A Bacterial mRNA Leader that Employs Different Mechanisms to Sense Disparate Intracellular Signals

Abstract

SUMMARY Bacterial mRNAs often contain leader sequences that respond to specific metabolites or ions by altering expression of the associated downstream protein coding sequences. Here we report that the leader RNA of the Mg2+ transporter gene mgtA of Salmonella enterica, which was previously known to function as a Mg2+-sensing riboswitch, harbors an 18-codon proline-rich open reading frame – termed mgtL – that permits intracellular proline to regulate mgtA expression. Interfering with mgtL translation by genetic, pharmacological or environmental means was observed to increase the mRNA levels from the mgtA coding region. Substitution of the mgtL proline codons by other codons abolished the response to proline and to hyperosmotic stress but not to Mg2+. Our findings show that mRNA leader sequences can consist of complex regulatory elements that utilize different mechanisms to sense separate signals and mediate an appropriate cellular response.

Mg 2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

“…The role of mgtL is reminiscent of the regulatory functions of short ORFs in the trp, his, and other amino acid biosynthetic operons, in which the efficiency of translation of the leader peptide regulates termination or read-through of transcription into the rest of the operon (14). However, opposing conclusions were reached in previous work for the role of Pro in the regulation of mgtA expression: Park et al (12) suggested that low levels of Pro-charged tRNA Pro increase expression of mgtA, whereas Zhao et al (13) concluded that Pro has no role in mgtA regulation. Subsequent Significance Mg 2+ is an important signal for the regulation of virulence and thermotolerance in Salmonella enterica.…”

“…Our observation that the replacement of three Pro codons nearly abolished expression of mgtA suggests that the 5′ LR mRNA favors the formation of stem loop B and enables Rho to terminate transcription. Park et al (12) and Zhao et al (13) constructed mutants in which various combinations of Pro codons in mgtL were substituted by other amino acid codons, and they also found that substitutions of Pro codons in mgtL diminished the sensitivity of the expression of mgtA to the Mg 2+ concentration.…”

“…In the wild-type strain carrying the mgtA9226::MudJ insertion, expression of lacZY is low on MacConkey agar, giving rise to the Lac − (white) phenotype. By selecting Lac + (red) mutants, we (12), which governs folding of the 5′ LR mRNA and Rho-dependent termination. At very low intracellular [Mg 2+ ], ribosomes stall during translation of mgtL (inset 2a), enabling the formation of stem loop C in the RNA, which sequesters the Rho-binding site rut and allows transcription to proceed into the mgtA coding region, turning mgtA transcription ON (inset 3a).…”

“…Pro codons and residues are highlighted in yellow. Predicted folding of the mgtA 5′ LR mRNA is based on the MFold web server (12,40). AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; G37, guanine at position 37 in tRNA Pro ; m 1 G37, methylation at N 1 position of G37 in tRNA Pro ; RNA-P, RNA polymerase; RpmA and RpmE, ribosomal proteins L27 and L31, respectively.…”

“…Unaccounted in this model was the presence of a short ORF, called mgtL, in the mgtA 5′ LR that encodes a proline (Pro)-rich leader peptide highly conserved in Enterobacteriaceae ( Fig. 1) (12,13). The role of mgtL is reminiscent of the regulatory functions of short ORFs in the trp, his, and other amino acid biosynthetic operons, in which the efficiency of translation of the leader peptide regulates termination or read-through of transcription into the rest of the operon (14).…”