Nucleic Acids Research, 1992, Vol. 20, No. 16 4283-4290
© 1992
MOLECULAR BIOLOGY |
Interaction of N-terminal domain of U1A protein with an RNA stem/loop
Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics Box 8231, 660 S. Euclid Avenue, St Louis, MO 63110, USA
Received April 24, 1992. Revised July 10, 1992. Accepted July 10, 1992.
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 ‘RRMrsquo; (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 KD = 2x10–11 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 10x , or about 10% of the total free energy.
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