Riboswitches are RNA elements that are involved in the regulation of genes. They bind small metabolites and undergo upon metabolite binding conformational changes that switches the corresponding genes on or off. The Guanidine-II Riboswitch binds guanidine and is composed out-off two stem loops, P1 and P2, connected via a single strand. X-ray crystallography on the isolated P1 and P2 stem loops revealed that they form homodimers (P1)2 and (P2)2 with the guanidine bound between the loops. This lead to the working hypothesis, that binding of the guanidine to the full-length guanidine-II riboswitch induces intraRNA P1P2 contacts with the guanidine bound between both loops.
We set out to investigate this in solution by a combination of site directed spin labelling and Pulsed Electron-Electron Double Resonance (PELDOR or DEER). The labelling was done via click chemistry in solution and RNA strand ligation in case of the full-length riboswitch. We find that the isolate stem loops P1 and P2 occur in form of duplexes or single strands in the absence of ligand. Adding guanidine leads in both cases to the formation of the homodimers (P1)2 and (P2)2. Mixing both isolated stem loops induces the formation of duplexes and of the heterodimeric (P1P2) complex after adding guanidine. In contrast, single and double nitroxide labelling of the full-length riboswitch reveals that predominantly P1P2 contacts are formed upon adding guanidine.
This shows that PELDOR in combination with site directed spin labelling and ligation strategies is a valuable tool to follow ligand induced conformational changes of riboswitches.
Acknowledgment: Funding from the DFG via SPP1601 is gratefully acknowledged.