A fully orthogonal system for protein synthesis in bacterial cells.

Aleksashin, Nikolay A; Szal, Teresa; d'Aquino, Anne E; Jewett, Michael C; Vázquez-Laslop, Nora; Mankin, Alexander S

Aleksashin, Nikolay A; Szal, Teresa; d'Aquino, Anne E; Jewett, Michael C; Vázquez-Laslop, Nora; Mankin, Alexander S.

Afiliación

Aleksashin NA; Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, 60607, USA.
Szal T; Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, 60612, USA.
d'Aquino AE; Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, 60607, USA.
Jewett MC; Interdisciplinary Biological Science Program, Northwestern University, 2145 Sheridan Rd, Evanston, IL, 60208, USA.
Vázquez-Laslop N; Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd, Evanston, IL, 60208, USA.
Mankin AS; Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd, Evanston, IL, 60208, USA.

Nat Commun ; 11(1): 1858, 2020 04 20.

Article en En | MEDLINE | ID: mdl-32313034

RESUMEN

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

Asunto(s)

Bacterias/metabolismo; Ingeniería de Proteínas/métodos; Ribosomas/química; Biología Sintética/métodos; Alelos; Sistema Libre de Células; Cristalografía por Rayos X; Modelos Moleculares; Modelos Teóricos; Conformación Molecular; Mutación; Péptidos/química; Peptidil Transferasas/química; Plásmidos/genética; Biosíntesis de Proteínas; Proteoma; ARN Mensajero/genética; ARN Ribosómico/genética; ARN Ribosómico 23S/genética; Thermus thermophilus/química

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ribosomas / Bacterias / Ingeniería de Proteínas / Biología Sintética Idioma: En Revista: Nat Commun Asunto de la revista: BIOLOGIA / CIENCIA Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido

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