Patel R C, Stanton P, Sen G C
Department of Molecular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
J Biol Chem. 1996 Oct 11;271(41):25657-63. doi: 10.1074/jbc.271.41.25657.
The amino-terminal region of the double-stranded (ds) RNA-dependent protein kinase, PKR, has been shown to mediate both dsRNA binding and protein dimerization. To critically examine if PKR dimerization is dependent on dsRNA binding, we generated a series of mutants that are incapable of binding dsRNA. Some, but not all, of these mutants retained the ability to dimerize, as shown by a two-hybrid transcriptional activation assay in vivo and a chemical cross-linking assay in vitro. These mutants were used further to demonstrate that the translational inhibitory activity of PKR in vivo requires dsRNA binding; PKR mutants that dimerized but did not bind dsRNA could not inhibit the translation of a transfected reporter gene.
双链（ds）RNA依赖的蛋白激酶PKR的氨基末端区域已被证明可介导dsRNA结合和蛋白二聚化。为了严格检验PKR二聚化是否依赖于dsRNA结合，我们构建了一系列无法结合dsRNA的突变体。如体内双杂交转录激活试验和体外化学交联试验所示，其中一些（但并非全部）突变体保留了二聚化能力。这些突变体进一步用于证明PKR在体内的翻译抑制活性需要dsRNA结合；能够二聚化但不结合dsRNA的PKR突变体无法抑制转染报告基因的翻译。
