Binding of the 3′ terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF‐G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3′ end of tRNAPhe, which considerably decrease the affinity of E‐site binding, lower the translocation rate up to 40‐fold. Furthermore, 3′‐end modifications lower or abolish the stimulation by P site‐bound tRNA of the GTPase activity of EF‐G on the ribosome. The results suggest that a hydrogen‐bonding interaction of the 3′‐terminal adenine of the leaving tRNA in the E site, most likely base‐pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF‐G on the ribosome. We propose the following molecular model of translocation: after the binding of EF‐G.GTP, the P site‐bound tRNA, by a movement of the 3′‐terminal single‐stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co‐operative interaction between the E site and the EF‐G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.