Abstract
Polypeptidyl-tRNAs are indispensible intermediates in protein synthesis. They are present in all productive translation reactions, in which the growing nascent chain remains attached to tRNA throughout elongation. In some cases, polypeptidyl-tRNA accumulates as the dead-end product of aberrant translation. Detection of polypeptidyl-tRNAs in the cell is of vital importance for studying the dynamics of translation. However, current experimental approaches largely disregard polypeptidyl-tRNAs as a target of analysis, even though co-translational protein targeting, folding, modification, and other maturation/quality control events draw increasing attention. Now may be the time to revisit the role of these intermediates to reveal snapshots of the elongation process, in which genetic information is translated into covalent connectivity of amino acids through dynamic and regulated molecular interactions involving the ribosome. In this short chapter, I propose that the chemical trait of having a covalently attached tRNA moiety can be used to detect and profile the global complement of nascent polypeptide chains in the cell, the “nascentome.” Accordingly, I describe a simple method to separate cellular proteins into two electrophoretically resolved lines, one containing completed polypeptides with no tRNA attachment (proteome members) and the other containing nascent polypeptides linked to tRNA in the cell at their C-terminus (nascentome members). Specific detection of nascent polypeptides enables the analysis of global patterns of polypeptide chain growth as well as elongation profiles of individual proteins of the cell.
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Acknowledgments
We thank Allen Buskirk for critical reading and editing of the manuscript, Yuhei Chadani for sharing the unpublished results of polypeptidyl-tRNA profiling of cloned E. coli genes, and Sinobu Chiba for discussion. The experiments conducted in the author’s laboratory were supported by grants from the Ministry of Education, Culture, Sports, Science & Technology, Japan (MEXT)-Japan Society for Promotion of Science (JSPS) Grants-in-Aid for Scientific Research and by funds from the Private University Strategic Research Foundation Support Program from MEXT.
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Ito, K. (2014). Analyzing the Nascentome (Polypeptidyl-tRNAs), the Dynamic Hub of Translation. In: Ito, K. (eds) Regulatory Nascent Polypeptides. Springer, Tokyo. https://doi.org/10.1007/978-4-431-55052-5_8
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