STRUCTURE AND FUNCTION OF ESCHERICHIA COLI RIBOSOMES .6. MECHANISM OF ASSEMBLY OF 30-S RIBOSOMES STUDIED IN VITRO
STRUCTURE AND FUNCTION OF ESCHERICHIA COLI RIBOSOMES .6. MECHANISM OF ASSEMBLY OF 30-S RIBOSOMES STUDIED IN VITRO
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DOI：
10.1016/0022-2836(69)90161-2
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发表时间：
1969-01-01
影响因子：
5.6
通讯作者：
NOMURA, M
中科院分区：
文献类型：
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作者：
TRAUB, P;NOMURA, M
研究了从 16 s 核糖体 RNA 和 30 s 核糖体蛋白混合物中重建 30 s 核糖体。研究了影响重构效率的几个因素。结果发现，重构混合物的离子强度存在明显的最佳值（I = 0.37）。重构混合物中镁离子的浓度必须为 0.01 m 或更高（测量最高 0.03 m）。在 40°C 下孵育 15 分钟后，pH 值在 6.5 至 8.0 之间（40°C 时）可获得最大效率。就活性 30 s 核糖体的形成而言，重构反应是一级反应。反应物总浓度的变化不会影响重构动力学，以最终值的百分比表示。因此，限速反应可能是单分子的。重构速率很大程度上取决于孵育混合物的温度。根据速率常数对温度的依赖性，获得了 37.8 kcal./mole 的阿累尼乌斯活化能。计算还给出了激活熵的正值。在 0° C 下孵育重构混合物不会产生 30 s 核糖体。通过离心从此类重构混合物中分离出的颗粒（假定的重构中间体，或RI颗粒）缺乏几种蛋白质成分（S蛋白），这些蛋白质成分在离心后从上清液级分中回收。 RI 颗粒在功能上不活跃。在 0°C 下，S 蛋白的功能活性恢复和 S 蛋白与 RI 颗粒的有效结合都无法发生，而只有在更高温度下孵育后才能发生。然而，在 40° C 下单独加热 RI 颗粒 20 分钟会产生颗粒（活化的重构中间体，或 RI* 颗粒），这些颗粒现在能够在 0° C 下结合 S 蛋白并成为功能性 30 s 核糖体。因此，重构以逐步的方式进行：16 s RNA→+ RI 蛋白 RI 颗粒→加热 RI* 颗粒→+ S 蛋白 30 s 核糖体。 RI 粒子→ RI* 粒子的反应是限速单分子反应，代表 RI 粒子的结构重排，需要高活化能。还描述了实验，其中证明了从未折叠的 30 年代核糖体形成功能活性的 30 年代核糖体。在所使用的条件下，这种重折叠过程的动力学已被证明与从 RNA 和蛋白质重建 30 s 核糖体的动力学几乎相同。结果表明，两个反应都涉及相同的中间体 RI 颗粒。
Reconstitution of 30 s ribosomes from 16 s ribosomal RNA and a mixture of 30 s ribosomal proteins was studied. Several factors affecting the efficiency of the reconstitution were examined. It was found that there is a sharp optimum (I= 0.37) for the ionic strength of the reconstitution mixture. Magnesium ions must be present in the reconstitution mixture at a concentration of 0.01 m or higher (measured up to 0.03 m). With a 15-minute incubation at 40° C, the maximum efficiency was obtained at pH values between 6.5 and 8.0 (at 40° C). The reconstitution reaction was first-order with respect to formation of active 30 s ribosomes. Changes in the total concentration of the reactants do not affect the kinetics of reconstitution, expressed as a percentage of the final value. Thus the rate-limiting reaction is probably unimolecular. The rate of reconstitution is strongly dependent on the temperature of the incubation mixture. From the dependence of the rate constant on temperature, an Arrhenius activation energy of 37.8 kcal./mole has been obtained. Calculation has also given a positive value for the entropy of activation. Incubation of the reconstitution mixture at 0° C does not produce 30 s ribosomes. The particles (presumed reconstitution intermediates, or RI particles) isolated by centrifugation from such reconstitution mixtures are deficient in several protein components (S proteins), which are recovered from the supernatant fraction after centrifugation. RI particles are functionally inactive. Neither restoration of their functional activity by S proteins nor efficient binding of S proteins to RI particles can take place at 0° C, but do so only after incubation at higher temperatures. However, heating RI particles alone at 40° C for 20 minutes produces particles (activated reconstitution intermediates, or RI∗ particles) which are now capable of binding S proteins at 0° C and becoming functional 30 s ribosomes. Thus, the reconstitution takes place in step-wise fashion: 16 s RNA→+ RI proteins RI particles→ heating RI∗ particles→+ S proteins 30 s ribosomes. The reaction RI particles→ RI∗ particles is the ratelimiting unimolecular reaction, representing a structural rearrangement of RI particles which requires high activation energy. Experiments are also described in which formation of functionally active 30 s ribosomes from unfolded 30 s ribosomes was demonstrated. The kinetics of this refolding process under the conditions used has been shown to be almost identical to those of reconstitution of 30 s ribosomes from RNA and proteins. It is shown that the same intermediate, the RI particle, is involved in both reactions.