Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation.
Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation.
复制标题
同期单分子力和荧光测量以确定核糖体易位的机制。
DOI：
10.1007/978-1-0716-2229-2_14
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Bustamante,CarlosJ
中科院分区：
文献类型：
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作者：
Desai,VarshaP;Frank,Filipp;Bustamante,CarlosJ
核糖体是生命中心信条的核心。它们通过将核苷酸密码子序列中的信息翻译成蛋白质的氨基酸序列来执行基因表达的最后一个主要步骤。这是一个复杂的机械力化学过程，需要协调核糖体内的多种动态事件，如精确的解码时间和随后沿mRNA的转位。我们之前已经使用了具有单分子荧光能力的高分辨率光镊仪(Fleezers)来研究核糖体如何将GTPase翻译延伸因子EF-G的结合与内部构象变化结合在一起，以解开和跨越mRNA二级结构构成的机械障碍。在这里，我们详细描述了通过单个主动翻译的核糖体实时监测两个正交通道(EF-G结合和易位)的过程，以揭示它们利用化学能产生机械力和位移的机制。
Ribosomes are at the core of the central dogma of life. They perform the last major step of gene expression by translating the information written in the nucleotide codon sequences into the amino acid sequence of a protein. This is a complex mechanochemical process that requires the coordination of multiple dynamic events within the ribosome such as the precise timing of decoding and the subsequent translocation along the mRNA. We have previously used a high-resolution optical tweezers instrument with single-molecule fluorescence capabilities (“fleezers”) to study how ribosomes couple binding of the GTPase translation elongation factor EF-G with internal conformational changes to unwind and progress across the mechanical barriers posed by mRNA secondary structures. Here, we present a detailed description of the procedures for monitoring two orthogonal channels (EF-G binding and translocation) by single actively translating ribosomes in real-time, to uncover the mechanism by which they harness chemical energy to generate mechanical force and displacement.