Refolding through a Linear Transition State Enables Fast Temperature Adaptation of a Translational Riboswitch.

The adenine-sensing riboswitch from the Gram-negative bacterium Vibrio vulnificus is an RNA-based gene regulatory element that acts in response to both its cognate low-molecular weight ligand and temperature. The combined sensitivity to environmental temperature and ligand concentration is maintained by an equilibrium of three distinct conformations involving two ligand-free states and one ligand-bound state. The key structural element that undergoes refolding in the ligand-free states comprises a 35-nucleotide temperature response module. Here, we present the structural characterization of this temperature response module. We employ high-resolution NMR spectroscopy and photocaged RNAs as molecular probes to decipher the kinetic and thermodynamic framework of the secondary structure transition in the apo state of the riboswitch. We propose a model for the transition state adopted during the thermal refolding of the temperature response module that connects two mutually exclusive long-lived and stable conformational states. This transition state is characterized by a comparatively low free activation enthalpy. A pseudoknot conformation in the transition state, as commonly seen in RNA refolding, is therefore unlikely. More likely, the transition state of the adenine-sensing riboswitch temperature response module features a linear conformation.

Characterization of conformational dynamics of bistable RNA by equilibrium and non-equilibrium NMR.

Unlike proteins, a given RNA sequence can adopt more than a single conformation. The two (or more) conformations are long-lived and have similar stabilities, but interconvert only slowly. Such bi- or multistability is often linked to the biological functions of the RNA. This unit describes how nuclear magnetic resonance (NMR) spectroscopy can be used to characterize the conformational dynamics of bistable RNAs.