Binding of the protein kinase PKR to RNAs with secondary structure defects: Role of the tandem A-G mismatch and noncontiguous helixes
Binding of the protein kinase PKR to RNAs with secondary structure defects: Role of the tandem A-G mismatch and noncontiguous helixes
复制标题
DOI：
10.1021/bi980113j
复制
发表时间：
1998-05-05
期刊：
影响因子：
2.9
通讯作者：
Cech, TR
中科院分区：
文献类型：
--
作者：
Bevilacqua, PC;George, CX;Cech, TR
人干扰素诱导的双链RNA（dsRNA）激活的蛋白激酶（PKR）是一种抗病毒药物，它能被长链dsRNA激活，也能被一系列含有非Watson-Crick基序的细胞和病毒RNA激活或抑制。PKR具有dsRNA结合结构域（dsRBD），其含有dsRNA结合基序（dsRBM）的两个串联拷贝。进行体外选择实验以寻找能够与含有dsRBD的PKR的截短形式结合的RNA，通过与His（6）-标记的蛋白质结合并在镍（II）次氮基三乙酸琼脂糖上层析来选择RNA配体。选择与模型dsRNA茎环类似或更紧密结合的一系列RNA。通过各种方法，包括序列比较，自由能最小化，结构映射，边界实验，定点诱变和足迹法，这些RNA的检查，揭示了由非连续螺旋组成的蛋白质结合位点。此外，所选RNA含有串联A-G错配（（5 'AG 3'）（3 'GA 5'）），但以与仅含有Watson-Crick碱基对的双链体相似的亲和力结合至截短蛋白。RNA螺旋中串联A-G错配的NMR结构（rGGCAGGCC）（2）揭示了在错配处具有微小扰动的整体A型螺旋[Wu，M.，SantaLucia，J.，小的，Turner，D. H.（1997）Biochemistry 36，4449-4460]。这支持了这样的观点，即含有dsRBM的蛋白质可以与具有二级结构缺陷的RNA结合，只要RNA具有总体A型几何形状。此外，选择的RNA能够激活或抑制野生型PKR自磷酸化以及其蛋白质合成起始因子eIF-2的磷酸化，表明全长PKR可以结合到含有串联A-G错配的RNA并受其调节。
The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA, PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif(dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD, RNA ligands were selected by binding to His(6)-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches ((5'AG3')(3'GA5')), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)(2) reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.