Cargando…

tRNA 3′-amino-tailing for stable amino acid attachment

Amino acids are attached to the tRNA 3′-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and t...

Descripción completa

Detalles Bibliográficos
Autores principales: Gamper, Howard, Hou, Ya-Ming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239183/
https://www.ncbi.nlm.nih.gov/pubmed/30217865
http://dx.doi.org/10.1261/rna.068015.118
_version_ 1783371519540330496
author Gamper, Howard
Hou, Ya-Ming
author_facet Gamper, Howard
Hou, Ya-Ming
author_sort Gamper, Howard
collection PubMed
description Amino acids are attached to the tRNA 3′-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3′-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3′-amino-3′-deoxy A76 in a 3′-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3′-end. This protocol for 3′-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.
format Online
Article
Text
id pubmed-6239183
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Cold Spring Harbor Laboratory Press
record_format MEDLINE/PubMed
spelling pubmed-62391832019-12-01 tRNA 3′-amino-tailing for stable amino acid attachment Gamper, Howard Hou, Ya-Ming RNA Method Amino acids are attached to the tRNA 3′-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3′-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3′-amino-3′-deoxy A76 in a 3′-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3′-end. This protocol for 3′-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities. Cold Spring Harbor Laboratory Press 2018-12 /pmc/articles/PMC6239183/ /pubmed/30217865 http://dx.doi.org/10.1261/rna.068015.118 Text en © 2018 Gamper and Hou; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Gamper, Howard
Hou, Ya-Ming
tRNA 3′-amino-tailing for stable amino acid attachment
title tRNA 3′-amino-tailing for stable amino acid attachment
title_full tRNA 3′-amino-tailing for stable amino acid attachment
title_fullStr tRNA 3′-amino-tailing for stable amino acid attachment
title_full_unstemmed tRNA 3′-amino-tailing for stable amino acid attachment
title_short tRNA 3′-amino-tailing for stable amino acid attachment
title_sort trna 3′-amino-tailing for stable amino acid attachment
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239183/
https://www.ncbi.nlm.nih.gov/pubmed/30217865
http://dx.doi.org/10.1261/rna.068015.118
work_keys_str_mv AT gamperhoward trna3aminotailingforstableaminoacidattachment
AT houyaming trna3aminotailingforstableaminoacidattachment