HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 28, 18953-18960, July 14, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/28/18953    most recent M602867200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schwer, B. Articles by Shuman, S. Search for Related Content PubMed PubMed Citation Articles by Schwer, B. Articles by Shuman, S. Social Bookmarking                      What's this?

Poxvirus mRNA Cap Methyltransferase

BYPASS OF THE REQUIREMENT FOR THE STIMULATORY SUBUNIT BY MUTATIONS IN THE CATALYTIC SUBUNIT AND EVIDENCE FOR INTERSUBUNIT ALLOSTERY^*

Beate Schwer¹, Stéphane Hausmann, Susanne Schneider, and Stewart Shuman²

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540–844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1 cells. We performed a genetic screen for mutations in the catalytic subunit that bypassed the requirement for the stimulatory subunit in vivo. We thereby identified missense changes in vicinal residues Tyr-752 (to Ser, Cys, or His) and Asn-753 (to Ile), which are located in the cap guanine-binding pocket. Biochemical experiments illuminated a mechanism of intersubunit allostery, whereby the vD12 subunit enhances the affinity of the catalytic subunit for AdoMet and the cap guanine methyl acceptor by 6- and 14-fold, respectively, and increases k_cat by a factor of 4. The bypass mutations elicited gains of function in both vD12-independent and vD12-dependent catalysis of cap methylation in vitro when compared with wild-type vD1-(540–844). These results highlight the power of yeast as a surrogate model for the genetic analysis of interacting poxvirus proteins and demonstrate that the activity of an RNA processing enzyme can be augmented through selection and protein engineering.

Received for publication, March 27, 2006 , and in revised form, May 12, 2006.

^* This work was supported by National Institutes of Health Grant GM42498. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: bschwer{at}med.cornell.edu. ² An American Cancer Society Research Professor. To whom correspondence may be addressed. E-mail: s-shuman{at}ski.mskcc.org.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. Benarroch, Z. R. Qiu, B. Schwer, and S. Shuman Characterization of a mimivirus RNA cap guanine-N2 methyltransferase RNA, April 1, 2009; 15(4): 666 - 674. [Abstract] [Full Text] [PDF]

				S. Zheng and S. Shuman Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site RNA, November 1, 2008; 14(11): 2297 - 2304. [Abstract] [Full Text] [PDF]

				S. Zheng and S. Shuman Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase RNA, April 1, 2008; 14(4): 696 - 705. [Abstract] [Full Text] [PDF]

				S. Zheng, S. Hausmann, Q. Liu, A. Ghosh, B. Schwer, C. D. Lima, and S. Shuman Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity J. Biol. Chem., November 24, 2006; 281(47): 35904 - 35913. [Abstract] [Full Text] [PDF]