Structure-function analysis of yeast tRNA ligase Journal Article
| Authors: | Wang, L. K.; Shuman, S. |
| Article Title: | Structure-function analysis of yeast tRNA ligase |
| 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 catalytic domains - an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5′-kinase/cyclic phosphodiesterase component (amino acids 389-827) - that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery. Copyright © 2005 The RNA Society. |
| Keywords: | unclassified drug; genetics; missense mutation; mutation; nonhuman; molecular genetics; protein domain; protein motif; proteome; animal; metabolism; animals; in vivo study; in vitro study; enzymology; enzyme activity; trypanosoma; structure activity relation; substitution reaction; structure-activity relationship; mutational analysis; chemistry; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; messenger rna; saccharomyces cerevisiae; nucleotide sequence; dna mutational analysis; yeast; saccharomyces cerevisiae proteins; alanine; saccharomyces cerevisiae protein; molecular biology; rna structure; sequence homology; transfer rna; protein family; polydeoxyribonucleotide synthase; glutamic acid; catalytic domain; fungi; trna splicing; lysine; polynucleotide 5' hydroxyl kinase; polynucleotide 5'-hydroxyl-kinase; monomer; rna splicing; amino acid motifs; enzyme active site; lethal gene; genes, lethal; amino acid analysis; phylogeny; adenylyltransferase; nucleotidyltransferase; polypeptide; rna analysis; rna capping; rna ligase; phosphodiesterase; phosphoric diester hydrolases; polynucleotide ligases; polynucleotide ligase; antifungal target; trna ligase 1 [1-388]; trna ligase 1 adenylyltransferase; trna ligase, yeast; leishmania |
| Journal Title: | RNA |
| Volume: | 11 |
| Issue: | 6 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2005-06-01 |
| Start Page: | 966 |
| End Page: | 975 |
| Language: | English |
| DOI: | 10.1261/rna.2170305 |
| PUBMED: | 15923379 |
| PROVIDER: | scopus |
| PMCID: | PMC1370781 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 15" - "Export Date: 24 October 2012" - "CODEN: RNARF" - "Source: Scopus" |
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