Spectrophotometric assays for monitoring tRNA aminoacylation and aminoacyl-tRNA hydrolysis reactions.
Methods
; 113: 3-12, 2017 01 15.
Article
en En
| MEDLINE
| ID: mdl-27780756
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tirosina
/
Tirosina-ARNt Ligasa
/
ARN de Transferencia de Tirosina
/
Adenosina Monofosfato
/
Aminoacilación de ARN de Transferencia
/
Pruebas de Enzimas
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2017
Tipo del documento:
Article