title: Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing.
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.
authors.affiliation: Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan; Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
authors.forename: Yuhei
authors.initials: Y
authors.lastname: Chadani
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Niwa
authors.affiliation: Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan;
authors.forename: Shinobu
authors.initials: S
authors.lastname: Chiba
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan kito@cc.kyoto-su.ac.jp taguchi@bio.titech.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Taguchi
authors.affiliation: Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan; kito@cc.kyoto-su.ac.jp taguchi@bio.titech.ac.jp.
authors.forename: Koreaki
authors.initials: K
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D014176:Protein Biosynthesis; D011691:Puromycin / Q000494:pharmacology; D012343:RNA, Transfer / Q000235:genetics
chemical_list: D011691:Puromycin; D012343:RNA, Transfer
keywords: nascent polypeptides | Escherichia coli; peptidyl–tRNA; translation pausing
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