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).

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

“…Over a period of several years, we have reported the reengineering of Escherichia coli ribosomes, allowing them to incorporate α -D-amino acids 8,9 and β -amino acids, 10,11 while still incorporating α -L-amino acids with good fidelity. More recently, we have extended this strategy to the selection of ribosomes capable of incorporating dipeptides and dipeptide analogues from an activated suppressor tRNA into a specific site in a protein in a single ribosomal bond-forming event by suppression of a nonsense codon.…”

Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids

“…12,13 Scheme 1 illustrates the preparation of a suppressor tRNA CUA misacylated with β-amino acid 4 . The preparation of the remaining β-aminoacyl-tRNA CUA s starting from free β-amino acids is described in the Supporting Information (Schemes S1–S3).…”

“…These clones were identified previously, on the basis of a selection employing β 3 -puromycin. 12,13 Among the four methyl-β-alanine isomers examined, the best incorporation results were obtained with 4 (up to 19% suppression yield) using clone 040217. The incorporation efficiency of 3( S )-methyl-β-alanine ( 4 ) was 2–4 times higher than that of 3( R )-methyl-β-alanine ( 3 ).…”

“…During our first selection experiments using β 3 -puromycin, clones showing some (but <40%) inhibition of cell growth in the presence of β 3 -puromycin were not used for any further experiments. 12 It seemed logical to think that this pool of clones might be a good source of clones more sensitive to β 2 -puromycin. Therefore, we used this pool of clones for a cell growth inhibition assay in the presence of β 2 -puromycin; six clones were found to exhibit >40% inhibition of cell growth (Figure 5A).…”

“…Inhibition of cell growth by β 2 - and β 3 -puromycin was estimated as described previously. 12 Clones with an inhibition value of >50% were used for verification experiments. Cultures were diluted with an assay solution (LB medium supplemented with 100 μg/mL ampicillin and 1 mM IPTG) until the OD 600 value was ∼0.01 and placed in eight wells of a 96-well plate, having six different dilutions of β 2 - or β 3 -puromycin (dilution from 200 to 6.25 μg/mL).…”