University of California, Los Angeles, Los Angeles, CA, USA.
Emory University, Atlanta, GA, USA.
Methods Mol Biol. 2022;2478:381-399. doi: 10.1007/978-1-0716-2229-2_14.
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.
核糖体是生命中心法则的核心。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，完成基因表达的最后一个主要步骤。这是一个复杂的机械化学过程，需要核糖体内部多个动态事件的协调，例如解码的精确时间安排以及随后沿着 mRNA 的易位。我们之前使用具有单分子荧光功能的高分辨率光镊仪器（“fleezers”）来研究核糖体如何将 GTP 酶翻译延伸因子 EF-G 的结合与内部构象变化联系起来，以解开并跨越 mRNA 二级结构所构成的机械障碍。在这里，我们详细描述了实时监测两个正交通道（EF-G 结合和易位）的单个活性翻译核糖体的程序，以揭示它们利用化学能产生机械力和位移的机制。
