Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites

Abstract Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAla ugc drop off in bioch...

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Autores principales:	Jinfan Wang, Anthony C. Forster
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4dc37467c9fb4614b130d381bb418576
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spelling	oai:doaj.org-article:4dc37467c9fb4614b130d381bb4185762021-12-02T12:32:59ZTranslational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites10.1038/s41598-017-06991-62045-2322https://doaj.org/article/4dc37467c9fb4614b130d381bb4185762017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06991-6https://doaj.org/toc/2045-2322Abstract Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAla ugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAla ugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAla ugc and the translocation of the P-site deacylated tRNAAla ugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.Jinfan WangAnthony C. ForsterNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jinfan Wang Anthony C. Forster Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
description	Abstract Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAla ugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAla ugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAla ugc and the translocation of the P-site deacylated tRNAAla ugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.
format	article
author	Jinfan Wang Anthony C. Forster
author_facet	Jinfan Wang Anthony C. Forster
author_sort	Jinfan Wang
title	Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
title_short	Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
title_full	Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
title_fullStr	Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
title_full_unstemmed	Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
title_sort	translational roles of the c75 2′oh in an in vitro trna transcript at the ribosomal a, p and e sites
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/4dc37467c9fb4614b130d381bb418576
work_keys_str_mv	AT jinfanwang translationalrolesofthec752ohinaninvitrotrnatranscriptattheribosomalapandesites AT anthonycforster translationalrolesofthec752ohinaninvitrotrnatranscriptattheribosomalapandesites
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Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites

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