β-puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids

Larisa Dedkova, Nour Eddine Fahmi, Rakesh Paul, Melissa Del Rosario, Liqiang Zhang, Shengxi Chen, Glen Feder, Sidney Hecht

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against β-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated β-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing β-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of β-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence β-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. β-Puromycin, a putative mimetic for β-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of β-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to β-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of β-alanyltRNA CUA.

Original languageEnglish (US)
Pages (from-to)401-415
Number of pages15
JournalBiochemistry
Volume51
Issue number1
DOIs
StatePublished - Jan 10 2012

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Puromycin
Ribosomes
Peptidyl Transferases
Amino Acids
Transfer RNA
Amino Acid-Specific Transfer RNA
Proteins
Bearings (structural)
Scorpions
Tetrahydrofolate Dehydrogenase
Terminator Codon
Mutagenesis
Protein Biosynthesis
In Vitro Techniques
Biosynthesis
Libraries
Escherichia coli
X-Rays
Messenger RNA
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

β-puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids. / Dedkova, Larisa; Fahmi, Nour Eddine; Paul, Rakesh; Del Rosario, Melissa; Zhang, Liqiang; Chen, Shengxi; Feder, Glen; Hecht, Sidney.

In: Biochemistry, Vol. 51, No. 1, 10.01.2012, p. 401-415.

Research output: Contribution to journalArticle

Dedkova, Larisa ; Fahmi, Nour Eddine ; Paul, Rakesh ; Del Rosario, Melissa ; Zhang, Liqiang ; Chen, Shengxi ; Feder, Glen ; Hecht, Sidney. / β-puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids. In: Biochemistry. 2012 ; Vol. 51, No. 1. pp. 401-415.
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