Blumberg B M, Nakamoto T, Goldberg I S
Biochemistry. 1975 Jul;14(13):2889-94. doi: 10.1021/bi00684a015.
The problem of whether the initiation of bacterial protein synthesis involves the obligatory formation of a 30S initiation complex intermediate was examined in a model system with N-acetylphenylalanyl-tRNA as initiator 5RNA nad poly(uridylic acid) as mRNA. The time courses of the formation of the 30S and 70S initiation complex with Escherichia coli ribosomes were measured simultaneously by stopping the reaction with dextran sulfate and differentiating the N-acetylphenylalanyl-tRNA bound to 30S ribosomal subunits from that bound to 70S ribosomes with RNase I, which hydrolyzes N-acetylphenylalanyl-tRNA bound to 30S subunits but not that bound to 70S ribosomes. A maximum in the 30S complex concentration was observed within the first 10-15 sec of the reaction, whereas 70S complex formed formed more slowly with a slight initial time lag. When an analog computer was programmed with rate constants determined separately for the formation of the 30S initiation complex from preformed 30S complex, kinetic curves very similar to the empirical curves were obtained for the entire time course of the reaction. The results show clearly that formation of the 70S complex obeys the kinetic laws for consecutive reactions, and the 30S complex is, therefore, an obligatory intermediate in the initiation of polyphenylalanine synthesis in the model system.
以N - 乙酰苯丙氨酰 - tRNA作为起始5RNA，聚（尿苷酸）作为mRNA的模型系统，研究了细菌蛋白质合成起始是否涉及30S起始复合物中间体的必然形成这一问题。通过用硫酸葡聚糖终止反应，并利用核糖核酸酶I区分与30S核糖体亚基结合的N - 乙酰苯丙氨酰 - tRNA和与70S核糖体结合的N - 乙酰苯丙氨酰 - tRNA，同时测量了大肠杆菌核糖体形成30S和70S起始复合物的时间进程。核糖核酸酶I可水解与30S亚基结合的N - 乙酰苯丙氨酰 - tRNA，但不能水解与70S核糖体结合的N - 乙酰苯丙氨酰 - tRNA。在反应的最初10 - 15秒内观察到30S复合物浓度达到最大值，而70S复合物形成得更慢，最初有轻微的时间延迟。当用分别为从预先形成的30S复合物形成30S起始复合物确定的速率常数对模拟计算机进行编程时，在反应的整个时间进程中获得了与经验曲线非常相似的动力学曲线。结果清楚地表明，70S复合物的形成遵循连续反应的动力学规律，因此，在该模型系统中，30S复合物是聚苯丙氨酸合成起始过程中的一个必然中间体。
