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

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-tRNA(Ala) (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 tRNA(Ala) (ugc). Rather, the peptidyl acceptor activity of the A-site Ala-tRNA(Ala) (ugc) and the translocation of the P-site deacylated tRNA(Ala) (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.