Pel H J, Moffat J G, Ito K, Nakamura Y, Tate W P
Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand.
RNA. 1998 Jan;4(1):47-54.
Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.
大肠杆菌释放因子3（RF3）是一种参与蛋白质合成终止的G蛋白，它能刺激终止信号解码释放因子RF1和RF2的活性。与G蛋白的情况相悖的是，据报道GDP和GTP在体外均对无核苷酸的RF3活性有负调节作用。通过直接核糖体结合试验，我们发现RF3的GTP类似物形式RF3xGDPCP对核糖体的亲和力比该蛋白的GDP形式高10倍，并且在没有解码释放因子的情况下，RF3xGDPCP能有效地与核糖体结合。这些结果表明RF3作为一种典型的翻译G蛋白与核糖体结合，并且提示GTP在体外对RF3活性产生的矛盾抑制作用很可能是由于核糖体介导的GTP水解不及时且无效。无核苷酸的RF3具有中等活性，其与核糖体的结合对RF2表现出正协同性。然而，在存在GDPαS的情况下，这种协同性不存在。观察到的无核苷酸RF3的活性表明它模拟了RF3的一种过渡状态，在此状态下该蛋白与解码释放因子相互作用，同时提高终止反应的效率。
