Recycling of Ribosomal Complexes Stalled at the Step of Elongation in Escherichia coli

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation.Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood.We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA- inigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters $tRNA^{Arg4}$ as a short-chain peptidyl-$tRNA^{Arg4}$ in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-$tRNA^{Arg4}$ from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on \lambda immP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs