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Nucleic Acids Research 2006 34(2):517-527; doi:10.1093/nar/gkj441
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Published online 20 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Structure–function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog

Li Kai Wang, Beate Schwer1, Markus Englert2, Hildburg Beier2 and Stewart Shuman*

Molecular Biology Program, Sloan-Kettering Institute New York, NY 10021, USA 1Department of Microbiology and Immunology, Weill Medical College of Cornell University New York, NY 10021, USA 2Institut für Biochemie, Universität Würzburg Biozentrum, D-97074, Würzburg, Germany

*To whom correspondence should be addressed. Tel: 212-639-7145; Fax: 212-717-3623; Email: s-shuman{at}ski.mskcc.org

Received November 22, 2005. Revised December 23, 2005. Accepted December 23, 2005.

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5'-kinase/2',3'-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2'-PO4 end for tRNA splicing in vivo.


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