Multiple Amino Acid-Excluded Genetic Codes for Protein Engineering Using Multiple Sets of tRNA Variants

Amikura, Kazuaki; Shibata, Yoko; Asami, Shun; Kiga, Daisuke

doi:10.1021/sb400144h

Cited by 6 publications

(6 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Some degree of mutation that leads to undesirable non‐alphabet codons is inevitable during library synthesis and propagation. However, techniques such as controlling translation conditions through cell‐free expression systems could further improve the adherrance of a library to its alphabet in future applications.…”

Section: Discussionsupporting

confidence: 73%

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

et al. 2019

View full text Add to dashboard Cite

The universal genetic code of 20 amino acids is the product of evolution. It is believed that earlier versions of the code had fewer residues. Many theories for the order in which amino acids were integrated into the code have been proposed, considering factors ranging from prebiotic chemistry to codon capture. Several meta‐analyses combined these theories to yield a feasible consensus chronology of the genetic code's evolution, but there is a dearth of experimental data to test the hypothesised order. We used combinatorial chemistry to synthesise libraries of random polypeptides that were based on different subsets of the 20 standard amino acids, thus representing different stages of a plausible history of the alphabet. Four libraries were comprised of the five, nine, and 16 most ancient amino acids, and all 20 extant residues for a direct side‐by‐side comparison. We characterised numerous variants from each library for their solubility and propensity to form secondary, tertiary or quaternary structures. Proteins from the two most ancient libraries were more likely to be soluble than those from the extant library. Several individual protein variants exhibited inducible protein folding and other traits typical of intrinsically disordered proteins. From these libraries, we can infer how primordial protein structure and function might have evolved with the genetic code.

show abstract

Section: Discussionsupporting

confidence: 73%

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Another possibility is suggested by the existence of simplified genetic codes 4 : if codes started with few amino acids, then optimality could be an emergent consequence of code expansion from early simple ones to the standard genetic code, whereby new amino acids were assigned to codons that had previously encoded chemically similar amino acids 5 . This theory appeals to mutation, and in some cases selection 6 , to account for the inclusion of new amino acids, and therefore still assigns a key role to vertical descent, but some of its versions are neutral with respect to the role of selection 7 .…”

Section: Introductionmentioning

confidence: 99%

Horizontal transfer of code fragments between protocells can explain the origins of the genetic code without vertical descent

Froese

Campos

Fujishima

et al. 2018

Sci Rep

Self Cite

View full text Add to dashboard Cite

Theories of the origin of the genetic code typically appeal to natural selection and/or mutation of hereditable traits to explain its regularities and error robustness, yet the present translation system presupposes high-fidelity replication. Woese’s solution to this bootstrapping problem was to assume that code optimization had played a key role in reducing the effect of errors caused by the early translation system. He further conjectured that initially evolution was dominated by horizontal exchange of cellular components among loosely organized protocells (“progenotes”), rather than by vertical transmission of genes. Here we simulated such communal evolution based on horizontal transfer of code fragments, possibly involving pairs of tRNAs and their cognate aminoacyl tRNA synthetases or a precursor tRNA ribozyme capable of catalysing its own aminoacylation, by using an iterated learning model. This is the first model to confirm Woese’s conjecture that regularity, optimality, and (near) universality could have emerged via horizontal interactions alone.

show abstract

“…This is an important issue as tRNAs are known to undergo more than 85 secondary modifications, with some of the identified tRNA modifications shown to affect tRNA folding, structure, and function. , However, only tRNAs for Ile, Glu, and Lys require modifications for efficient aminoacylation by their respective aaRSes . T7 RNA polymerase-synthesized tRNAs (t7tRNAs) are widely used in cell-free translation systems for genetic reprogramming or genetic code simplification . In the latter case, several codons were reassigned to Ser or Ala by grafting the heterologous anticodons into Ser or Ala tRNAs respectively, , and the results strongly suggested that, at least in some cases, t7tRNAs could functionally replace endogenous tRNAs.…”

Section: Introductionmentioning

confidence: 99%

Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis

et al. 2015

View full text Add to dashboard Cite

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

show abstract

Multiple Amino Acid-Excluded Genetic Codes for Protein Engineering Using Multiple Sets of tRNA Variants

Cited by 6 publications

References 27 publications

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

Horizontal transfer of code fragments between protocells can explain the origins of the genetic code without vertical descent

Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis

Contact Info

Product

Resources

About