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Serendipitously, the precise position of stalled ribosomes is revealed within our 5\u2032 RNA-seq datasets used to identify and validate the tRNA target of the toxin, precluding the need for Ribo-seq. Here we show how mapping of stalled ribosomes can be harnessed as an innovative tool for reliable detection of new Cys-containing Mtb open reading frames (ORFs). Using proteogenomics we unmasked 96 unannotated ORFs; of which 54% are small ORFs \u226450 amino acids. We validated 69% of the 96 ORFs by mass spectrometry, including four whose spectra was matched to synthetic controls Also, 25% of these unannotated ORFs were identified by previously published Ribo-RET. Some of the 96 ORFs are Cys-responsive attenuators or encode stable Cys-containing proteins that map immediately before, or within, genes in the opposite, or same, orientation. These ORF sequences can also reveal functional clues, e.g. zinc-binding motifs or encode novel EsxB-like proteins. Our findings demonstrate that toxin-mediated ribosome stalling can serve as a robust genome annotation tool that is applicable to mycobacteria and other bacteria, with unique advantages that complement existing genome annotation methods.<\/jats:p>","DOI":"10.1093\/nar\/gkag252","type":"journal-article","created":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T15:53:22Z","timestamp":1773330802000},"source":"Crossref","is-referenced-by-count":1,"title":["Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs"],"prefix":"10.1093","volume":"54","author":[{"given":"Eduardo A","family":"Troian","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School , Piscataway, NJ 08854 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valdir C","family":"Barth","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School , Piscataway, NJ 08854 ,","place":["United States"]},{"name":"Division of Infectious Diseases, Department of Pediatrics, Boston Children\u2019s Hospital, Harvard Medical School , Boston, MA 02115 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Unnati","family":"Chauhan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School , Piscataway, NJ 08854 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiyan","family":"Zheng","sequence":"additional","affiliation":[{"name":"Biological Mass Spectrometry Facility of Robert Wood Johnson Medical School and Rutgers, Rutgers University , Piscataway, NJ 08854 ,","place":["United 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