Research ArticleStructural Biology

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

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Science Signaling  01 Mar 2016:
Vol. 9, Issue 417, pp. ra24
DOI: 10.1126/scisignal.aad4805

Identifying the active site

A phosphatase responsible for dephosphorylating specific residues of RNA polymerase II, the enzyme that catalyzes gene transcription, has been an enigma. This phosphatase, Rtr1 in yeast, lacks a consensus active site found in other types of phosphatases, and crystal structures have so far not yielded clues. Irani et al. performed detailed biochemical enzyme kinetic analysis of Saccharomyces cerevisiae Rtr1, the enzyme believed to have this function. Because when synthesized in and purified from bacteria, Rtr1 had very little activity, the authors relied on a highly sensitive fluorescent chemical assay and identified key residues necessary for activity and a minimum truncated form of the protein with activity. Crystals obtained with this smallest active truncated form enabled the identification of a putative phosphate-binding site, providing structural data for an active site and insight into the potential reaction mechanism for this family of enzymes.


Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

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