Translation of a histone H3 tail as a model system for studying peptidyl-tRNA drop-off

► The site of peptidyl-tRNA drop-off could be determined using histone H3 tail. ► Peptidyl-tRNA drop-off followed by translation continuation is suggested. ► Peptidyl-tRNA drop-off depends heavily on the amino acid contexts. We found that the synthesis of histone H3 N-terminal peptide (tail) in a reconstituted protein synthesis system yielded fragmented peptides along with the full-length product. With the combined use of MALDI-TOF analysis and peptidyl-tRNA hydrolase cleavage of the Flag tagged product species, we concluded that the fragments were generated by peptidyl-tRNA drop-off at specific sites and subsequent translation continuation. Using the histone H3 tail we also found that peptidyl-tRNA drop-off is strongly correlated with the amino acid context. We envision that the system described here would be useful as a model system for studying peptidyl-tRNA drop-off events.