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Title: Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

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 inmore » 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. In conclusion, 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.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [4] ;  [3] ;  [5] ;  [6] ;  [7] ;  [7] ;  [7] ;  [1]
  1. Harvard Medical School, Boston, MA (United States)
  2. Harvard Medical School, Boston, MA (United States); Ecole des Mines de Paris, Paris (France)
  3. Harvard Medical School, Boston, MA (United States); Harvard Univ., Cambridge, MA (United States)
  4. National Univ. of Singapore (Singapore)
  5. Harvard Medical School, Boston, MA (United States); Harvard Univ., Boston, MA (United States)
  6. Harvard Medical School, Boston, MA (United States); Harvard-MIT Health Sciences and Technology, Cambridge, MA (United States)
  7. Yale Univ., New Haven, CT (United States)
Publication Date:
Grant/Contract Number:
FG02-02ER63445
Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 38; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Harvard Medical School, Boston, MA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; codon choice; genome editing; recoded genomes
OSTI Identifier:
1321025
Alternate Identifier(s):
OSTI ID: 1469662

Napolitano, Michael G., Landon, Matthieu, Gregg, Christopher J., Lajoie, Marc J., Govindarajan, Lakshmi, Mosberg, Joshua A., Kuznetsov, Gleb, Goodman, Daniel B., Vargas-Rodriguez, Oscar, Isaacs, Farren J., Söll, Dieter, and Church, George M.. Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli. United States: N. p., Web. doi:10.1073/pnas.1605856113.
Napolitano, Michael G., Landon, Matthieu, Gregg, Christopher J., Lajoie, Marc J., Govindarajan, Lakshmi, Mosberg, Joshua A., Kuznetsov, Gleb, Goodman, Daniel B., Vargas-Rodriguez, Oscar, Isaacs, Farren J., Söll, Dieter, & Church, George M.. Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli. United States. doi:10.1073/pnas.1605856113.
Napolitano, Michael G., Landon, Matthieu, Gregg, Christopher J., Lajoie, Marc J., Govindarajan, Lakshmi, Mosberg, Joshua A., Kuznetsov, Gleb, Goodman, Daniel B., Vargas-Rodriguez, Oscar, Isaacs, Farren J., Söll, Dieter, and Church, George M.. 2016. "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli". United States. doi:10.1073/pnas.1605856113.
@article{osti_1321025,
title = {Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli},
author = {Napolitano, Michael G. and Landon, Matthieu and Gregg, Christopher J. and Lajoie, Marc J. and Govindarajan, Lakshmi and Mosberg, Joshua A. and Kuznetsov, Gleb and Goodman, Daniel B. and Vargas-Rodriguez, Oscar and Isaacs, Farren J. and Söll, Dieter and Church, George M.},
abstractNote = {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. In conclusion, 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.},
doi = {10.1073/pnas.1605856113},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 38,
volume = 113,
place = {United States},
year = {2016},
month = {9}
}

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