A fully orthogonal system for protein synthesis in bacterial cells

Nikolay A Aleksashin,Anne E D’Aquino,Michael C Jewett,Nora Vázquez-Laslop,Alexander S Mankin,Teresa Szal

doi:10.1038/s41467-020-15756-1

Nikolay A Aleksashin, Anne E D’Aquino + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-15756-1

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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.

Highlights

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus
The growing amino acid chain advances from the peptidyl transferase center (PTC) into the nascent peptide exit tunnel (NPET) on its way out from the ribosome[3,4]
The ribosome has evolved to operate with its natural substrates enabling it to synthesize genetically encoded proteins

Summary

Introduction

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. 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. In Ribo-T and in subsequent similar a rrn ribo-T cp23S

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