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Originally published In Press as doi:10.1074/jbc.M100588200 on February 1, 2001

J. Biol. Chem., Vol. 276, Issue 18, 14996-15002, May 4, 2001
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Importance of Homodimerization for the in Vivo Function of Yeast RNA Triphosphatase*

Kevin Lehman, C. Kiong Ho, and Stewart ShumanDagger

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta -barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys330 and Val331 and a cross-dimer hydrogen bond of Asp280 with the backbone amide of Gln329. The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe272 and Leu273. Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.

* This work was supported by NIH Grant GM52470.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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