Patel R C, Sen G C
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195-5285, USA.
Cell Mol Biol Res. 1994;40(7-8):671-82.
The interferon-inducible protein kinase, PKR, requires double-stranded (ds) RNA for its activation. We have previously mapped its dsRNA-binding domain (DRBD) to the amino terminal 170 residues (Patel and Sen, 1992). In the present study, we have characterized in detail the interactions between dsRNA and DRBD. For this purpose, DRBD was produced in bacteria as a polyhistidine-tagged protein and purified by affinity chromatography. A polyclonal antibody was raised against purified DRBD. For studying dsRNA-DRBD interactions, a Northwestern assay and an electrophoretic mobility shift assay (EMSA) using a radiolabeled in vitro transcribed 82 bp dsRNA probe was developed. The antiserum reacted with both DRBD and PKR but did not prevent their interactions with dsRNA. DRBD, on the other hand, blocked the activation of PKR by dsRNA. DRBD and the dsRNA probe formed multimeric complexes which were separable by EMSA. The antibody could interact with these complexes and supershift their mobility. Competition with unlabeled dsRNA revealed that the dimeric DRBD-dsRNA complex was much more stable than the monomeric complex. Similar competition assays using 11 different synthetic and natural RNA molecules revealed that only authentic dsRNA molecules could effectively compete with the probe for binding DRBD in a sequence-independent fashion.
干扰素诱导蛋白激酶PKR的激活需要双链（ds）RNA。我们之前已将其dsRNA结合结构域（DRBD）定位到氨基末端的170个残基上（帕特尔和森，1992年）。在本研究中，我们详细表征了dsRNA与DRBD之间的相互作用。为此，DRBD在细菌中作为带多聚组氨酸标签的蛋白产生，并通过亲和层析进行纯化。制备了针对纯化后的DRBD的多克隆抗体。为了研究dsRNA-DRBD相互作用，开发了一种蛋白质印迹法（Northwestern分析法）和一种使用放射性标记的体外转录82 bp dsRNA探针的电泳迁移率变动分析（EMSA）。抗血清与DRBD和PKR都发生反应，但不阻止它们与dsRNA的相互作用。另一方面，DRBD可阻断dsRNA对PKR的激活。DRBD与dsRNA探针形成多聚体复合物，可通过EMSA分离。该抗体可与这些复合物相互作用并使其迁移率发生超迁移。与未标记的dsRNA进行竞争实验表明，二聚体DRBD-dsRNA复合物比单体复合物稳定得多。使用11种不同的合成和天然RNA分子进行的类似竞争实验表明，只有真正的dsRNA分子能够以序列无关的方式有效地与探针竞争结合DRBD。
