Yin Heng, Xu Shoujun, Wang Yuhong
Department of Chemistry, University of Houston.
Department of Chemistry, University of Houston;
J Vis Exp. 2019 Jul 8(149). doi: 10.3791/59918.
The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS). With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.
核糖体易位是指核糖体在信使核糖核酸（mRNA）上精确移动三个核苷酸（nt）的过程，这是蛋白质合成的核心步骤。为了研究其机制，有两个基本的技术要求。首先是单核苷酸分辨率，它能够区分正常易位和移码，移码过程中核糖体会移动超过3个核苷酸。其次是能够探测mRNA的入口和出口两侧，以便阐明易位的全貌。我们报道了一种基于通过力诱导剩余磁化光谱法（FIRMS）获得的DNA-mRNA双链体临界解离力的双DNA尺测定法。该双尺测定法具有2-4皮牛（pN）的力分辨率，足以区分不同的易位步骤。通过在探测DNA上使用长连接子，它们可以到达核糖体另一侧的mRNA，从而能够确定mRNA两侧的位置。因此，双尺测定法特别适合研究核糖体易位以及一般的核酸运动。我们展示了具有代表性的结果，这些结果表明了mRNA呈环状构象，并区分了正常易位和移码。
