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Protein synthesis by ribosomes with tethered subunits

机译：具有束缚亚基的核糖体的蛋白质合成

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摘要

The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation(1). Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.
机译：核糖体是负责蛋白质合成的核糖核蛋白机器。在所有生命王国中，它都由两个亚基组成，每个亚基都建立在自己的核糖体RNA（rRNA）支架上。亚基的独立但协调的功能，包括它们在启动时缔合，在延伸过程中旋转以及在蛋白质释放后解离的能力，是蛋白质合成的公认模型。此外，由于专用的装配因子使未成熟的核糖体亚基分开并阻止了它们的翻译起始，因此核糖体的二分性质被认为对生物发生至关重要。（1）亚基的自由交换限制了专门的正交遗传系统的发展，该系统可以为新功能而发展，而不会干扰本地翻译。在这里，我们显示具有拴系的亚单位的核糖体，因此不可分离的亚基（称为Ribo-T）能够成功进行蛋白质合成。通过工程化由大小亚基rRNA序列组成的杂交rRNA，我们制备了功能性核糖体，其中亚基通过短RNA接头共价连接成单个实体。值得注意的是，Ribo-T不仅在体外具有功能，而且即使在没有野生型核糖体的情况下也能够支持大肠杆菌细胞的生长。我们使用Ribo-T创建了第一个完全正交的核糖体信使RNA系统，并通过在肽基转移酶中心选择否则具有致命性的rRNA突变来证明其可进化性，该突变有利于有问题的蛋白质序列的翻译。 Ribo-T可用于探索人们对核糖体功能了解甚少的功能，启用正交遗传系统以及工程化具有新功能的核糖体。

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  • 来源
    《Nature》 |2015年第7563期|119-124|共6页
  • 作者

    Orelle Cedric; Carlson Erik D.; Szal Teresa; Florin Tanja; Jewett Michael C.; Mankin Alexander S.;

  • 作者单位

    Univ Illinois, Ctr Pharmaceut Biotechnol MC 870, Chicago, IL 60607 USA;

    Northwestern Univ, Dept Chem & Biol Engn, Evanston, IL 60208 USA|Northwestern Univ, Chem Life Proc Inst, Evanston, IL 60208 USA;

    Univ Illinois, Ctr Pharmaceut Biotechnol MC 870, Chicago, IL 60607 USA;

    Univ Illinois, Ctr Pharmaceut Biotechnol MC 870, Chicago, IL 60607 USA;

    Northwestern Univ, Dept Chem & Biol Engn, Evanston, IL 60208 USA|Northwestern Univ, Chem Life Proc Inst, Evanston, IL 60208 USA;

    Univ Illinois, Ctr Pharmaceut Biotechnol MC 870, Chicago, IL 60607 USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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