Terao K, Uchiumi T, Ogata K
Biochim Biophys Acta. 1982 Apr 26;697(1):20-4. doi: 10.1016/0167-4781(82)90040-9.
To cross-link the 3'-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 125I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the alpha-chymotrypsin digest of this 125I-labeled protein-oligonucleotide complex was similar to that of the digest of 125I-labeled L5 protein. The results indicate that L5 protein binds to the 3'-terminal region of 5 S RNA in rat liver 60 S subunits.
为了将5 S RNA的3'-末端与相邻蛋白质交联，用高碘酸钠氧化大鼠肝脏的核糖体60 S亚基，并用硼氢化钠还原。然后通过EDTA处理，接着进行蔗糖密度梯度离心分离5 S RNP，并进行SDS-聚丙烯酰胺凝胶电泳。迁移率比L5蛋白慢的蛋白质，被认为是交联的5 S RNP，用125I标记，用RNA酶处理，并通过二维聚丙烯酰胺凝胶电泳分析，随后进行放射自显影。观察到一个位于L5蛋白阳极的放射性斑点，表明它是L5蛋白-寡核苷酸复合物。当通过SDS平板聚丙烯酰胺凝胶电泳分析并随后进行放射自显影时，这种125I标记的蛋白-寡核苷酸复合物的α-胰凝乳蛋白酶消化产物的肽谱与125I标记的L5蛋白消化产物的肽谱相似。结果表明，L5蛋白与大鼠肝脏60 S亚基中5 S RNA的3'-末端区域结合。
