A fully orthogonal system for protein synthesis in bacterial cells

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.

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

“…5d ). Mutating U2585 partially releases translation termination arrest on tnaC , in agreement with the proposed functional role for this residue 34 . Finally, the side chain of TnaC residue Phe23 would clash with methylated Gln252 of the RF2 GGQ loop 35 , likely preventing RF2 from accommodating fully into the PTC (Fig.…”

Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro

“…In addition, in vitro ribosome assembly 49 and selection 50 platforms could evolve ribosomes with altered properties that increase incorporation efficiency of the backbone-extended monomers into peptides (i.e., form less truncated products) and facilitate the synthesis of polymers comprised solely of such monomers. Finally, extension to cellular systems with orthogonal engineered tethered, or stapled, ribosomes 51 – 55 offers another exciting direction. However, the lack of aminoacyl tRNA-synthetases (aaRS) that charge the monomers into tRNA in the cell will need to be addressed.…”

A call for caution in analysing mammalian co-transfection experiments and implications of resource competition in data misinterpretation

“…Such designs have been demonstrated not only to provide stable and reliable expression, but also to reduce the burden imposed on the native machinery, thus representing a win-win approach that can prove useful to reduce experimental variability. Examples in bacteria from Cameron et al 23 , Aleksashin et al 24 and Carlson et al 25 show how orthogonal protease-based or ribosome-based decoupling of circuit function can lead to controlled expression. Similarly, we speculate that other orthogonal components may prove beneficial, such as unnatural nucleotides that go on to generate polypeptides containing unnatural amino acids 26 , thus relieving the endogenous pool of natural precursors from feeding into the synthetic system.…”