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Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site

Jaikaran, Dominic C. J. and Collins, Cynthia H. and MacMillan, Andrew M. (2002) Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site. Journal of Biological Chemistry, 277 (40). pp. 37624-37629. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:JAIjbc02

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Abstract

RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by, the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the RIG site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be crosslinked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2.RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.


Item Type:Article
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http://www.jbc.org/cgi/content/abstract/277/40/37624OtherUNSPECIFIED
Additional Information:© 2002 the American Society for Biochemistry and Molecular Biology. Received for publication, April 29, 2002, and in revised form, July 31, 2002. Published, JBC Papers in Press, August 5, 2002, DOI 10.1074/jbc.M204126200. We thank W. Keller for providing the P. pastoris hADAR2a expression construct, H. Shaikh, A. Franko, and K. Chilibeck for contributions to this work, and A. Gerber, D. Woolley, S. Chaulk, O. Kent, R. Zamel, R. Collins, and members of the Collins lab for helpful discussions.
Subject Keywords:PRE-MESSENGER-RNA; GLUTAMATE-RECEPTOR RNA; IN-VITRO; UNWINDING ACTIVITY; BINDING DOMAINS; DEAMINASE; CLONING; GENE; PURIFICATION; CONVERSION
Issue or Number:40
Record Number:CaltechAUTHORS:JAIjbc02
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:JAIjbc02
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4951
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:15 Sep 2006
Last Modified:02 Oct 2019 23:17

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