Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli
Name
Napolitano-2016-Emergent rules for c.pdf
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Author(s)
Napolitano, Michael G.
Landon, Matthieu
Gregg, Christopher J.
Lajoie, Marc J.
Govindarajan, Lakshmi
Mosberg, Joshua A.
Kuznetsov, Gleb
Vargas-Rodriguez, Oscar
Isaacs, Farren J.
Söll, Dieter
Date Issued
September 2016
Journal
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences (U.S.)
Citation
Napolitano, Michael G.; Landon, Matthieu; Gregg, Christopher J.; Lajoie, Marc J.; Govindarajan, Lakshmi; Mosberg, Joshua A.; Kuznetsov, Gleb et al. “Emergent Rules for Codon Choice Elucidated by Editing Rare Arginine Codons inEscherichia Coli.” Proceedings of the National Academy of Sciences 113, no. 38 (September 2016): E5588–E5597. © National Academy of Sciences
Version
Final published version
Abstract
The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 “recalcitrant” AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.
MIT Department
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Harvard University--MIT Division of Health Sciences and Technology
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DOI of Published Version
https://doi.org/10.1073/pnas.1605856113
