Takahashi Y, Ogata K
Eur J Biochem. 1985 Oct 15;152(2):279-86. doi: 10.1111/j.1432-1033.1985.tb09195.x.
An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.
在稀疏霉素存在的情况下，将异源无细胞系统与125I标记的珠蛋白mRNA一起温育，形成80S起始复合物。然后用微球菌核酸酶消化80S起始复合物。通过EDTA处理释放的核糖体5S-RNA×L5蛋白（5S RNP）组分含有125I标记的mRNA片段。通过（a）CsCl等密度离心、（b）通过蔗糖密度梯度再离心以及（c）对通过（b）纯化的5S RNP进行丙烯酰胺凝胶电泳，显示了标记的mRNA片段与5S RNP的结合。用十二烷基硫酸钠或链霉蛋白酶处理可从5S RNP中释放出标记片段，表明蛋白L5参与了这种结合。所附着的mRNA片段长度为23 - 25个核苷酸。使用兔β珠蛋白mRNA的cDNA限制性片段进行的杂交实验表明，所附着的mRNA片段源自珠蛋白mRNA的5'部分。在用125I标记的珠蛋白mRNA温育网织红细胞裂解物形成的80S起始复合物中也观察到125I标记的mRNA片段与5S RNP的结合，但在标记的多核糖体组分中未观察到。这些发现可能表明5S RNP与珠蛋白mRNA的5'部分相互作用，该部分在80S起始复合物中包含珠蛋白mRNA的翻译起始密码子。讨论了这些结果的生化意义。
