Hall K B
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1994 Aug 23;33(33):10076-88. doi: 10.1021/bi00199a035.
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.
人U1A蛋白分离出的102个氨基酸的N端RNA结合结构域（RBD）与含有U1 snRNA茎/环II序列的短RNA发夹特异性相互作用。这种识别是核苷酸特异性的，通过硝酸纤维素滤膜结合实验测定，RNA环中关键核苷酸的取代会使结合亲和力降低多达10^6倍。保守的GCA序列中单核苷酸取代导致的结合自由能损失程度表明，RBD与RNA之间的相互作用是通过复合物中许多相互依赖的特异性接触发生的。使用同位素标记的RNA和未标记的蛋白质进行的13C和15N NMR实验表明，结合RNA中许多质子的化学位移与游离RNA的化学位移有很大不同，尤其是在发夹的环区域。所有这些数据表明，RBD-RNA复合物形成时RNA会发生构象变化。
