title: Spectrophotometric assays for monitoring tRNA aminoacylation and aminoacyl-tRNA hydrolysis reactions.
abstract: Aminoacyl-tRNA synthetases play a central role in protein synthesis, catalyzing the attachment of amino acids to their cognate tRNAs. Here, we describe a spectrophotometric assay for tyrosyl-tRNA synthetase in which the Tyr-tRNA product is cleaved, regenerating the tRNA substrate. As tRNA is the limiting substrate in the assay, recycling it substantially increases the sensitivity of the assay while simultaneously reducing its cost. The tRNA aminoacylation reaction is monitored spectrophotometrically by coupling the production of AMP to the conversion of NAD+to NADH. We have adapted the tyrosyl-tRNA synthetase assay to monitor: (1) aminoacylation of tRNA by l- or d-tyrosine, (2) cyclodipeptide formation by cyclodipeptide synthases, (3) hydrolysis of d-aminoacyl-tRNAs by d-tyrosyl-tRNA deacylase, and (4) post-transfer editing by aminoacyl-tRNA synthetases. All of these assays are continuous and homogenous, making them amenable for use in high-throughput screens of chemical libraries. In the case of the cyclodipeptide synthase, d-tyrosyl-tRNA deacylase, and post-transfer editing assays, the aminoacyl-tRNAs are generated in situ, avoiding the need to synthesize and purify aminoacyl-tRNA substrates prior to performing the assays. Lastly, we describe how the tyrosyl-tRNA synthetase assay can be adapted to monitor the activity of other aminoacyl-tRNA synthetases and how the approach to regenerating the tRNA substrate can be used to increase the sensitivity and decrease the cost of commercially available aminoacyl-tRNA synthetase assays.
authors: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, 1501 Kings Highway, Shreveport, LA 71130, United States. Electronic address: efirst@lsuhsc.edu.
authors: Eric A
authors: EA
authors: First
authors: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, 1501 Kings Highway, Shreveport, LA 71130, United States.
authors: Charles J
authors: CJ
authors: Richardson
mesh_terms: D000249:Adenosine Monophosphate / Q000096:biosynthesis*; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009243:NAD / Q000378:metabolism; D010456:Peptides, Cyclic / Q000096:biosynthesis; D012365:RNA, Transfer, Tyr / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013237:Stereoisomerism; D046249:Transfer RNA Aminoacylation; D014443:Tyrosine / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
chemical_list: D010456:Peptides, Cyclic; D012365:RNA, Transfer, Tyr; D011994:Recombinant Proteins; D009243:NAD; D000249:Adenosine Monophosphate; D014443:Tyrosine; D019881:Aminoacyltransferases; C120167:D-tyrosine tRNA(Tyr) deacylase; D014448:Tyrosine-tRNA Ligase
keywords: Aminoacyl-tRNA synthetase; Cyclodipeptide synthase; High-throughput screen; Post-transfer editing; Spectrophotometric assay; d-tyrosyltRNA deacylase