Single-molecule mechanical unfolding kinetics of unmodified Saccharomyces cerevisiae tRNAPhe: a hint to the tRNA chaperone-tRNA interaction mechanism

ABSTRACT The biological activity of tRNA is closely related to its mechanical folding properties. Although previous studies focused on the folding and unfolding mechanism of tRNA, its kinetics are largely unknown. In this study, combining optical tweezers and molecule dynamics simulations, we characterized the mechanical folding and unfolding processes of a single unmodified Saccharomyces cerevisiae tRNA phe . We identified the intermediates and pathways for tRNA mechanical folding and unfolding in the presence of Mg 2+ , discovering that the folding/unfolding kinetics of D stem-loop and T stem-loop but not the anti-codon stem-loop significantly affected by their upstream and downstream structures. The cooperative unfolding of the tRNA in the presence of Mg 2+ lead to a large hysteresis between the folding and unfolding pathway, and such hysteresis and unfolding cooperativity are significantly reduced by lowering the Mg 2+ concentration or mutating the nucleotides forming the ‘elbow’ structure. Moreover, both steered molecular dynamics simulation and optical tweezers experiment results support that, formation of tertiary interactions in the elbow region increases energy barriers of the mechanical unfolding pathway, including those in between intermediates, and determines the overall unfolding cooperativity. Our studies may shed light on the detailed tRNA chaperone mechanism of TruB and TrmA.