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

Beate Schwer, Agnidipta Ghosh, Ana M. Sanchez, Christopher D. Lima, Stewart Shuman

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS2PTSPS repeat. The SpFcp1 catalytic domain (aa 140-580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1-139) and its putative metal-binding motif C99H101Cys109C112 are essential; (iii) the carboxyterminal domain (aa 581-723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330-393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T1PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg2+ and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction.

Original languageEnglish (US)
Pages (from-to)1135-1146
Number of pages12
JournalRNA
Volume21
Issue number6
DOIs
StatePublished - Jun 1 2015
Externally publishedYes

Keywords

  • CTD code
  • Spt5
  • Ssu72

ASJC Scopus subject areas

  • Molecular Biology

Fingerprint Dive into the research topics of 'Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1'. Together they form a unique fingerprint.

  • Cite this