Measuring the tolerance of the genetic code to altered codon size

  1. Erika Alden DeBenedictis  Is a corresponding author
  2. Dieter Söll
  3. Kevin M Esvelt
  1. Massachusetts Institue of Technology, United States
  2. Yale University, United States
  3. Massachusetts Institute of Technology, United States


Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the Escherichia coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

Data availability

All luminescence raw data are compiled in Figure 5A and provided as Source Data 1. Raw spectra have been deposited in the PRIDE database, dataset identifier PXD031925 and 10.6019/PXD031925.

The following data sets were generated

Article and author information

Author details

  1. Erika Alden DeBenedictis

    Department of Biological Engineering, Massachusetts Institue of Technology, Cambridge, United States
    For correspondence
    Competing interests
    Erika Alden DeBenedictis, filed US Patent 16405380 on tRNA sequences engineered in this work...
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7933-2651
  2. Dieter Söll

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3077-8986
  3. Kevin M Esvelt

    Department of Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    Kevin M Esvelt, filed US Patent 16405380 on tRNA sequences engineered in this work...
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8797-3945


National Institute of General Medical Sciences (R35GM122560)

  • Dieter Söll

National Institute of General Medical Sciences (3R35GM122560-05W1)

  • Dieter Söll

National Institute of Allergy and Infectious Diseases (F31 AI145181-01)

  • Erika Alden DeBenedictis

National Institute of Diabetes and Digestive and Kidney Diseases (R00 DK102669-01)

  • Kevin M Esvelt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Timothy W Nilsen, Case Western Reserve University, United States

Version history

  1. Preprint posted: April 26, 2021 (view preprint)
  2. Received: January 10, 2022
  3. Accepted: March 15, 2022
  4. Accepted Manuscript published: March 16, 2022 (version 1)
  5. Version of Record published: May 11, 2022 (version 2)


© 2022, DeBenedictis et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.


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  1. Erika Alden DeBenedictis
  2. Dieter Söll
  3. Kevin M Esvelt
Measuring the tolerance of the genetic code to altered codon size
eLife 11:e76941.

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