Initiating protein synthesis with noncanonical monomers in vitro and in vivo.

Autor:	Tharp JM; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, United States., Walker JA; Department of Chemistry, University of California, Berkeley, CA, United States., Söll D; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, United States; Department of Chemistry, Yale University, New Haven, CT, United States. Electronic address: dieter.soll@yale.edu., Schepartz A; Department of Chemistry, University of California, Berkeley, CA, United States; Department of Molecular & Cell Biology, University of California, Berkeley, CA, United States. Electronic address: schepartz@berkeley.edu.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2021; Vol. 656, pp. 495-519. Date of Electronic Publication: 2021 Jun 02.
DOI:	10.1016/bs.mie.2021.05.002
Abstrakt:	With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNA fMet . By mis-acylating tRNA fMet , we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNA fMet , prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers. (Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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