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A fully orthogonal system for protein synthesis in bacterial cells.
- Source :
-
Nature communications [Nat Commun] 2020 Apr 20; Vol. 11 (1), pp. 1858. Date of Electronic Publication: 2020 Apr 20. - Publication Year :
- 2020
-
Abstract
- 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.
- Subjects :
- Alleles
Cell-Free System
Crystallography, X-Ray
Models, Molecular
Models, Theoretical
Molecular Conformation
Mutation
Peptides chemistry
Peptidyl Transferases chemistry
Plasmids genetics
Protein Biosynthesis
Proteome
RNA, Messenger genetics
RNA, Ribosomal genetics
RNA, Ribosomal, 23S genetics
Thermus thermophilus chemistry
Bacteria metabolism
Protein Engineering methods
Ribosomes chemistry
Synthetic Biology methods
Subjects
Details
- Language :
- English
- ISSN :
- 2041-1723
- Volume :
- 11
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Nature communications
- Publication Type :
- Academic Journal
- Accession number :
- 32313034
- Full Text :
- https://doi.org/10.1038/s41467-020-15756-1