Ribosome profiling analyses on WT and RRFTs strain
In the early stage of translation elongation, peptidyl-tRNA (pep-tRNA) with short nascent chain frequently dissociates from the ribosome (pep-tRNA drop-off). Inactivation of PTH (peptidyl-tRNA hydrolase) causes rapid accumulation of pep-tRNAs and depletion of aminoacyl(aa)-tRNAs, leading to defective protein synthesis. We established a highly sensitive method for direct profiling of pep-tRNAs accumulated in the cell and identified more than 700 peptide sequences of the pep-tRNAs. In this experiment, to examine the involvement of RRF in pep-tRNA drop-off in the early elongation stage, we performed ribosome profiling of WT and temperature-sensitive RRF strain incubated at 43Â°C for 30 min.