In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

体外重组是研究核糖体功能和生物发生以及发现新核糖体特征的有力工具。在这项研究中，我们整合了大肠杆菌所需的所有过程小核糖体亚基组装。在我们的方法中，称为完全基于重组的综合合成、组装和翻译 (R-iSAT)，小核糖体亚基的组装和评估与重组无细胞蛋白质合成系统中的核糖体 RNA (rRNA) 合成相结合。通过改变 R-iSAT 的成分，包括重组核糖体蛋白的组成，我们将核糖体组装与核糖体蛋白合成结合起来，实现了核糖体蛋白的功能合成和随后的亚基组装。此外，我们组装并评估了在 rRNA 和核糖体蛋白中都有突变的亚基。该研究表明，我们的方案提供了全面分析小核糖体亚基的任何元素的新方法，目的是提高我们对核糖体生物发生、功能、