Genetic Analysis of Riboswitch-mediated Transcriptional Regulation Responding to Mn2+ in Salmonella

Riboswitches are a class of cis -acting regulatory RNAs normally characterized from the 5′-UTR of bacterial transcripts that bind a specific ligand to regulate expression of associated genes by forming alternative conformations. Here, we present a riboswitch that contributes to transcriptional regulation through sensing Mn 2+ in Salmonella typhimurium . We characterized a 5′-UTR (UTR1) from the mntH locus encoding a Mn 2+ transporter, which forms a Rho-independent terminator to implement transcription termination with a high Mn 2+ selectivity both in vivo and in vitro . Nucleotide substitutions that cause disruption of the terminator interfere with the regulatory function of UTR1. RNA probing analyses outlined a specific UTR1 conformation that favors the terminator structure in Mn 2+ -replete condition. Switch sequence GCUAUG can alternatively base pair duplicated hexanucleotide CAUAGC to form either a pseudoknot or terminator stem. Mn 2+ , but not Mg 2+ , and Ca 2+ , can enhance cleavage at specific nucleotides in UTR1. We conclude that UTR1 is a riboswitch that senses cytoplasmic Mn 2+ and therefore participates in Mn 2+ -responsive mntH regulation in Salmonella . This riboswitch domain is also conserved in several Gram-negative enteric bacteria, indicating that this Mn 2+ -responsive mechanism could have broader implications in bacterial gene expression. Additionally, a high level of cytoplasmic Mn 2+ can down-regulate transcription of the Salmonella Mg 2+ transporter mgtA locus in a Mg 2+ riboswitch-dependent manner. On the other hand, these two types of cation riboswitches do not share similarity at the primary or secondary structural levels. Taken together, characterization of Mn 2+ -responsive riboswitches should expand the scope of RNA regulatory elements in response to inorganic ions. Background: Divalent cation binding to riboswitch RNAs regulates expression of their transporter genes in bacteria. Results: Mn 2+ interacts with Salmonella riboswitches characterized from Mn 2+ transporter mntH and Mg 2+ transporter mgtA to modulate transcription of the downstream coding region. Conclusion: Specific riboswitches control gene expression in response to Mn 2+ in bacteria. Significance: This is the discovery of a Mn 2+ riboswitch.