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

Sci Rep 7 (1) 6709 [2017-07-27; online 2017-07-27]

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.

PubMed 28751745

DOI 10.1038/s41598-017-06991-6

Crossref 10.1038/s41598-017-06991-6

pmc: PMC5532260
pii: 10.1038/s41598-017-06991-6