Refolding through a linear transition state enables fast temperature adaptation of a translational riboswitch.

The adenine-sensing riboswitch from the gram-negative bacterium V. vulnificus is an RNA-based gene regulatory element that acts both in response to its cognate low molecular weight ligand and to temperature. The combined sensitivity to environ-mental temperature and ligand concentration is maintained by an equilibrium of three distinct con-formations involving two ligand-free and one lig-and-bound state. The key structural element that undergoes refolding in the ligand-free states com-prises a 35-nucleotide long temperature response module. Here, we present the structural characteri-zation of this temperature response module. We employ high-resolution NMR spectroscopy and photo-caged 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 conforma-tional 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 un-likely. More likely, the transition state of the ade-nine-sensing riboswitch temperature response mod-ule features a linear conformation.