Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing
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Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved January 6, 2016 (received for review October 16, 2015)

Significance
The synthesis of a protein takes tens of seconds to a few minutes, in which amino acids are polymerized linearly. Nonuniform progression of this elongation process is thought to be important for the subsequent fates of newly synthesized proteins. However, there have been few attempts to profile elongation intermediates, polypeptidyl–tRNAs, directly. Here we attempted to detect systematically the accumulation of tRNA-linked nascent chain intermediates during the translation of Escherichia coli proteins in vivo and in vitro. The results revealed the widespread occurrence of translational pausing in a manner correlated with the subcellular localization and solubility properties of proteins. Our in vivo/in vitro integrated nascent chain profiling provides groundwork information for our understanding of genetic message translation into functional proteins.
Abstract
Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.
Footnotes
- ↵1To whom correspondence may be addressed. Email: kito{at}cc.kyoto-su.ac.jp or taguchi{at}bio.titech.ac.jp.
Author contributions: Y.C., T.N., S.C., H.T., and K.I. designed research; Y.C., T.N., and K.I. performed research; Y.C., T.N., S.C., H.T., and K.I. analyzed data; and Y.C. and K.I. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
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