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-tRNAAlaugc 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 tRNAAlaugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAlaugc and the translocation of the P-site deacylated tRNAAlaugc 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.
Highlights
The 2′-hydroxyl group (2′OH) of the ribose in the backbone of ribonucleic acids (RNAs) is the main difference between RNAs and deoxyribonucleic acids (DNAs)
The ribosome catalyzes the polymerization of amino acids (AAs) in the sequence encoded by the messenger RNA; the site in the ribosome where peptide bond formation occurs, the peptidyl transferase center (PTC), is composed entirely of RNA; AAs are linked to transfer RNAs via ester bonds with the terminal A76 of the tRNAs before they can serve as the substrates of ribosomes[2]
The loss of the P-site H bonds with W2 and with the 2′OH of ribose C2073 of 23S rRNA may be predicted to affect correct positioning of the electrophile and translocation of the A site peptidyl-tRNA to the P site
Summary
The 2′-hydroxyl group (2′OH) of the ribose in the backbone of ribonucleic acids (RNAs) is the main difference between RNAs and deoxyribonucleic acids (DNAs). The lack of modifications and the C75 2′H in the tRNA body showed negligible effect on single incorporation yield of an AA into polypeptide[8,9,10], and such changes were believed to play only minor roles in translation. When incorporating multiple AAs consecutively with the unmodified AA-tRNAAlaugc bearing the 2′H in C75, we found that these supposedly benign changes caused a big reduction in full-length peptide yields[10] by allowing more time for peptidyl-tRNA drop off[11]. This reduction was not due to the lack of tRNA modifications as the www.nature.com/scientificreports/. We use recent ribosome-substrate crystal structures to predict the function of the 2′OH in C75 of the A-, P- and E-site tRNAAlaugc and test these structure-function models by fast kinetics
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