Do the P1 and P2 hairpins of the Guanidine-II riboswitch interact?

Abstract

Riboswitches regulate genes by adopting different structures in responds to metabolite binding. The guanidine-II riboswitch is the smallest representative of the ykkC class with the mechanism of its function being centred on the idea that its two stem loops P1 and P2 form a kissing hairpin interaction upon binding of guanidinium (Gdm+). This mechanism is based on in-line probing experiments with the full-length riboswitch and crystal structures of the truncated stem loops P1 and P2. However, the crystal structures reveal only the formation of the homodimers P1 | P1 and P2 | P2 but not of the proposed heterodimer P1 | P2. Here, site-directed spin labeling (SDSL) in combination with Pulsed Electron-Electron Double Resonance (PELDOR or DEER) is used to study their structures in solution and how they change upon binding of Gdm+. It is found that both hairpins adopt different structures in solution and that binding of Gdm+ does indeed lead to the formation of the heterodimer but alongside the homodimers in a statistical 1:2:1 fashion. These results do thus support the proposed switching mechanism.