A protonated cytidine stabilizes the ligand-binding pocket in the preQ1 riboswitch in thermophilic bacteria.

Abstract

Riboswitches are bacterial mRNA structure elements regulating either transcription or translation of downstream genes in response to high-affinity binding of a low molecular weight ligand. Among this diverse group of RNA structures, the class-I preQ1sensing riboswitches (QSW) stand out since they are the smallest known natural riboswitches. QSW combine ligand sensing and functional control within a single structural domain that adopts a pseudoknot conformation encapsulating both the cognate ligand and the ribosome binding site. QSW also occur in thermophilic bacteria. In these cases, their tertiary structures have to be stable even at temperatures above 60 °C to be functional at the organism's optimal growth temperatures. Despite the available high-resolution structures of these riboswitches, it is not yet understood which tertiary interactions are primarily responsible for their exceptional temperature stability. Here, we show that an intricate three-dimensional network of non-canonical interactions involving various non-neighboring nucleobases is the origin of the riboswitch's thermostability. An essential part of this network is a so far undetected stably protonated cytidine. It is characterized by an exceptional high pKA value of >9.7 and could be unambiguously identified through the application of modern heteronuclear detected NMR experiments.