RIBOSOME RELEASE FACTOR RF4 AND TERMINATION FACTOR RF3 ARE INVOLVED IN DISSOCIATION OF PEPTIDYL-TRANSFER-RNA FROM THE RIBOSOME

Peptidyl-tRNA dissociation from ribosomes is an energetically costly b ut apparently inevitable process that accompanies normal protein synth esis, The drop-off products of these events are hydrolysed by peptidyl -tRNA hydrolase, Mutant selections have been made to identify genes in volved in the drop-off of peptidyl-tRNA, using a thermosensitive pepti dyl-tRNA hydrolase mutant in Escherichia coli, Transposon insertions u pstream of the frr gene, which encodes RF4 (ribosome release or recycl ing factor), restored growth to this mutant, The insertions impaired e xpression of the frr gene, Mutations inactivating prfC, encoding RF3 ( release factor 3), displayed a similar phenotype, Conversely, producti on of RF4 from a plasmid increased the thermosensitivity of the peptid yl-tRNA hydrolase mutant, In vitro measurements of peptidyl-tRNA relea se from ribosomes paused at stop signals or sense codons confirmed tha t RF3 and RF4 were able to stimulate peptidyl-tRNA release from riboso mes, and showed that this action of RF4 required the presence of trans location factor EF2, known to be needed for the function of RF4 in rib osome recycling, When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displac e peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.