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

In an earlier study, beta(3)-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different beta-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate beta-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the beta(3)-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNACUA activated with each of four methyl-beta-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-beta-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-beta-alanine (beta-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated beta-tyrosine moiety of beta(3)-puromycin. Also conducted were a selection of clones that are responsive to beta(2)-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-beta-alanine (beta-mAla; 4) into a short a-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.