Interaction of RNA Hairpins with the Human U1A N-terminal RNA Binding Domain

The isolated 102 amino acid N-terminal RNA binding domain (RBD) of the human U1A protein specifically interacts with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. This recognition is nucleotide-specific, for substitutions of critical nucleotides in the RNA loop decrease binding affinity up to 10(6)-fold, as measured by nitrocellulose filter binding experiments. The magnitude of the loss of binding free energy with single-nucleotide substitution in the conserved GCA sequence suggests that the interaction between the RBD and RNA occurs through a number of interdependent specific contacts in the complex. 13C and 15N NMR experiments, using isotopically-labeled RNA together with unlabeled protein, show that the chemical shifts of many protons from the bound RNA are substantially different from those of the free RNA, especially in the loop region of the hairpin. All these data suggest that there is a conformational change in the RNA upon formation of the RBD-RNA complex.