Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex
Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex
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DOI：
10.1017/s1355838298971576
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发表时间：
1998-08-01
期刊：
影响因子：
4.5
通讯作者：
Kaji, A
中科院分区：
文献类型：
--
作者：
Grentzmann, G;Kelly, PJ;Kaji, A
RF 3最初被表征为在体外测定中刺激翻译终止的因子。该因子有一个GTP结合位点，并显示出与延伸因子EF-Tu和EF-G的序列相似性，特别是，在经典的终止试验中，使用终止三联体，加入GTP消除了RF 3的刺激。应用一种新的终止试验，使用最小信使RNA代替单独的三联体，我们表明，在RF 3存在下，GTP在RF 1的限速浓度下刺激终止。我们表明，在RRF依赖的体外核糖体回收反应中，RF 3可以取代EF-G，这种活性是GTP依赖的。此外，在GTP存在下，过量的RF 3和RRF引起非水解的fmet-tRNA的释放，这支持了先前的遗传实验，表明RF 3可能参与肽基-tRNA的核糖体脱落。与GTP参与上述反应相反，RF 3对RF 2终止的刺激与GTP的存在无关。这与以前的研究一致，表明RF 3增强了RF 2对终止复合物的亲和力，而没有GTP水解。基于我们的结果，我们提出了RF 3如何在翻译终止和核糖体回收中发挥作用的模型。
RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G, Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets.We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1, We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro, This activity is GTP-dependent, In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA, This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis, Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.