A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell

Abstract

AbstractLife depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains; i.e., polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods for profiling nascent chains such as ribosome profiling.Significance StatementIn the central dogma of biology, RNA and protein are usually regarded as two completely independent molecular species. However, they are combined into a single species called peptidyl-tRNA (pep-tRNA) during the translation process in the ribosome. Despite the importance of pep-tRNAs as precursors of all proteins in the cell, a general method to analyze pep-tRNAs on a large scale was lacking. Taking advantage of the properties of pep-tRNAs as RNA and protein, we developed a method to enrich the pep-tRNAs by organic solvent extraction and silica column separation. The method, termed PETEOS, not only provides a unique approach to examine the nascent state of proteins but also may be effective in capturing snapshots of translation status in the cell.