Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, 60607, USA.
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia.
Nat Commun. 2019 Feb 25;10(1):930. doi: 10.1038/s41467-019-08892-w.
Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
Ribo-T 是一种经过工程改造的核糖体，其小亚基和大亚基通过在单个分子中连接 16S rRNA 和 23S rRNA 而连接在一起。尽管 Ribo-T 可以在没有野生型核糖体的情况下支持细胞增殖，但 Ribo-T 细胞的生长速度比具有野生型核糖体的细胞慢。在这里，我们表明细胞生长缺陷可能主要是由于 Ribo-T 组装缓慢而不是其功能不完善所致。Ribo-T 的成熟在晚期组装阶段停滞。在积累的组装中间体和 rRNA 末端中，几个转录后 rRNA 修饰和一些核糖体蛋白的表达水平较低，并且 rRNA 末端未完全修剪。对 Ribo-T 细胞的核糖体谱分析显示翻译延伸没有缺陷，但 Ribo-T 对起始密码子的占有率略高，对终止密码子的占有率略低，这表明亚基连接轻微影响翻译的起始和终止阶段。了解 Ribo-T 系统的局限性为其未来的发展提供了途径。
