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 Journal Article

   

Authors:	Wang, L. K.; Schwer, B.; Englert, M.; Beier, H.; Shuman, S.
Article Title:	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
Abstract:	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. © The Author 2006. Published by Oxford University Press. All rights reserved.
Keywords:	controlled study; unclassified drug; gene mutation; nonhuman; chemical analysis; protein domain; amino acid substitution; in vivo study; in vitro study; enzyme activity; structure activity relation; structure-activity relationship; amino acid sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; dna mutational analysis; yeast; saccharomyces cerevisiae proteins; alanine; sequence homology; rna, transfer; enzyme structure; catalytic domain; phosphate; aspartic acid; genetic conservation; fungi; polynucleotide 5' hydroxyl kinase; polynucleotide 5'-hydroxyl-kinase; rna ligase (atp); fungal protein; arginine; histidine; rna splicing; genetic complementation; amino acid analysis; cloning; arabidopsis; genetic complementation test; rna ligase; protein trl1; 2',3'-cyclic-nucleotide phosphodiesterases; arabidopsis proteins; plant proteins
Journal Title:	Nucleic Acids Research
Volume:	34
Issue:	2
ISSN:	0305-1048
Publisher:	Oxford University Press
Date Published:	2006-02-01
Start Page:	517
End Page:	527
Language:	English
DOI:	10.1093/nar/gkj441
PUBMED:	16428247
PROVIDER:	scopus
PMCID:	PMC1345694
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-32644444603&partnerID=40&md5=1dc5b5ce8f5728fbbbdef7bf2108fb77
Notes:	--- - "Cited By (since 1996): 20" - "Export Date: 4 June 2012" - "CODEN: NARHA" - "Source: Scopus"

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