Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming

Abstract

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.

Continuous Fluorescence Assay for In Vitro Translation Compatible with Noncanonical Amino Acids

“…34 One notable difference between our study and his is our use of error-restrictive or hyperaccurate ribosomes. 42,48 These ribosomes enable enhanced discrimination against tRNAs within a single codon box, 49 which bodes well for downstream experiments aimed to extensively break the degeneracy of the genetic code.…”

“…The multiple peaks made interpretation of the fidelity difficult. To solve this problem, we turned to error-restrictive ribosomes which are more stringent toward near-cognate AA-tRNAs and have been shown to eliminate these issues . Here, we used the well-characterized S12 ribosomal protein mutant K42T …”

“…To solve this problem, we turned to error-restrictive ribosomes which are more stringent toward near-cognate AA-tRNAs and have been shown to eliminate these issues. 42 Here, we used the well-characterized S12 ribosomal protein mutant K42T. 43 It is notable that fully modified tRNAs have widened codon reading preferences relative to their in vitro transcribed counterparts.…”

“…The corresponding MALDI experiments are shown on the right. We initially performed these experiments with wild-type ribosomes; however, we noticed that MALDI analysis showed multiple peaks indicative of His to Gln mutations, which have been observed previously in short his-tagged templates. The multiple peaks made interpretation of the fidelity difficult.…”

SynBio 2.0, a new era for synthetic life: Neglected essential functions for resilience

“…The most advanced work is with Mycoplasmas, with entirely recoded genomes (see Talenton et al, 2022, for an example of the most recent trends), and this type of work has also been extended to the unicellular eukaryote, Saccharomyces cerevisiae (Zhang et al, 2020), with shuffling genes and merging its chromosomes into a smaller number (Wang et al, 2018). A widely explored line is the follow-up of research into the general exploration of the genetic code alteration (see, e.g., Shakya et al, 2022). This goes hand in hand with orthogonal implementation of specific replication/transcription and translation pathways.…”