Schmedt C, Green S R, Manche L, Taylor D R, Ma Y, Mathews M B
Cold Spring Harbor Laboratory, NY 11724, USA.
J Mol Biol. 1995 May 26;249(1):29-44. doi: 10.1006/jmbi.1995.0278.
The double-stranded (ds) RNA-activated protein kinase, DAI (also known as PKR), contains an RNA-binding domain comprising two tandem repeats of a motif, the dsRBM, which is shared with a number of other proteins that interact with structured RNAs. We have expressed the entire domain and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to study their interactions with RNA and with the intact kinase enzyme. Both p20 and p10 bound preferentially to structured RNA molecules. Competition assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than the entire domain. Measurement of the dissociation constants for dsRNA by quantitative gel mobility shift analysis gave apparent Kd values of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively. The binding of p20 molecules to dsRNA appeared to be cooperative. Multiple complexes were formed between the intact domain and dsRNA, saturating at a density of about one p20 molecule/11.25 base-pairs (or one turn) of duplex, whereas p10 achieved only about half of this packing density. The apparent Kd for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinct complexes were visualized for the interaction between p10 and VA RNA. Both p20 and p10 inhibited autophosphorylation of intact DAI, probably by binding the dsRNA activator. Once activated, DAI could phosphorylate both p10 and p20, suggesting that intermolecular phosphorylation can occur.
双链（ds）RNA激活蛋白激酶DAI（也称为PKR）含有一个RNA结合结构域，该结构域由一个基序的两个串联重复序列组成，即dsRBM，它与许多其他与结构化RNA相互作用的蛋白质共有。我们在大肠杆菌中表达了整个结构域和该基序的第一个拷贝，并将两种蛋白质p20和p10纯化至表观均一，以便研究它们与RNA以及完整激酶的相互作用。p20和p10都优先结合结构化RNA分子。竞争分析表明，在两种情况下，亲和力顺序均为dsRNA＞VA RNA＞tRNA，但分离的基序结合得比整个结构域松散得多。通过定量凝胶迁移率变动分析测量dsRNA的解离常数，p20和p10的表观Kd值分别为4×10⁻⁹ M和3.8×10⁻⁷ M。p20分子与dsRNA的结合似乎具有协同性。完整结构域与dsRNA之间形成了多个复合物，饱和度约为每11.25个碱基对（或一圈）双链有一个p20分子，而p10仅达到该堆积密度的一半左右。p20与VA RNA相互作用的表观Kd估计为3.5×10⁻⁷ M，检测到至少三种复合物，但未观察到p10与VA RNA相互作用形成明显的复合物。p20和p10都抑制完整DAI的自磷酸化，可能是通过结合dsRNA激活剂。一旦被激活，DAI可以磷酸化p10和p20，这表明分子间磷酸化可以发生。
