Chemical probing profiling of 2'-deoxyguanosine sensing RNA reveals magnesium ion-dependent conformational switching.

Abstract

Maintaining the homeostasis of cellular metabolites is essential for all living organisms. Noncoding mRNA in procaryotes strategically regulates the expression of downstream genes which are linked to the expression of proteins involved in specific metabolite salvage and biosynthesis, in response to binding to that specific metabolite. Such noncoding mRNAs are known as the Riboswitches. A class of riboswitches that recognizes purine analogs were identified. In this present contribution, we report the influence of magnesium and temperature on the conformation of the aptamer domain (the ligand binding domain) of 2’-deoxyguanosine riboswitch from Mesoplasma florum. Our comprehensive SHAPE (Selective 2′ Hydroxyl Acylation analyzed by Primer Extension) probing experiments have shown that the aptamer domain exhibited a strong dependence on the Mg2+ ion concentration for the efficient ligand binding and the concomitant structural change. Mg2+ ions reduce the conformational flexibility mainly by organizing P2 and P3 helices and thereby facilitate ligand binding by shifting the conformational ensemble of the ligand-free aptamer towards a pre-organized, ligand-binding competent conformation. The subtle conformational switching in the presence and absence of 2’-deoxyguanosine is observed at the (A) regulatory helix (P1) and (B) the interhelical junctions (which serve as the ligand binding pocket) of the aptamer. We have observed the formation of a regulatory helix, the folding of the binding pocket, and the subsequent encapsulation of the ligand is accomplished only in the simultaneous presence of Mg2+ and 2’-deoxyguanosine. We have also identified the existence of two conformations in equilibrium in a ligand-free state. Finally, we have shown that for the 2’-dG sensing riboswitch from Mesoplasma forum, the conformation of the full-length transcripts populates the functional OFF-state regardless of the presence or absence of ligand, consistent with a kinetic riboswitch mechanism.