Quality control of protein synthesis in the early elongation stage

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pth(ts) strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation. Peptidyl-tRNAs (pep-tRNAs) frequently dissociate from ribosome, called as pep-tRNA drop-off. But, its function remained unclear. The authors proposed a new quality control mechanism of protein synthesis by active rejection of miscoded pep-tRNAs in the early stage of translation.

Automated summary

The researchers established a highly sensitive method to profile pep-tRNAs using mass spectrometry, and detected a large number of nascent peptides from pep-tRNAs in Escherichia coli pth(ts) strain. Molecular mass analysis revealed that about 20% of the peptides had single amino-acid substitutions in the N-terminal sequences. Further analysis showed that most of the substitutions occurred at the C-terminal drop-off site, and the miscoded pep-tRNAs rarely participated in the next round of elongation but dissociated from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism for the ribosome to reject miscoded pep-tRNAs in early elongation and contribute to quality control of protein synthesis.