Endo Y, Chan Y L, Lin A, Tsurugi K, Wool I G
Department of Biochemistry, Yamanashi Medical School, Japan.
J Biol Chem. 1988 Jun 15;263(17):7917-20.
An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.
一种模拟真核生物28 S rRNA的α-肌动蛋白和蓖麻毒素区域的寡核糖核苷酸（35聚体），通过T7 RNA聚合酶从合成模板体外转录得到，并用于测试毒素水解的特异性是否得以保留。低浓度的α-肌动蛋白在合成寡聚物中对应于28 S rRNA中G-4325（毒素在完整核糖体中的作用位点）的鸟苷残基3'侧切割一个磷酸二酯键。在高浓度的α-肌动蛋白作用下，底物（35聚体）的每个嘌呤之后都会发生水解。α-肌动蛋白对一种合成RNA（20聚体）没有影响，该合成RNA再现了毒素结构域环中几乎通用的核苷酸序列，但缺少茎部。因此，α-肌动蛋白对28 S rRNA精确区域的攻击特异性似乎取决于核苷酸序列和结构域的结构。蓖麻毒素使合成寡聚物（35聚体）中的一个核苷酸脱嘌呤，并且在苯胺存在的情况下，磷酸核糖主链在与28 S rRNA中A-4324（毒素在体内的作用位点）一致的位置被切割。
