β-Puromycin Selection of Modified Ribosomes for in Vitro Incorporation of β-Amino Acids

Ribosomally mediated protein biosynthesis is limited to alpha-L-amino acids. A strong bias against beta-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated beta-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing beta-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of beta-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 beta-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to alpha-L-amino acids. beta-Puromycin, a putative mimetic for beta-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of beta-L-amino acids. Eight types of modified rib osomes altered within the PTC have been selected by monitoring improved sensitivity to beta-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 beta-alanyl-tRNA(CUA).