Single-molecule mechanical folding and unfolding kinetics of armless mitochondrial tRNAArg from Romanomermis culicivorax

The mechanical stability of tRNAs contributes to their biological activities. The mitochondrial tRNAArg from Romanomermis culicivorax is the shortest tRNA ever known. This tRNA lacks D- and T- arms, represents a stem-bulge-stem architecture but still folds into a stable tertiary structure. Although its structure had been reported, studies on its mechanical folding and unfolding kinetic characteristics are lacking. Here, we directly measured the single-molecule mechanical folding and unfolding kinetics of the armless mt tRNAArg by using optical tweezers in different solution conditions. We revealed a two-step reversible unfolding pathway: the first and large transition corresponds to the unfolding of acceptor stem and bulge below 11 pN, and the second and small transition corresponds to the unfolding of anticodon arm at 12-14 pN. Moreover, the free energy landscapes of the unfolding pathways were reconstructed. We also demonstrated that amino acid-chelated Mg2+(aaCM), which mimics the intracellular solution condition, stabilizes the bulge of mitochondrial tRNAArg possibly by reducing the topological constraints or stabilizing the possible local non-canonical base pairings within the bulge region. Our study revealed the solution-dependent mechanical stability of an armless mt tRNA, which may shed light on future mt tRNA studies.