RELEASE FACTOR RF3 ABOLISHES COMPETITION BETWEEN RELEASE FACTOR RF1 AND RIBOSOME RECYCLING FACTOR (RRF) FOR A RIBOSOME BINDING-SITE

The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on t he concentrations of release factor RF1 and the ribosome recycling fac tor (RRF) has been studied in vitro. High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduc tion depended on stop codon context. The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF con centration. This indicates that RF1 and RRF have mutually exclusive an d perhaps overlapping binding sites on the ribosome. Addition of relea se factor RF3 to the translation system abolishes the inhibitory effec t of high RF1 concentration and increases the overall rate of ribosome recycling. These data can be explained by a three-step model for term ination where the first step is RF1-promoted hydrolysis of peptidyl-tR NA. The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3. The third step, catalysed by RRF and elongatio n factor G, leads to mobility of the ribosome on mRNA allowing it to e nter a further round of translation. In the absence of RF3, RF1 can re -associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, p reventing RRF from entering the ribosomal A-site and thereby inhibitin g ribosomal recycling. The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vit ro experiments. (C) 1997 Academic Press Limited.