The ribosomal elongation cycle: tRNA binding, translocation and tRNA release.

Abstract

Experiments concerning the ribosomal elongation cycle have been performed; the following results were obtained. A measurements of the percentage of tightly coupled ribosomes participating in a tRNA binding reaction is obtained by determining both the stoichiometry of binding and the site location. In the case of AcPhe‐tRNA it is sufficient to determine the maximal binding since at moderate Mg2+ concentration (≤ 15 mM) one ribosome cannot bind more than one AcPhe‐tRNA molecule, which can be present at either the A or P site (exclusion principle). tRNA binding to poly(U)‐programmed ribosomes does not follow the predictions of simple models with rigid tRNA binding sites as postulated by the classical A/P site model. For example, 2.7 molecules of tRNAPhe are found per ribosome at a ribosome concentration of 0.33 μM and a [14C]tRNAPhe excess over ribosomes of about 40‐fold, in agreement with our previous report. At a 70S ribosome concentration of 3.3 μM and a sevenfold excess of tRNAPhe a binding of 2.5 tRNA molecules/ribosome would be expected according to the binding constants reported by us, but in fact a binding of only 1.8 molecules/70S ribosome is found. These findings explain the current discrepancies concerning the number of tRNA sites on the ribosome and confirm previous reports that three tRNA molecules can be bound simultaneously to one ribosome. The ternary complex EF‐Tu · GTP · Phe‐tRNAPhe binds to the A site at 0°C even if the P site is not occupied. Only if the P site is free does the addition of EF‐G provoke a translocation to the P site at 0°C, whereas at 37°C the Phe‐tRNAPhe‘slides’ from the A to the P site without EF‐G. This EF‐G‐independent sliding is not observed if the P site is filled by a deacylated tRNAPhe. Thus, EF‐G is directly involved in the translocation reaction, and its role is not restricted to tRNA release. Kinetics of the puromycin reaction with Phe‐tRNA and AcPhe‐tRNA, respectively, were compared. The initial rate of AcPhe‐puromycin formation exceeds that of Phe‐puromycin formation by a factor of 3.6 (0°C, 15 mM Mg2+). Poly(U)‐programmed ribosomes carrying only [14C]tRNA release tRNA upon addition of EF‐G. tRNA release is observed only if at least two tRNAPhe molecules are present per ribosome. The tRNA bound first is released with a higher probability than that bound second. Such a complex is not related to a classical pretrans‐location complex carrying a peptidyl‐tRNA at the A site. Therefore, when tRNA release during the course of an EF‐G‐induced translocation reaction is studied, care must be taken that no significant amounts of the complex 70S · mRNA · (deacylated tRNA) are present.