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DNA Res. 2016 Jun;23(3):193-201. doi: 10.1093/dnares/dsw008. Epub 2016 Mar 23.

Comprehensive identification of translation start sites by tetracycline-inhibited ribosome profiling.

Author information

1
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan knakahig@pobox.com hmori@gtc.naist.jp.
2
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
3
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
4
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
5
Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Tokyo 156-8502, Japan.
6
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.
7
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
8
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan knakahig@pobox.com hmori@gtc.naist.jp.

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.

KEYWORDS:

N-terminal; TetRP; genome annotation; ribosome profiling; translation initiation

PMID:
27013550
PMCID:
PMC4909307
DOI:
10.1093/dnares/dsw008
[Indexed for MEDLINE]
Free PMC Article

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