Metal Dependence of Ligand Binding and Heavy-Atom Derivatization of Evolutionarily Distinct PreQ1 Riboswitches

Abstract

Riboswitches are functional RNA elements located most frequently within the 5′-leader sequences of bacterial mRNAs. By directly binding to small molecules via an aptamer domain, a riboswitch can adapt quickly to changes in the concentration of a specific intracellular ligand, thereby establishing a feedback loop that controls gene expression. Here we discuss methods utilized in the structure determination of evolutionarily distinct classes of preQ1 riboswitches known as class I and II, respectively. These riboswitches “sense” the pyrrolopyrimidine metabolite preQ1—an intermediate on the biosynthetic pathway that produces the hypermodified tRNA base queuosine, which imparts translational fidelity. Herein, we describe (1) the use of isothermal titration calorimetry (ITC) to explore metal ion requirements for ligand binding and (2) modifications to crystallization media containing SO42 − or Na+ that were necessary for phase determination using site-bound Os(NH3)53 + or Cs+ ions, respectively. Our experience has shown that simple manipulations to the mother liquor can lead to favorable binding of the latter ions without the need to engineer metal-binding sites, thus making our methods a first-choice approach that is broadly applicable to functional RNAs.