TY - JOUR
T1 - A fully orthogonal system for protein synthesis in bacterial cells
AU - Aleksashin, Nikolay A.
AU - Szal, Teresa
AU - d’Aquino, Anne E.
AU - Jewett, Michael C.
AU - Vázquez-Laslop, Nora
AU - Mankin, Alexander S.
N1 - Funding Information:
We thank R. Green (Johns Hopkins University) for providing plasmids with some of the rRNA mutations; M. O’Connor (University of Missouri-Kansas City) for providing the plasmid with tRNA genes and technical advice; J. Williamson (Scripps Research Institute) for providing the plasmids with LuxR-based transcription system; R. Cruz-Vera for providing the tnaC plasmids and L-PSA; A. Saxena, X. Jiang and K. Gallik (University of Illinois at Chicago) for help with imaging of the GFP-expressing cells; M.Svetlov (University of Illinois at Chicago) for help with some experiments; Y.Polikanov (University of Illinois at Chicago) for support, discussions and critical reading of the manuscript. This work was supported by the National Science Foundation (grant MCB 1615851 to ASM and NV-L and grant MCB-1716766 to M.C.J.); the Human Frontiers Science Program (Grant RGP0015/2017 to M.C.J.), the Northwestern University Presidential Fellowship and the National Science Foundation Graduate Research Fellowship Program (both—to A.E.D.).
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-020-15756-1
DO - 10.1038/s41467-020-15756-1
M3 - Article
C2 - 32313034
AN - SCOPUS:85083802792
VL - 11
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 1858
ER -