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 synthetases, In this system, ribosomes perform repeated rounds of translation 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 factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is similar to 40 s. This time is reduced 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 factor RF3 is seen to promote RF1 cycling between different ribosomes, The 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 mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome, It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.