nascentome analysis uncovers futile protein synthesis in escherichia colinascentome分析揭示了这些徒劳的大肠杆菌的蛋白质合成.pdfVIP

Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli 1 2 1 1 3 2 Koreaki Ito *, Yuhei Chadani , Kenta Nakamori , Shinobu Chiba , Yoshinori Akiyama , Tatsuhiko Abo 1 Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan, 2 Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, 3 Institute for Virus Research, Kyoto University, Kyoto, Japan Abstract Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a ‘‘nascentome.’’ We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDSin two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied