Simulations Of Optical Tweezers Experiments Reveal Details Of Rna Structure Unfolding

Genomic RNA can fold into alternative structures with distinct biological functions, such as replication and translation. We have previously identified and characterized a structure of a 3’ cap-independent translation enhancer within the 3′ UTR of the positive strand RNA turnip crinkle virus (TCV). This T-shaped Structure (TSS) is similar in shape and size to a tRNA, despite a distinct secondary structure involving three hairpins and two co-axially stacked helix-pseudoknot motifs at the 5’-end and the 3’-end of the structure. TSS binds ribosomes and enhances translation, but upon binding to the RNA-dependent RNA-polymerase (RdRp), it can change its conformation and foster viral replication. We present a study of the unfolding pathway of the extended TCV TSS, combining steered molecular dynamics simulations (SMD) and optical tweezers (OT) experiments. Developing an SMD protocol for a structure of this size and complexity was an important and novel part of the study. Simulations and experiments indicate that the TSS unfolding starts with the 3’-side structures, followed by the long central hairpin, and ends with the 5’-side pseudoknot-hairpin motif. This order could not be deduced from the ranking of the free energies of the TSS motifs. The SMD simulations explained this sequence of unfolding based on the 3D organization of the TSS and formation of transient tertiary interactions that maintain the 5’-end pseudoknot topology and delay its full unfolding until all other structures have opened. The SMD results also explained the differences between the estimated and measured contour lengths for the TCV TSS domains, while mutational studies pinpointed the RdRp binding site. Thus, combined biochemical analysis, single molecule force spectroscopy and SMD simulations yielding complementary information at multiple levels of resolution provide a means for studying complex structures. Funded in part by HHSN261200800001E.