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Protein synthesis by ribosomes with tethered subunits

2015; Nature Portfolio; Volume: 524; Issue: 7563 Linguagem: Inglês

10.1038/nature14862

1476-4687

Cédric Orelle, Erik D. Carlson, Teresa Szal, Tanja Florin, Michael C. Jewett, Alexander S. Mankin,

Bacterial Genetics and Biotechnology

A ribosome with tethered subunits, ‘Ribo-T’, is engineered by making a hybrid RNA composed of ribosomal RNA of large and small subunits; Ribo-T can support cell growth in vivo in the absence of wild-type ribosomes, and is used to establish a fully orthogonal ribosome–mRNA system. The ribosome consists of two subunits, each of which contains a long ribosomal RNA (rRNA) on which dozens of proteins assemble. Reconstitution of the full ribosome occurs once one subunit locates a translation initiation signal. As the ribosome translocates on the mRNA, the two subunits undergo ratcheting movements that shift the tRNAs and nascent chain through the complex. This paper reports the synthesis of a ribosome with tethered subunits, achieved by making a hybrid rRNA. The new complex, termed Ribo-T, is able to support cell growth in vivo in the absence of wild-type ribosomes, and is used to establish a fully orthogonal ribosome–mRNA system. 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 initiation1. 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.