Johnson A E, Miller D L, Cantor C R
Proc Natl Acad Sci U S A. 1978 Jul;75(7):3075-9. doi: 10.1073/pnas.75.7.3075.
Complex formation between elongation factor Tu, GTP, and Nepsilon-bromoacetyl-Lys-tRNA results in the cross-linking of the protein and the modified Lys-tRNA. The efficiency of affinity labeling is greater than 50%. In the presence of unmodified Lys-tRNA, the amount of crosslinking is greatly decreased. There is no covalent reaction with elongation factor Tu in the absence of complex formation. Substantial purification of the crosslinked ternary complex can be achieved by gel filtration at low Mg2+ concentration and passage through nitrocellulose filters. The crosslinked complex exhibits message-dependent binding to ribosomes which is accompanied by the hydrolysis of the associated GTP, as shown by both filter assays and gel filtration profiles. The crosslinked complex therefore appears to function normally except for its inability to dissociate. These experiments demonstrate that the ternary complex is the true intermediate in the binding of aminoacyl-tRNA to the ribosomes.
延伸因子Tu、GTP与Nε-溴乙酰-Lys-tRNA之间形成复合物会导致蛋白质与修饰的Lys-tRNA发生交联。亲和标记的效率大于50%。在未修饰的Lys-tRNA存在的情况下,交联量会大幅减少。在没有复合物形成的情况下,不会与延伸因子Tu发生共价反应。通过在低Mg2+浓度下进行凝胶过滤以及通过硝酸纤维素滤膜,可以实现交联三元复合物的大量纯化。如滤膜分析和凝胶过滤图谱所示,交联复合物表现出与核糖体的依赖于信使的结合,同时伴随着相关GTP的水解。因此,除了无法解离外,交联复合物似乎功能正常。这些实验表明,三元复合物是氨酰-tRNA与核糖体结合过程中的真正中间体。