Interaction of N-terminal domain of U1A protein with an RNA stem/loop

The U1A protein is a sequence-specific RNA binding protein found in the U1 snRNP particle where it binds to stem/loop II of U1 snRNA. U1A contains two 'RNP' or 'RRM' (RNA Recognition Motif) domains, which are common to many RNA-binding proteins. The N-terminal RRM has been shown to bind specifically to the U1 RNA stem/loop, while the RNA target of the C-termlnal domain is unknown. Here, we describe experiments using a 102 amino acid N-termlnal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:proteln complex. Using nitrocellulose filter binding, we measure a dissociation constant K_D = 2×10^-11 M in 250 mM NaCI, 2 mM MgC₂, and 10 mM sodium cacodylate, pH 6 at room temperature, and a half-life for the complex of 5 minutes. The free energy of association (AG°) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ionpairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10× , or about 10% of the total free energy.