title: Protein synthesis by ribosomes with tethered subunits.
abstract: 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. 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.
authors.affiliation: Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
authors.forename: Cédric
authors.initials: C
authors.lastname: Orelle
authors.affiliation: 1] Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, Illinois 60208, USA [2] Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, USA.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Carlson
authors.affiliation: Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
authors.forename: Teresa
authors.initials: T
authors.lastname: Szal
authors.affiliation: Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
authors.forename: Tanja
authors.initials: T
authors.lastname: Florin
authors.affiliation: 1] Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, Illinois 60208, USA [2] Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.affiliation: Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mankin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D057005:Bioengineering / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; D010458:Peptidyl Transferases