A Mycobacterium tuberculosis Mbox controls a conserved, small upstream ORF via a translational expression platform and Rho-dependent termination of transcription.

Magnesium is vital for bacterial survival, and its homeostasis is tightly regulated. Intracellular pathogens like Mycobacterium tuberculosis (Mtb) often face host-mediated magnesium limitation, which can be counteracted by upregulating the expression of Mg2+ transporters. This upregulation may be via Mg2+-sensing regulatory RNA such as the Bacillus subtilis ykoK Mbox riboswitch, which acts as a transcriptional "OFF-switch" under high Mg2+ conditions. Mtb encodes two Mbox elements with strong similarity to the ykoK Mbox. In the current study, we characterize the Mbox encoded upstream of the Mtb pe20 operon, which is required for growth in low Mg2+/low pH. We show that this switch operates via a translational expression platform and Rho-dependent transcription termination, which is the first such case reported for an Mbox. Moreover, we show that the switch directly controls a small ORF encoded upstream of pe20 We have annotated this highly conserved uORF rv1805A, but its role remains unclear. Interestingly, a homologous gene exists outside the Mbox-regulated context, suggesting functional importance beyond magnesium stress. Overall, this study uncovers a dual mechanism of riboswitch-regulation in Mtb, combining translational control with Rho-mediated transcription termination. These findings expand our understanding of RNA-based gene regulation in mycobacteria, with implications for pathogenesis and stress adaptation.