Irreversible chemical steps control intersubunit dynamics during translation
Irreversible chemical steps control intersubunit dynamics during translation
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DOI：
10.1073/pnas.0805299105
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发表时间：
2008-10-07
影响因子：
11.1
通讯作者：
Puglisi, Joseph D.
中科院分区：
文献类型：
--
作者：
Marshall, R. Andrew;Dorywalska, Magdalena;Puglisi, Joseph D.
核糖体是一种二亚基大分子机器，可破译遗传密码并催化肽键形成。核糖体亚基之间的动态旋转运动可能是有效和准确的蛋白质合成所必需的，但对亚基间动力学的直接观察已被翻译的重复性、多步骤性质所掩盖。在这里，我们报告了一系列单分子荧光共振能量转移测定，这些测定揭示了起始和第一轮延伸过程中实时的核糖体亚基间构象循环。在亚基连接并将正确的氨酰基-tRNA递送至核糖体后，肽键形成导致快速构象变化，这与先前的结构和生化研究暗示的30S亚基相对于50S亚基的逆时针旋转一致。随后延伸因子 G 和 GTP 水解的结合导致 30S 亚基相对于 50S 亚基顺时针旋转，为核糖体下一轮 tRNA 选择和肽键形成做好准备。因此，核糖体利用不可逆肽基转移和 GTP 水解的自由能来克服翻译过程中大规模构象变化的激活障碍。亚基间旋转可能是易位过程中 tRNA 和 mRNA 底物协同运动的必要条件。
The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.