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 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.
Katsamba
P S
PS
Norris Cancer Center/Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9176, USA.
Myszka
D G
DG
Laird-Offringa
I A
IA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity
D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; D001120:Arginine; D008239:Lysine