Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):E5588-97. doi: 10.1073/pnas.1605856113. Epub 2016 Sep 6.

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

Author information

1
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115;
2
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Systems Biology Graduate Program, Harvard Medical School, Boston, MA 02115; Ecole des Mines de Paris, Mines Paristech, 75272 Paris, France;
3
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115;
4
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Program in Chemical Biology, Harvard University, Cambridge, MA 02138; gchurch@genetics.med.harvard.edu mlajoie@uw.edu.
5
National University of Singapore, School of Computing, Singapore 117417;
6
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Program in Chemical Biology, Harvard University, Cambridge, MA 02138;
7
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Program in Biophysics, Harvard University, Boston, MA 02115;
8
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; Harvard-MIT Health Sciences and Technology, Cambridge, MA 02139;
9
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06516;
10
Molecular, Cellular, Developmental and Systems Biology Institute, Yale University, New Haven, CT 06516;
11
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06516; Department of Chemistry Yale University, New Haven, CT 06516.
12
Department of Genetics, Harvard Medical School, Boston, MA 02115; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115; gchurch@genetics.med.harvard.edu mlajoie@uw.edu.

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.

KEYWORDS:

codon choice; genome editing; recoded genomes

PMID:
27601680
PMCID:
PMC5035903
DOI:
10.1073/pnas.1605856113
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center