Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs

The Mycobacterium tuberculosis (Mtb) VapC4 endoribonuclease toxin exclusively cleaves and inactivates tRNACys, which leads to extensive ribosome stalling at Cys codons. Serendipitously, the precise position of stalled ribosomes is revealed within our 5' 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 ≤50 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.