ABSTRACT FROM AUTHOR]

Fcp 1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y' S2P3T4S5p6s7. The CTD of the microsporidian parasite Encephalitzoon cuniculi Consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculj encodes a minimized 411-aa Fcpl-like protein (EcFcpl), which consists of a DxDx(TtV) phosphatase domain and a BRCA I carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp 1 enzymes. Nonetheless, EcFcp I can function in lieu of Saccharomyces cerevisjae Fcp I to sustain yeast cell growth. Recombinant EcFcp 1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-P04) substrates. EcFcp 1 dephosphorylates CTD positions Ser2 and SerS with similar efficacy in vitro. We exploit synthetic CTD Ser2-P04 and SerS-P()4 peptides to define minimized substrates for EcFcp 1 and to illuminate the importance of CTD primary structure in Ser2 and SerS phosphatase activity. [ABSTRACT FROM AUTHOR]

Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Sera of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y¹S²P³T4S5P6S7 . Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser²-PO4 heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S5P6S7Y¹S²P³T4 4; and (iii) single alanine mutations of flanking residues Tyr¹ or Pro³ result in 6-fold decrements in CTD phosphatase activity. Fcp1 belongs to the DXDX(TN) family of phosphotransferases that act via an acyl-phosphoenzyme intermediate. An alanine scan of 11 conserved positions of S. pombe Fcpl identifies Thr174, Tyr237, Thr243, and Tyr249 as important for phosphatase activity. Structure-activity relationships at these positions were determined by introducing conservative substitutions. Our results, together with previous mutational studies, highlight a constellation of 11 amino acids that are conserved in all Fcp1 orthologs and likely comprise the active site. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic β-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp[sup 280] as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile[sup 470], Leu[sup 519], Ile[sup 520], Phe[sup 523], Leu[sup 524], and Ile[sup 530]) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment [sup 41]VPKIEMNFLN[sup 50] located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain. [ABSTRACT FROM AUTHOR]

Describes a minimal phosphatase domain spanning amino acids 156-580. Dephosphorylation of the C-terminal of RNA polymerase II; Location of candidate catalytic residues; Determination of structure-activity relationships.