Wintermeyer W, Gualerzi C
Biochemistry. 1983 Feb 1;22(3):690-4. doi: 10.1021/bi00272a025.
The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.
通过荧光停流动力学测量，研究了N-乙酰苯丙氨酰-tRNA苯丙氨酸（酵母）与聚尿苷酸编程的大肠杆菌30S核糖体亚基的结合机制，以及单个起始因子（IF-1、IF-2和IF-3）和GTP对该过程的影响。三元复合物的形成伴随着位于tRNA反密码子环中的原黄素标签荧光强度和偏振度的增加。起始因子和GTP的作用是提高三元复合物形成的速度（在7 mM Mg2+时约提高400倍）。在存在三种起始因子和GTP的情况下，三元复合物的形成可分解为两个部分反应：一个快速的明显二级步骤（k12 = 5 x 10(6) M-1 s-1，k21 = 1.4 s-1），随后是一个缓慢的重排步骤（k23 ≤ 0.1 s-1）。数据表明一种机制，其中三元复合物是由最初形成的前体三元复合物的至少两次重排形成的。IF-2和IF-3的加速作用可以通过假设这些因子对30S核糖体亚基的协同变构作用来理解，而IF-1似乎通过影响其他两个因子间接起作用。
