sb2c00150_si_001.pdf (1.6 MB)
Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming
journal contribution
posted on 2022-05-12, 17:16 authored by Bipasana Shakya, Olivia G. Joyner, Matthew C. T. HartmanThe 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.
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peptide drug discoverynoncanonical amino acidsexpanded anticodon stemenabled exciting advancesquadruplet codon readthrougherror restrictive ribosomescognate readthroughsynthetic biologysurprising extentsignificant improvementsmutation alongsideimpressive improvementsimportant advancehigh efficiencygenetic codedisubstituted ncaacompeting processescompeting pathwayscanonical aasalso showaffinity tags