Dynamically probing ATP-dependent RNA helicase A-assisted RNA structure conversion using single molecule fluorescence resonance energy transfer.

RNA helicase A (RHA) as a member of DExH-box subgroup of helicase superfamily II, participates in diverse biological processes involved in RNA metabolism in organisms, and these RNA-mediated biological processes rely on RNA structure conversion. However, how RHA regulate the RNA structure conversion was still unknown. In order to unveil the mechanism of RNA structure conversion mediated by RHA, single molecule fluorescence resonance energy transfer was adopted to in our assay, and substrates RNA were from internal ribosome entry site of foot-and-mouth disease virus genome. We first found that the RNA structure conversion by RHA against thermodynamic equilibrium in vitro, and the process of dsRNA YZ converted to dsRNA XY through a tripartite intermediate state. In addition, the rate of the RNA structure conversion and the distribution of dsRNA YZ and XY were affected by ATP concentrations. Our study provides real-time insight into ATP-dependent RHA-assisted RNA structure conversion at the single molecule level, the mechanism displayed by RHA may help in understand how RHA contributes to many biological functions, and the basic mechanistic features illustrated in our work also underlay more complex protein-assisted RNA structure conversions.