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

   

Authors:	Schwer, B.; Ghosh, A.; Sanchez, A. M.; Lima, C. D.; Shuman, S.
Article Title:	Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1
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-PO<inf>4</inf> 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-PO<inf>4</inf> 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-PO<inf>4</inf> 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 AlF<inf>3</inf> that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction. © 2015 Schwer et al.
Keywords:	unclassified drug; gene mutation; nonhuman; genetic analysis; protein domain; protein motif; serine; carboxy terminal sequence; in vivo study; enzyme activity; enzyme substrate; mutational analysis; enzyme phosphorylation; amino terminal sequence; crystal structure; threonine; protein dephosphorylation; catalysis; structure analysis; enzyme structure; rna polymerase ii; hydrolysis; cysteine; fungus growth; schizosaccharomycetaceae; phosphoprotein phosphatase; schizosaccharomyces pombe protein; schizosaccharomyces pombe; transcription elongation factor; metal binding; vegetative growth; ssu72; priority journal; article; phosphorane derivative; spt5; ctd code; rna polymerase ii carboxy terminal domain phosphatase fcp1
Journal Title:	RNA
Volume:	21
Issue:	6
ISSN:	1355-8382
Publisher:	Cold Spring Harbor Laboratory Press
Date Published:	2015-06-01
Start Page:	1135
End Page:	1146
Language:	English
DOI:	10.1261/rna.050286.115
PROVIDER:	scopus
PMCID:	PMC4436666
PUBMED:	25883047
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-84940390579&partnerID=40&md5=70beba8dc75b598bf188286c11d74767
Notes:	Export Date: 2 October 2015 -- Source: Scopus

https://synapse.mskcc.org/synapse/works/has_related_data_chk/81351