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

Rumit Maini, Sandipan Roy Chowdhury, Larisa Dedkova, Basab Roy, Sasha M. Daskalova, Rakesh Paul, Shengxi Chen, Sidney Hecht

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

In an earlier study, β3-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-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 β3-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-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β3-puromycin. Also conducted were a selection of clones that are responsive to β2-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-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

Original languageEnglish (US)
Pages (from-to)3694-3706
Number of pages13
JournalBiochemistry
Volume54
Issue number23
DOIs
StatePublished - Jun 16 2015

ASJC Scopus subject areas

  • Biochemistry

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