Cosentino G P, Venkatesan S, Serluca F C, Green S R, Mathews M B, Sonenberg N
Department of Biochemistry, McGill University, Montreal, QC Canada.
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9445-9. doi: 10.1073/pnas.92.21.9445.
The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.
酵母双杂交系统和Far-Western蛋白质印迹分析被用于证明人双链RNA（dsRNA）依赖性蛋白激酶（PKR）在体内和体外的二聚化。发现具有单个氨基酸取代（K296R）的PKR催化失活突变体在体内二聚化，并且缺失PKR催化结构域的突变体保留了二聚化能力。相反，PKR N端调节结构域中两个dsRNA结合基序的缺失消除了二聚化。dsRNA结合结构域的体外二聚化需要dsRNA的存在。这些结果表明PKR与dsRNA的结合对于二聚化是必需的。最初基于其与人免疫缺陷病毒RNA反式激活区域（TAR）结合的能力而鉴定的哺乳动物dsRNA结合蛋白TRBP，在酵母分析中也自身二聚化并与PKR二聚化。综上所述，这些结果表明体内可能存在由dsRNA结合蛋白不同组合组成的复合物。这样的复合物可以介导对基因表达的不同影响和细胞生长的控制。
