Gast F U, Peters F, Pingoud A
J Biol Chem. 1987 Sep 5;262(25):11920-6.
The ribosomal translocation, as measured in vitro by peptide formation on poly(U)-programmed Escherichia coli ribosomes in the presence of ternary complex, deacylated tRNA or N-acetyl-Phe-tRNA, and elongation factor G, is the rate-limiting step of protein synthesis. Elongation factor G stimulates the spontaneous translocation by a factor of about 500. N-Acetyl-Phe-Phe-tRNA(Phe E. coli) is translocated with a rate constant of 1-2 s-1 at 25 degrees C. Translocation of N-acetyl-Phe-Phe-tRNA(Phe yeast) and N-acetyl-Phe-Leu-tRNA(Leu E. coli) under identical conditions proceeds with a rate by about a factor of 2 and 10, respectively, more slowly. The translocation rate, therefore, is influenced by the nature of the tRNAs in the A-site. We can show, furthermore, that also the tRNA in the P-site, and presumably in the E-site as well, influences the rate of translocation. Reduced rates of translocation of noncognate peptidyl-tRNAs are accompanied by preferential dissociation of these tRNAs at the beginning of the translation of a mRNA.
在体外，通过在三元复合物、脱酰基tRNA或N-乙酰苯丙氨酰-tRNA存在的情况下，在聚（U）编程的大肠杆菌核糖体上形成肽来测量核糖体转位，而延伸因子G参与其中，核糖体转位是蛋白质合成的限速步骤。延伸因子G将自发转位速率提高约500倍。N-乙酰苯丙氨酰-苯丙氨酰-tRNA（苯丙氨酸 大肠杆菌）在25℃下以1 - 2 s⁻¹的速率常数进行转位。在相同条件下，N-乙酰苯丙氨酰-苯丙氨酰-tRNA（苯丙氨酸 酵母）和N-乙酰苯丙氨酰-亮氨酰-tRNA（亮氨酸 大肠杆菌）的转位速率分别慢约2倍和10倍。因此，转位速率受A位点tRNA性质的影响。此外，我们可以证明，P位点的tRNA，可能还有E位点的tRNA，也会影响转位速率。非同源肽基-tRNA转位速率降低伴随着这些tRNA在mRNA翻译开始时优先解离。
