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

ABSTRACT: In an earlier study, ?³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different ?-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate ?-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 ?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 tRNA(CUA) activated with each of four methyl-?-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-?-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-?-alanine (?-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated ?-tyrosine moiety of ?³-puromycin. Also conducted were a selection of clones that are responsive to ?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-?-alanine (?-mAla; 4) into a short ?-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

SUBMITTER: Maini R 

PROVIDER: S-EPMC4472090 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature