Abstract

Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome–mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.

Details

Title
Engineered ribosomes with tethered subunits for expanding biological function
Author
Carlson, Erik D 1 ; Anne E d’Aquino 2 ; Kim, Do Soon 3 ; Fulk, Emily M 3 ; Hoang, Kim 4 ; Szal, Teresa 5 ; Mankin, Alexander S 5   VIAFID ORCID Logo  ; Jewett, Michael C 2   VIAFID ORCID Logo 

1  Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA; Center for Synthetic Biology, Northwestern University, Evanston, IL, USA; Department of Chemical Engineering, Stanford University, Stanford, CA, USA 
2  Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA; Center for Synthetic Biology, Northwestern University, Evanston, IL, USA; Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL, USA 
3  Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA; Center for Synthetic Biology, Northwestern University, Evanston, IL, USA 
4  Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA; Department of Biology, Johnson and Wales University, Providence, RI, USA 
5  Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL, USA 
Pages
1-13
Publication year
2019
Publication date
Sep 2019
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41467-019-11427-y
ProQuest document ID
2283279167
Copyright
© 2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.