Unexpected Preference of the <i>E. coli</i> Translation System for the Ester Bond during Incorporation of Backbone-Elongated Substrates

Sando, Shinsuke; Abe, Kenji; Sato, Naomi; Shibata, Toshihiro; Mizusawa, Keigo; Aoyama, Yasuhiro

doi:10.1021/ja068033n

Cited by 18 publications

(14 citation statements)

References 47 publications

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“…This is consistent with our finding that β9 is able to serve as an initiator amino acid, albeit inefficiently. Very recently, Sando and coworkers have found that β−hydroxy acids are competent translation substrates and have suggested that the elevated pKa of the amine in β−amino acids prevents deprotonation and peptidyl transfer [53]. If this interpretation is correct, ribosome engineering may be required to enable the efficient incorporation of β-amino acids into peptides.…”

Section: Resultsmentioning

confidence: 99%

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An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides

et al. 2007

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BackgroundThe application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA.Methodology/Principal FindingsWe have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few α,α disubstituted, N-methyl and α-hydroxy derivatives, but no β-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids.Conclusions/SignificanceOur results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides.

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Section: Resultsmentioning

confidence: 99%

“…By default, poor efficiency of tRNA charging may be the limiting factor. In contrast, the β-amino acids are likely to be discriminated against by the active site of the peptidyl transferase center, possibly due to a higher pKa of the amino group [53].…”

Section: Discussionmentioning

confidence: 99%

An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides

et al. 2007

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“…A related, but technically more complex challenge would involve the assembly of proteins in which the linkages between “amino acid” residues were altered. This has been achieved using native ribosomes for the synthesis of proteins having some ester linkages, − and for other functional groups as well, , albeit generally only in low yield. Even for linkages that probably cannot be realized directly, such as thioamides, the use of dipeptidomimetic cassettes could be useful to obtain the desired products, as exemplified in Figure .…”

Section: Opportunities and Challengesmentioning

confidence: 99%

Expanding the Scope of Protein Synthesis Using Modified Ribosomes

Dedkova

Hecht

2019

J. Am. Chem. Soc.

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The ribosome produces all of the proteins and many of the peptides present in cells. As a macromolecular complex comprised of both RNAs and proteins, it employs a constituent RNA to catalyze the formation of peptide bonds rapidly and with high fidelity. Thus the ribosome can be argued to represent the key link between the RNA World, in which RNAs were the primary catalysts, and present biological systems in which protein catalysts predominate. In spite of the well known phylogenetic conservation of ribosomal RNAs through evolutionary history, ribosomal RNAs can be altered readily when placed under suitable pressure, e.g. in the presence of antibiotics which bind to functionally critical regions of rRNAs. While the structures of rRNAs have been altered intentionally for decades to enable the study of their role(s) in the mechanism of peptide bond formation, it is remarkable that the purposeful alteration of rRNA structure to enable the elaboration of proteins and peptides containing non-canonical amino acids has occurred only recently. In this Perspective, we summarize the history of rRNA modifications, and demonstrate how the intentional modification of 23S rRNA in regions critical for peptide bond formation now enables the direct ribosomal incorporation of D-amino acids, β-amino acids, dipeptides and dipeptidomimetic analogues of the normal proteinogenic L-α-amino acids. While proteins containing metabolically important functional groups such as carbohydrates and phosphate groups are normally elaborated by the post-translational modification of nascent polypeptides, the use of modified ribosomes to produce such polymers directly is also discussed. Finally, we describe the elaboration of such modified proteins both in vitro and in bacterial cells, and suggest how such novel biomaterials may be exploited in future studies.

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“…Stimulated by Foster's experiment, several groups have started work on further development of geneticcode reprogramming. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] For nearly ten years, we have engaged in a project to develop artificial ribozymes, called "flexizymes", which are capable of charging amino acids onto tRNA. [16,[26][27][28][29][30][31] The latest version of the flexizyme system enables us to charge virtually any amino acid, including nonproteinogenic ones, onto tRNA that bear various anticodons.…”

Section: Wwwchembiochemorgmentioning

confidence: 99%

Polymerization of α‐Hydroxy Acids by Ribosomes

2008

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Over 30 years ago, Fahnestock and Rich reported intriguing data showing the capability of the ribosome to polymerize phenyllactic acid. Although the polymerization was initiated and terminated randomly on polyuridic acids, the given data convincingly suggested that the generated polymer was composed of an approximately 7:3 mixture of phenyllactic acid and phenylalanine. Despite the fact that Fahnestock's conclusion was very likely correct, there have been no reports to follow up the ribosome-catalyzed polymerization of alpha-hydroxy acids until very recently. At the end of 2007, we reported messenger RNA (mRNA)-directed polyester synthesis by using the new emerging method of genetic-code reprogramming in which alpha-hydroxy acids with various kinds of side-chains are assigned to arbitrarily chosen codons. In this work, we have achieved the ribosomal synthesis of polyesters with the sequence composition and length in a fully controlled manner according to the sequence of mRNA. This Concept article describes the background of the method development and its application to the synthesis of polyesters.

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Unexpected Preference of the E. coli Translation System for the Ester Bond during Incorporation of Backbone-Elongated Substrates

Cited by 18 publications

References 47 publications

An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides

An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides

Expanding the Scope of Protein Synthesis Using Modified Ribosomes

Polymerization of α‐Hydroxy Acids by Ribosomes

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