Mg²⁺ facilitates leader peptide translation to induce riboswitch-mediated transcription termination

We have characterized a 17-residue peptide, MgtL, which is translated specifically in high Mg²⁺ from an open reading frame (ORF) embedded in the Mg²⁺ riboswitch domain, previously identified in the 5′ leader region of Mg²⁺ transporter gene mgtA in Salmonella. We demonstrate that mgtL translation is required to prematurely terminate mgtA transcription. Abrogation of mgtL translation by mutation of its start codon results in transcription of the mgtA-coding region in high Mg²⁺, suggesting that ribosome stalling is not required for preventing premature transcription termination. Consistently, the Mg²⁺ riboswitch responds to cytoplasmic Mg²⁺, but not to proline or arginine, both repeatedly present in the MgtL sequence, to mediate mgtL translation-coupled regulation. RNA structural probing and nucleotide substitution analysis show that the riboswitch loop A region alters base pairing in response to Mg²⁺, and favours stem-loop A1 in high Mg²⁺, subsequently opening the ribosome-binding sequence for mgtL translation. Presumably, mgtL ORF directs translation to localize a ribosome in cis to act on downstream RNA in a manner similar to some upstream ORFs in prokaryotes and eukaryotes.