During translation, ribosomes stall on
mRNA when the aminoacyl-
tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA
codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (
peptides of up to 6
amino acid residues, oligopep-)-
tRNA or polypeptidyl (
peptides of more than 300
amino acids in length, polypep-)-
tRNA associated with ribosomes. Stalling was achieved by
starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-
galactosidase) reporter gene harboring contiguous AGA
codons close to the
initiation codon or at internal
codon positions together with minigene ATGAGATAA accompanied by reduced
peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of
peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3),
ribosome recycling factor (RRF), Pth, and transfer-
messenger RNA (
tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the
peptide in the
peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of
peptidyl-tRNA, while RF1, RF2 (in the absence of
termination codon), or Pth may rescue by hydrolyzing the associated
peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of
tRNA and
mRNA by RRF and
elongation factor G.