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Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

ABSTRACT: Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for tRNA capture and applied it to the isolation of roughly half of the Escherichia coli tRNA isoacceptors. We then performed codon competition experiments between the five captured wild-type leucyl-tRNAs and their synthetic counterparts, revealing a strong preference for wild-type tRNA in an in vitro translation system. Finally, we compared the ability of wild-type and synthetic leucyl-tRNA to break the degeneracy of the leucine codon box, showing that only captured wild-type tRNAs are discriminated with enough fidelity to accurately split the leucine codon box for the encoding of three separate amino acids. Wild-type tRNAs are therefore enabling reagents for maximizing the reassignment potential of the genetic code.

SUBMITTER: McFeely CAL

PROVIDER: S-EPMC9638912 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

McFeely Clinton A L CAL Dods Kara K KK Patel Shivam S SS Hartman Matthew C T MCT

Nucleic acids research 20221001 19

Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for ...[more]

PMID: 36300637

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Project description:The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal Methanocaldococcus jannaschii tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the E. coli translation machinery decoding tyrosine codons. Mutations to the M. jannaschii aaRS that were found to improve tyrosine incorporation were transplanted onto a M. jannaschii aaRS evolved for the incorporation of para-azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, E. coli strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory E. coli strain in sense codon reassignment.

Dataset Information

Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

Publications

Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

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