Three-state mechanism couples ligand and temperature sensing in riboswitches

In the human pathogen Vibrio vulnificus, the riboswitch regulating gene expression of the adenosine deaminase is shown to exist in three distinct stable conformational states; this three-state mechanism allows control of gene expression over a broad temperature range, which is essential for Vibrio adaptation. Riboswitches are defined sequences (aptamers) within an mRNA that bind a ligand, such as a metabolite, in a way that changes the mRNA''s structure and affects expression of the mRNA. It has been thought that riboswitches had only two states, on or off. A new NMR study indicates this view to be too simplistic. Using a riboswitch found in organisms that exist in environments over a range of temperatures, Harald Schwalbe and colleagues found that the adenine-sensing riboswitch encoded by the add gene of the human pathogen Vibrio vulnificus can exist in three states: on, off and a third position that allows tighter control of expression by simultaneously monitoring both temperature and ligand concentration. Riboswitches are cis-acting gene-regulatory RNA elements that can function at the level of transcription, translation and RNA cleavage1,2,3. The commonly accepted molecular mechanism for riboswitch function proposes a ligand-dependent conformational switch between two mutually exclusive states4. According to this mechanism, ligand binding to an aptamer domain induces an allosteric conformational switch of an expression platform, leading to activation or repression of ligand-related gene expression5. However, many riboswitch properties cannot be explained by a pure two-state mechanism. Here we show that the regulation mechanism of the adenine-sensing riboswitch, encoded by the add gene on chromosome II of the human Gram-negative pathogenic bacterium Vibrio vulnificus6, is notably different from a two-state switch mechanism in that it involves three distinct stable conformations. We characterized the temperature and Mg2+ dependence of the population ratios of the three conformations and the kinetics of their interconversion at nucleotide resolution. The observed temperature dependence of a pre-equilibrium involving two structurally distinct ligand-free conformations of the add riboswitch conferred efficient regulation over a physiologically relevant temperature range. Such robust switching is a key requirement for gene regulation in bacteria that have to adapt to environments with varying temperatures. The translational adenine-sensing riboswitch represents the first example, to our knowledge, of a temperature-compensated regulatory RNA element.