title: β-Puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids.
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-alanyl-tRNA(CUA).
authors.affiliation: Center for BioEnergetics, Biodesign Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Larisa M
authors.initials: LM
authors.lastname: Dedkova
authors.forename: Nour Eddine
authors.initials: NE
authors.lastname: Fahmi
authors.forename: Rakesh
authors.initials: R
authors.lastname: Paul
authors.forename: Melissa
authors.initials: M
authors.lastname: del Rosario
authors.forename: Liqiang
authors.initials: L
authors.lastname: Zhang
authors.forename: Shengxi
authors.initials: S
authors.lastname: Chen
authors.forename: Glen
authors.initials: G
authors.lastname: Feder
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry; D000818:Animals; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010458:Peptidyl Transferases / Q000737:chemistry; D011692:Puromycin Aminonucleoside / Q000031:analogs & derivatives* / Q000737:chemistry*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D058928:Riboswitch / Q000235:genetics; D012605:Scorpions / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics; D012407:rRNA Operon / Q000235:genetics
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; D058928:Riboswitch; D011692:Puromycin Aminonucleoside; D013762:Tetrahydrofolate Dehydrogenase; D010458:Peptidyl Transferases; D000604:Amino Acyl-tRNA Synthetases