Faculty of Biology, Technion - Israel Institute of Technology.
Faculty of Biology, Technion - Israel Institute of Technology;
J Vis Exp. 2021 Oct 7(176). doi: 10.3791/62878.
In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis. Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.
近年来，人们已经认识到核糖体不仅可以解码我们的 mRNA，还可以引导多肽链进入拥挤的细胞环境。核糖体为空间和动力学控制的膜靶向因子、修饰酶和折叠伴侣的结合提供了平台。甚至组装成高阶寡聚体复合物以及蛋白质-蛋白质网络形成步骤最近也被发现与合成相协调。在这里，我们描述了选择性核糖体分析，这是一种开发来在体内捕获共翻译相互作用的方法。我们将详细介绍捕获核糖体-新生链复合物以及共翻译相互作用物所需的各种亲和纯化步骤，以及提取 mRNA、大小排除、反转录、深度测序和大数据分析所需的步骤，以近乎密码子分辨率解析共翻译相互作用。
