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Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains

Apostol, Barbara L. and Westaway, Shawn K. and Abelson, John and Greer, Chris L. (1991) Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains. Journal of Biological Chemistry, 266 (12). pp. 7445-7455. ISSN 0021-9258.

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Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires the action of two enzymes: a site specific endonuclease and a tRNA ligase. The tRNA ligase contains three distinct enzymatic activities: a polynucleotide kinase, a cyclic phosphodiesterase, and an RNA ligase. The polypeptide also has a high affinity pre-tRNA binding site based on its ability to form stable complexes with pre-tRNA substrates. To investigate the organization of functional enzymatic and binding elements within the polypeptide a series of defined tRNA ligase gene deletions were constructed and corresponding proteins were expressed in Escherichia coli as fusions with bacterial dihydrofolate reductase (DHFR). The DHFR/ligase derivative proteins were then efficiently purified by affinity chromatography. The complete ligase fusion protein retained enzymatic and binding activities which were unaffected by the presence of the DHFR segment. Examination of tRNA ligase deletion derivatives revealed that the amino-terminal region was required for adenylylation, while the carboxyl-terminal region was sufficient for cyclic phosphodiesterase activity. Deletions within the central region affected kinase activity. Pre-tRNA binding activity was not strictly correlated with a distinct enzymatic domain. A DHFR/ligase-derived protein lacking kinase activity efficiently joined tRNA halves. We postulate that this variant utilizes a novel RNA ligation mechanism.

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Additional Information:© 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, December 12, 1990. We thank Arek Avedian for the construction and initial characterization of certain deletion derivatives and Richard Ogden and Jennifer Normanly for help with DHFR expression systems. This work was supported by National Science Foundation Grant DMB-8917393 (to C. L. G.) and by National Institutes of Health Grant GM-32637 (to J. A.). 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.
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Issue or Number:12
Record Number:CaltechAUTHORS:20131022-112052861
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:42008
Deposited By: Tony Diaz
Deposited On:24 Oct 2013 23:50
Last Modified:03 Oct 2019 05:54

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