title: Comprehensive identification of translation start sites by tetracycline-inhibited ribosome profiling.
abstract: Tetracycline-inhibited ribosome profiling (TetRP) provides a powerful new experimental tool for comprehensive genome-wide identification of translation initiation sites in bacteria. We validated TetRP by confirming the translation start sites of protein-coding genes in accordance with the 2006 version of Escherichia coli K-12 annotation record (GenBank U000962) and found ∼150 new start sites within 60 nucleotides of the annotated site. This analysis revealed 72 per cent of the genes whose initiation site annotations were changed from the 2006 GenBank record to the newer 2014 annotation record (GenBank U000963), indicating a high sensitivity. Also, results from reporter fusion and proteomics of N-terminally enriched peptides showed high specificity of the TetRP results. In addition, we discovered over 300 translation start sites within non-coding, intergenic regions of the genome, using a threshold that retains ∼2,000 known coding genes. While some appear to correspond to pseudogenes, others may encode small peptides or have previously unforeseen roles. In summary, we showed that ribosome profiling upon translation inhibition by tetracycline offers a simple, reliable and comprehensive experimental tool for precise annotation of translation start sites of expressed genes in bacteria.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan knakahig@pobox.com hmori@gtc.naist.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Nakahigashi
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takai
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Abe
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayashiki
authors.affiliation: Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Tokyo 156-8502, Japan.
authors.forename: Yuh
authors.initials: Y
authors.lastname: Shiwa
authors.affiliation: Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Tokyo 156-8502, Japan Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yoshikawa
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Barry L
authors.initials: BL
authors.lastname: Wanner
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan knakahig@pobox.com hmori@gtc.naist.jp.
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
mesh_terms: D018387:Codon, Initiator; D004926:Escherichia coli / Q000235:genetics*; D058977:Molecular Sequence Annotation; D016366:Open Reading Frames; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D018387:Codon, Initiator; D011500:Protein Synthesis Inhibitors; D013752:Tetracycline
keywords: N-terminal; TetRP; genome annotation; ribosome profiling; translation initiation