Mg 2+ On-Off Switch

Abstract

Mg 2+ , a ubiquitous divalent cation, is required for the activity of many enzymes and stabilizes RNA structures and membranes. Because of its importance, organisms have mechanisms for controlling the intracellular concentration of Mg 2+ ([Mg 2+ ] i ). In Salmonella enterica serovar Typhimurium, decreased periplasmic Mg 2+ concentrations are sensed by the PhoQ protein, which triggers the phosphorylation of the PhoP transcription factor, thereby promoting the expression of genes encoding Mg 2+ transporters, mtgA and mtgB . However, expression of mtgA is also stimulated by low [Mg 2+ ] i in strains defective in the PhoP/PhoQ system. Cromie et al. show that Mg 2+ triggers changes in the structure of the 5' untranslated region (5′-UTR) of the mtgA transcript, which in high [Mg 2+ ] block transcription elongation and in low [Mg 2+ ] allow transcription through the coding region. Using quantitative RT-PCR (reverse transcription polymerase chain reaction) experiments, the authors showed that the 5′-UTR was detectable at higher [Mg 2+ ] than was the coding region. Substitution of 100 base pairs of the 5′-UTR with an irrelevant sequence resulted in constitutive mtgA expression in a strain in which PhoP was constitutively active. Transfer of the 5′-UTR from mtgA to a LacZ reporter resulted in stimulation of β-galactosidase activity in low [Mg 2+ ]. Secondary structure modeling suggested that a two-loop structure (stem loops A and B) was adopted under high [Mg 2+ ] conditions and a single-loop structure (stem loop C) was adopted under low [Mg 2+ ] conditions. These structures were supported by RNase and chemical adduct experiments, which showed differential accessibility to residues depending on [Mg 2+ ]. Further disruption of specific stem loop structures by placement of a derivative of the lac promoter at different positions in the 5′-UTR of mtgA in front of LacZ showed that when the formation of stem loop C was blocked all expression was blocked, and when the formation of stem loop A or all three stem loops was blocked, then expression was independent of [Mg 2+ ]. Point mutations in the 5′-UTR to disrupt specific stem loops also supported the model that stem loop A formed the Mg 2+ sensor such that when [Mg 2+ ] was high, stem loops A and B were present (and transcription was blocked) and when [Mg 2+ ] was low, stem loop C was present (and transcription proceeded). These proposed structures were also predicted for mtgA 5′-UTR from six other Gram-negative bacteria, suggesting that this Mg 2+ switch may be a common mechanism for regulating the abundance of these Mg 2+ transporters. M. J. Cromie, Y. Shi, T. Latifi, E. A. Groisman, An RNA sensor for intracellular Mg 2+ . Cell 125 , 71-84 (2006). [Online Journal]