Li S W, Moskow J J, Suhadolnik R J
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
J Biol Chem. 1990 Apr 5;265(10):5470-4.
The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.
光亲和标记技术已应用于双链RNA（dsRNA）依赖性酶2',5'-寡腺苷酸（2-5A）合成酶，为研究该酶dsRNA变构结合域中的RNA-蛋白质相互作用提供了一种手段。本文描述了光亲和探针聚[（32P）I,8-叠氮基I]·聚（C）及其错配类似物聚[（32P）I,8-叠氮基I]·聚（C12U）的合成、表征及生物学特性，它们模拟了母体分子聚（I）·聚（C）和聚（I）·聚（C12U）。使用从兔网织红细胞裂解物和干扰素处理的HeLa细胞提取物中高度纯化的2-5A合成酶，证明了聚[（32P）I,8-叠氮基I]·聚（C）和聚[（32P）I,8-叠氮基I]·聚（C12U）作为变构位点定向激活剂的功效。dsRNA光探针激活这两种2-5A合成酶。在0℃光解20 s后，在6×10（-4）g/ml聚[（32P）I,8-叠氮基I]·聚（C）时观察到2-5A合成酶的饱和。聚[（32P）I,8-叠氮基I]·聚（C）的光掺入是特异性的，天然聚（I）·聚（C）可阻止其光掺入即证明了这一点。DNA、聚（I）和聚（C）不是聚[（32P）I,8-叠氮基I]·聚（C）的竞争者。用聚[（32P）I,8-叠氮基I]·聚（C）对2-5A合成酶进行紫外线照射后，用微球菌核酸酶处理反应混合物，以水解未与酶交联的叠氮基dsRNA。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影后，观察到一条110 kDa的放射性条带（与天然兔网织红细胞裂解物2-5A合成酶报道的相同）。2-5A合成酶的特异性光标记表明叠氮基dsRNA是变构结合域所固有的。讨论了聚[（32P）I,8-叠氮基I]·聚（C）在检测dsRNA依赖性结合蛋白以及分离变构结合位点或其附近肽段方面的实用性。
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