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

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Abstract
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-terminal domain is unknown. Here, we describe experiments using a 102 amino acid N-terminal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:protein complex. Using nitrocellulose filter binding, we measure a dissociation constant KD = 2 x 10(-11) M in 250 mM NaCl, 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 (delta G degrees) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ion-pairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10 x, or about 10% of the total free energy.