Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA

Binding of the U1A protein to its RNA target U1A.U1 hairpin II has been ext ensively studied as a model for a high affinity RNA/protein interaction. Ho wever, the mechanism and kinetics by which this complex is formed remain la rgely unknown. Here we use real-time biomolecular interaction analysis to d issect the roles various protein and RNA structural elements play in the fo rmation of the U1A.U1 hairpin II complex. We show that neutralization of po sitive charges on the protein or increasing the salt concentration slows th e association rate, suggesting that electrostatic interactions play an impo rtant role in bringing RNA and protein together. In contrast, removal of hy drogen bonding or stacking interactions within the RNA/protein interface, o r reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electr ostatic interactions and a subsequent locking step based on close-range int eractions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for hig h affinity RNA/protein interactions.