Probing Life's Genetic Limits

Life, at some point, instead decided that a triplet code is sufficient. A new study breaks down this evolutionary path in greater detail.

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I only work on big problems.
H. Gobind Khorana

The early 1960s was a transformative period in biochemistry. Khorana, Nirenberg, Holley (and many others) were busy cracking the genetic code: namely, how a cell 'reads' the message in an RNA polymer, and converts its four-letter language into a dazzling assortment of proteins.

In a span of about four years, they solved the problem and, together, earned the 1968 Nobel Prize in Physiology or Medicine. Their result was the "64-codon" table that so many biochemistry students are familiar with today — AGA encodes arginine, CUU leucine, UAG stop.

Khorana ended his Nobel acceptance speech with a look towards the future:

" is not inconceivable that the laboratory synthesis of specific proteins will be carried out using nucleic acid templates. For this purpose, protected trinucleotides representing different codons will be made in quantity and on a commercial basis, and these will be used in the synthesis of nucleic acid templates for proteins, the approach offering flexibility and selectivity in amino acid substitutions at the template level."

A prescient observation, to be sure, and a foundation on which all of synthetic biology is built.

Still, there appears to be no fundamental reason as to why life evolved triplet codons, as opposed to a 'quadruplet' code. The latter, at least, would enable more permissive incorporation of amino acids into proteins, expanding the theoretical number of available codons to 256. There would be hundreds of new codons, in other words, that could encode a vast milieu of amino acids, thus creating proteins with alien-like functions.

Life, at some point, instead decided that a triplet code is sufficient. A new study breaks down this evolutionary path in greater detail.

Credit: National Human Genome Research Institute

The Case for Quadruplet Codons

To read the genetic code, mRNA strands are shuttled through a ribosome. tRNAs crash and tumble into that protein bohemoth, carefully checking whether the three letters stamped on their anticodon loop matches up to the mRNA triplet presented in the ribosome.

If the triplets pair up, the tRNA releases its amino acid and adds it to a growing protein chain. A class of proteins called aminoacyl-tRNA synthetases keep this process running by grabbing amino acids and ligating them to depleted tRNA carriers.

From a bird's eye view, then, only a few ingredients are required to read life's genetic code: mRNA sequences, ribosomes, tRNAs and aminoacyl-tRNA synthetases.

For a new paper in eLife, researchers at MIT and Yale University — Erika Alden DeBenedictis, Dieter Söll and Kevin Esvelt — probed the limits of this triplet code.

The researchers "systematically explored whether quadruplet codon translation can arise through simple point insertions in each of the tRNA anticodon loops," for instance, and "then used directed evolution to determine how often additional mutations throughout the tRNA can improve translation of the  resulting quadruplet" tRNAs. Often, tRNAs could be converted to a 'quadruplet form' by introducing just a few mutations (often, at positions 32, 37 and 38).

The researchers found that 12 of the 20 tRNA classes could be easily converted to a quadruplet form in E. coli bacteria, albeit with low efficiency. Indeed, "the presence of a single quadruplet codon in an mRNA transcript can reduce total protein yield to less than 3% relative to an all-triplet mRNA," the researchers wrote. Changing a tRNA in this way, unsurprisingly, caused severe growth defects.

In a further experiment, the researchers took their new tRNAs and used them to translate an mRNA sequence encoding green fluorescent protein. This mRNA had a quadruplet codon at residue 151, and mass spectral analysis was used to determine which amino acid, if any, was incorporated into the protein at that position.

Eight of the quadruplet tRNAs ended up incorporating the same amino acid that they would have introduced with a triplet code. In other words, changing them into a quadruplet tRNA didn't change anything. An additional four quadruplet tRNAs incorporated Arg, rather than their normal amino acid. Another two quadruplet tRNAs had a mixture; sometimes they carried their normal amino acid, and other times they became charged with Arg.

So why this preference, in the quadruplet code, for arginine? Well, the aminoacyl-tRNA synthetase that ligates argnine to tRNAs is responsible for the recognition of six codons in the triplet code, "including tRNAs that differ at both the first and last position of the anticodon," the authors wrote. "As a consequence, the identity of just one anticodon position is invariant. Examination of the [quadruplet tRNAs] charged with Arg shows that all satisfy this anticodon identity element, causing widespread promiscuous charging with Arg."

This study is broad and comprehensive; a benchmark for future efforts to expand the genetic code.

Summary: A majority of tRNAs can be easily converted to a form that recognizes quadruplet codons in E. coli. Often, this shift can be made by introducing just a few mutations into the anticodon loop.

Read more in eLife.

Other Papers

(* = open access, † = review article)

Basic Research

Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Dhainaut M...Brown BD. Cell. Link

A genome-scale screen for synthetic drivers of T cell proliferation. Legut M...Sanjana NE. Nature. Link

High-throughput biochemical profiling reveals functional adaptation of a bacterial Argonaute. Ober-Reynolds B...Greenleaf WJ. Molecular Cell. Link

Effects of Hydrophobic Residues on the Intracellular Self-Assembly of De Novo Designed Peptide Tags and Their Orthogonality. Miki T...Mihara H. ACS Synthetic Biology. Link


*†Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases. Kabay G...Dincer C. Advanced Materials. Link

*PeroxiHUB: a modular cell-free biosensing platform using H2O2 as signal integrator. Soudier P...Faulon J. bioRxiv (preprint). Link

*Identification and development of a glucaric acid biosensor in Saccharomyces cerevisiae. Su R...Deng Y. Systems Microbiology and Biomanufacturing. Link

*A synthetic switch based on orange carotenoid protein to control blue-green light responses in chloroplasts. Piccinini L...Licausi F. Plant Physiology. Link

Cell-Free Systems

*Expression of a gene-encoded FtsZ-based minimal machinery to drive synthetic cell division. Godino E. Thesis (TU Delft). Link


Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression. Benzinger D, Ovinnikov S & Khammash M. Cell Systems. Link

*CRISPR signal conductor 2.0 for redirecting cellular information flow. Zhan Y...Liu Y. Cell Discovery. Link

*Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics. Ma D...Green AA. Nature Biomedical Engineering. Link

Computation & Models

*Machine learning discovery of missing links that mediate alternative branches to plant alkaloids. Vavricka CJ...Hasunuma T. Nature Communications. Link

*A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity. Eslami-Mossallam B...Depken M. Nature Communications. Link

*Predicting exon criticality from protein sequence. Desai J...Hoss A. Nucleic Acids Research. Link

*Predicting base editing outcomes using position-specific sequence determinants. Pallaseni A...Parts L. Nucleic Acids Research. Link

CRISPR & Genetic Engineering

*FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity. Cui Z...Hu Z. Nature Communications. Link

*Rational design of Cas9 ribonucleoprotein with a “gRNA-shRNA” for multidimensional genome manipulation and enhanced homology-directed repair. Qiao J...Liu Y. bioRxiv (preprint). Link

Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum. Ye Y...Wang Y. ACS Synthetic Biology. Link

*A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants. Xiong X...Li J. Nucleic Acids Research. Link

Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells. Lu D...Hathaway NA. ACS Synthetic Biology. Link

*Self-Targeting Type IV CRISPR interference in Pseudomonas oleovorans. Guo X...Randau L. Research Square (preprint). Link

A CRISPR–Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola. Shi Y...Chen X. ACS Synthetic Biology. Link

DNA Assembly

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms. Pang Q...Qi Q. ACS Synthetic Biology. Link

Medicine & Diagnostics

Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A. Ozelo MC, et al. The New England Journal of Medicine. Link

CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver. Kaltenbacher T...Rad R. Nature Protocols. Link

*A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Harimoto T...Danino T. Nature Biotechnology. Link

*Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors. Wang Y...Liu J. Nature Communications. Link

In vivo prime editing of a metabolic liver disease in mice. Böck D...Schwank G. Science Translational Medicine. Link

In vitro analysis of genome-engineered muscle-derived stem cells for autoregulated anti-inflammatory and antifibrotic activity. Pferdehirt L...Huard J. Journal of Orthopaedic Research. Link

*†Carrier strategies boost the application of CRISPR/Cas system in gene therapy. Xu Z...Yang Y. Exploration. Link

Metabolic Engineering

*DNA-based platform for efficient and precisely targeted bioorthogonal catalysis in living systems. You Y...Qu X. Nature Communications. Link

*Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters. Seo H...Trinh CT. bioRxiv (preprint). Link

*Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage. Praznik A...Jerala R. Nature Communications. Link

Engineered Production of Strictosidine and Analogues in Yeast. Misa J...Tang Y. ACS Synthetic Biology. Link

*Modular engineering of E. coli coculture for efficient production of resveratrol from glucose and arabinose mixture. Li J, Qiu Z & Zhao G. Synthetic and Systems Biotechnology. Link

*Engineering microbial consortia of Elizabethkingia meningoseptica and Escherichia coli strains for the biosynthesis of vitamin K2. Yang Q...Wang P. Microbial Cell Factories. Link

Tools & Technology

*On-Ramp: a tool for rapid, multiplexed validation of plasmids using nanopore sequencing. Mumm C...Boyle AP. bioRxiv (preprint). Link

*clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ. Dardani I...Raj A. bioRxiv (preprint). Link

*Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR. Hernández-Rollán C, Ehrmann AK & Nørholm MH. bioRxiv (preprint). Link


†Protein degradation on the global scale. Rusilowicz-Jones EV, Urbé S & Clague MJ. Molecular Cell. Link

*Population genetics in microchannels. Koldaeva A...Pigolotti S. PNAS. Link

Thanks for reading.

— Niko // @NikoMcCarty