Multistep Kinetics of the U1A–SL2 RNA Complex Dissociation

The U1A–SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A–SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of ∼ 100 μs, ∼ 50 ms, and ∼ 2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A–SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM–RNA complex.

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► We studied the dissociation of the U1A protein–SL2 RNA complex using T-jump and stopped-flow fluorescence methods. Our kinetic data suggest a three-step mechanism of dissociation. Each step corresponds to a conformational change of the U1A protein. Our results contribute to an understanding of the complex dynamic processes involved in RRM–RNA association and dissociation.