A <i>Mycobacterium tuberculosis</i> 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 <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) 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 <i>Bacillus subtilis ykoK</i> Mbox riboswitch, which acts as a transcriptional "OFF-switch" under high Mg²⁺ conditions. <i>Mtb</i> encodes two Mbox elements with strong similarity to the ykoK Mbox. In the current study, we characterize the Mbox encoded upstream of the <i>Mtb pe20</i> 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 <i>pe20</i> We have annotated this highly conserved uORF <i>rv1805A</i>, 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 <i>Mtb</i>, 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.