Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center

Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions + beta-alanyl-tRNA(CUA), the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted beta-amino acids, and direct evidence for beta-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted beta-amino acids, including alpha-methyl-beta-alanine, beta,beta-dimethyl-beta-alanine, beta-phenylalanine, and beta-(p-bromophenyl)alanine. The beta-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of beta-alanine and representative alpha-L-amino acids. The isolated proteins containing the modified beta-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the beta-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of beta,beta-dimethyl-beta-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the beta-amino acid. (C) 2013 Elsevier Ltd. All rights reserved.