Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes

Flanagan, John F.; Namy, Olivier; Brierley, Ian; Gilbert, Robert J. C.

Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site, and from P-site to exit (E) site is catalysed in eukaryotes by the translocase elongation factor 2 (EF-2), and in prokaryotes by its homologue EF-G. During tRNA movement one or more “hybrid” states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy (cryo-EM) that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally-distorted tRNAs in two different A/P hybrid states. In one (A/P’) the tRNA is in contact with the translocase EF-2 which induces it. In the other (A/P”) the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation.