Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Program in Biophysics, Stanford University, Stanford, CA 94305-5126, USA.
Nucleic Acids Res. 2023 Jun 23;51(11):5774-5790. doi: 10.1093/nar/gkad286.
In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.
在细菌中，核糖体翻译终止时新合成蛋白质的释放是由 I 类释放因子（RF）RF1 或 RF2 催化的，分别读取 UAA 和 UAG 或 UAA 和 UGA 密码子。I 类 RF 通过 GTP 酶 RF3 从翻译后核糖体中循环回收，RF3 加速核糖体亚基间的旋转和 I 类 RF 的解离。核糖体构象状态如何与 RF 的结合和解离偶联仍然不清楚，并且核糖体催化的鸟嘌呤核苷酸交换对 RF3 在体内进行 RF3 循环回收的重要性一直存在争议。在这里，我们使用单分子荧光测定法来描绘这些分子事件，以阐明触发 I 类 RF 解离、GTP 水解和 RF3 解离的 RF3 结合和核糖体亚基间旋转的时间。这些发现与细胞内终止流的定量建模相结合，揭示了快速的核糖体依赖性鸟嘌呤核苷酸交换对于 RF3 在体内的作用至关重要。
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