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外文期刊>The biochemical journal
>Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue
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Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue
pReversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Sersup5/sup of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of iDrosophila melanogaster/i Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 ? (1 ?=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Metsup17/sup, Prosup46/sup, Aspsup51/sup, Tyrsup77/sup and Metsup85/sup) that are essential specifically for substrate recognition./p
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