Hall K B, Stump W T
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110.
Nucleic Acids Res. 1992 Aug 25;20(16):4283-90. doi: 10.1093/nar/20.16.4283.
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.
U1A蛋白是一种序列特异性RNA结合蛋白，存在于U1 snRNP颗粒中，它与U1 snRNA的茎环II结合。U1A包含两个“RNP”或“RRM”（RNA识别基序）结构域，这在许多RNA结合蛋白中很常见。已证明N端RRM能特异性结合U1 RNA茎环，而C端结构域的RNA靶标未知。在此，我们描述了使用U1A的1个102个氨基酸的N端RRM（102A）和一个25个核苷酸的RNA茎环来测量该RNA：蛋白质复合物的结合常数和热力学参数的实验。使用硝酸纤维素滤膜结合法，我们在室温下于250 mM NaCl、2 mM MgCl2和10 mM二甲胂酸钠（pH 6）中测得解离常数KD = 2×10^(-11) M，复合物的半衰期为5分钟。在这些条件下，该复合物的缔合自由能（ΔG°）约为 -14 kcal/mol。对结合的盐依赖性的测定表明，复合物形成时至少形成了8个离子对。RNA环序列中的一个突变使亲和力降低了10倍，约占总自由能的10%。
