Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code

  title={Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code},
  author={J. D. Bain and Christopher Y. Switzer and Richard Chamberlin and Steven A. Bennert},
ONE serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a… 
Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins
The current status of methods to incorporate new amino acids into proteins by in vitro and in vivo translation systems are introduced, by focusing on the creation of new codon-anticodon interactions, including unnatural base pair systems for genetic alphabet expansion.
Five-base codons for incorporation of nonnatural amino acids into proteins.
A novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression is provided by using five-base codon-anticodon pairs.
Strategies for in vitro and in vivo translation with non-natural amino acids.
  • M. Ibba
  • Biology, Chemistry
    Biotechnology & genetic engineering reviews
  • 1996
Recent progress in the understanding of aminoacyl-tRNA synthetase-catalyzed tRNA charging suggests that it may ultimately be possible to extend site-directed mutagenesis to growing cells.
Expanding the genetic code.
Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids.
Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs
The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.
Expanding the Genetic Code of Escherichia coli
A unique transfer RNA/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli and should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.
Incorporation of non-natural amino acids into proteins.
An unnatural base pair for incorporating amino acid analogs into proteins
This coupled transcription–translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position in an Escherichia coli cell-free system.
Reprogramming the genetic code
The ability to reprogramme cellular translation and genomes to produce non-canonical biopolymers has wide-ranging applications, including in therapeutics, but has yet to be fully realized.


A general method for site-specific incorporation of unnatural amino acids into proteins.
The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.
Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.
A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine, which resulted in the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation.
Enzymatic incorporation of a new base pair into DNA and RNA
D-iso-G was found at the correct position in the product oligonucleotide by a “nearest-neighbor” analysis9 and by the “minus” sequencing method of Sanger to determine the specificity with which the new bases pair.
Translational frameshifting generates the gamma subunit of DNA polymerase III holoenzyme.
The results suggest that a -1 frameshift during translation allows the use of this UGA codon to terminate translation of the gamma polypeptide.
A general and efficient route for chemical aminoacylation of transfer RNAs
These protocols greatly simplify the use of chemically misacylated tRNAs in the synthesis of proteins containing unnatural amino acids, as well as in studies of protein biosynthesis.
Ribosome-catalyzed formation of an abnormal peptide analogue.
The peptidyl-tRNA analogue N-(chloroacetyl)phenylalanyl-tRNAPhe was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid), which constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity.
Template activity of modified terminator codons.
"Chemical aminoacylation" of tRNA's.
Comparing chromatographic properties on benzolated diethylaminoethyl-cellulose, rates of chemical deacylation, and affinities for elongation factor Tu revealed the ability of misacylated tRNA's so derived to be deacylated chemically and then reactivated enzymatically with their cognate amino acids.