FAST RECYCLING OF ESCHERICHIA-COLI RIBOSOMES REQUIRES BOTH RIBOSOME RECYCLING FACTOR (RRF) AND RELEASE FACTOR RF3

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl-tRNA syn thetases, In this system, ribosomes perform repeated rounds of transla tion of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured, The system has been used to study the influence of release factor RF3 and of ribosome recycling f actor RRF on the rate of recycling of ribosomes. In the absence of bot h RF3 and RRF, the recycling time is similar to 40 s. This time is red uced to similar to 30 s by the addition of RF3 alone and to similar to 15 s by the addition of RRF alone, When both RF3 and RRF are added to the translation system, the recycling time drops to < 6 s. Release fa ctor RF3 is seen to promote RF1 cycling between different ribosomes, T he action of RRF is shown to depend on the concentration of elongation factor-G, Even in the presence of RRF, ribosomes do not leave the mRN A after termination, but translate the same mRNA several times. This s hows that RRF does not actively eject mRNA from the terminating riboso me, It is proposed that terminating ribosomes become mobile on mRNA an d ready to enter the next translation round only after two distinct st eps, catalysed consecutively by RF3 and RRF, which are slow in the abs ence of these factors.