Unexpected experimental outcomes led us to hypothesize that synonymously recoding segments of the M. tuberculosis (Mtb) rpoB gene (rv0667) caused the translation of many smaller protein isoforms from translation initiations sites within the recoded open reading frame (ORF). To test this hypothesis, we labeled the N-termini of Mtb rpoB expressed heterologously in the closely related model system M. smegmatis and identified peptides of rpoB carrying the N-terminal label by mass spectrometry. We found an increase in the detection of N-terminally labeled peptides that mapped to recoded regions of the rpoB gene variants, and thus inferred that recoding was linked to the generation of translation initiation sites within the ORF. We also observed a smaller number of N-terminally labeled peptides mapping to the middle of the wild-type ORF, which we interpreted to indicate that some intragenic translation initiation could possibly occur in the native gene or, more likely, arise from heterologous gene expression.
[doi:10.25345/C5Z02ZD1D]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: mycobacterium ; intragenic transcription initiation ; N-terminal labeling ; RpoB
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Beatrix Ueberheide, NYU School of Medicine, USA |
Submitting User: | Trixi |
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