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Multiple Nucleotide Cofactor Use by Yeast Ligase in tRNA Splicing

Belford, Heather G. and Westaway, Shawn K. and Abelson, John and Greer, Chris L. (1993) Multiple Nucleotide Cofactor Use by Yeast Ligase in tRNA Splicing. Journal of Biological Chemistry, 268 (4). pp. 2444-2450. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:20140328-131713485

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Abstract

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://www.jbc.org/content/268/4/2444.abstract?sid=cb51ec56-fe4d-40f7-a9d2-97b0bcf56d16PublisherAritcle
Additional Information:© 1993 American Society for Biochemistry and Molecular Biology, Inc. Received for publication, July 1, 1992. We thank Eric M. Phizicky and Craig L. Peebles for many helpful discussions and Reinhard Rauhut for the generous gift of highly purified tRNA endonuclease.
Funders:
Funding AgencyGrant Number
NSFDMB-8971393
NIHGM-35955
Issue or Number:4
Record Number:CaltechAUTHORS:20140328-131713485
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20140328-131713485
Official Citation:Belford, H. G., Westaway, S. K., Abelson, J., & Greer, C. L. (1993). Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. Journal of Biological Chemistry, 268(4), 2444-2450
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:44549
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:28 Mar 2014 23:04
Last Modified:03 Oct 2019 06:19

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