Two Functionally Distinct Steps Mediate High Affinity Binding of U1A Protein to U1 Hairpin II RNA

David G. Myszka,Phinikoula S. Katsamba,Ite A. Laird-Offringa

doi:10.1074/jbc.m101624200

David G. Myszka, Phinikoula S. Katsamba + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m101624200

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Abstract

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or 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 electrostatic interactions and a subsequent locking step based on close-range interactions 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 high affinity RNA/protein interactions.

Highlights

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction
Using the previously solved structure of the U1A1⁄7U1hpII complex, we have engineered a series of mutants designed to individually examine the roles of electrostatics, hydrogen bonding, aromatic stacking, and RNA loop length, all of which have been implicated in formation of the U1A1⁄7U1hpII complex (5–16)
We used a 101-amino acid N-terminal U1A fragment (referred to here as U1A; previously shown to be required and sufficient for specific, high affinity binding to U1hpII RNA (27, 28); see Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Construction of the U1A Mutants and Protein Purification—The expression plasmid for the human recombinant U1A protein (amino acids 1–101) was described previously (20). Using this plasmid, a U1A clone with a collection of engineered restriction sites throughout the coding region (U1A-MSHEB) was made by site-directed mutagenesis. All engineered restriction sites were silent at the amino acid level, except a BssHII site that resulted in a Lys Arg substitution Proteins from both plasmids had identical binding properties (data not shown). Data were fit to a simple 1:1 Langmuir interaction model with a correction for mass transport (23) using the global data analysis program CLAMP (24)

RESULTS AND DISCUSSION

Effect of NaCl on binding

RNA loop deletions