Interaction of ribosome recycling factor and elongation factor EF-G with E-coli ribosomes studied by the surface plasmon resonance technique
Interaction of ribosome recycling factor and elongation factor EF-G with E-coli ribosomes studied by the surface plasmon resonance technique
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DOI：
10.1046/j.1365-2443.2000.00382.x
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发表时间：
2000-12-01
期刊：
影响因子：
2.1
通讯作者：
Nakamura, Y
中科院分区：
文献类型：
--
作者：
Ishino, T;Atarashi, K;Nakamura, Y
背景：核糖体回收因子(RRF)与延伸因子EF-G共同作用，是多肽释放后核糖体终止后复合体的分解所必需的。长期以来，RRF如何解开这个复合体一直令人费解。最近RRF分子的晶体结构已被解决，并被显示为模拟转移RNA(TRNA)的形状，这促使我们研究RRF是否像tRNA那样与核糖体结合。结果：利用基于表面等离子体共振技术的Biacore 2000仪器实时监测了大肠杆菌表面偶联RRF和延伸因子EF-G上核糖体复合体的形成。RRF与70S核糖体以及50S和30S亚基相互作用，但它优先与50S亚基相互作用，这在高但生理离子条件下明显可见。这种50S相互作用被RRF的Arg132的单一氨基酸取代所减弱，这对蛋白质折叠没有明显的影响，但在体内和体外都使其活性丧失。此外，一组抑制RRF-50S相互作用的抗生素在体外也对RRF的多聚体破裂活性产生抑制作用。Biacore技术也很好地显示了通过冻结EF-G催化的转位前和转位后的中间产物来抑制RRF功能的抗生素硫链菌素和夫西地酸的作用。结论：RRF与50S亚基的优先相互作用可能具有生物学意义，可能反映了RRF的作用方式。Biacore技术被证明对实时监测核糖体和翻译因子之间的相互作用以及筛选核糖体循环因子的潜在抑制剂很有用。
Background: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does.Results: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G.Conclusions: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.