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

25 Citations (Scopus)

Abstract

In an earlier study, β<sup>3</sup>-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 β<sup>3</sup>-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<inf>CUA</inf> 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 β<sup>3</sup>-puromycin. Also conducted were a selection of clones that are responsive to β<sup>2</sup>-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

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Puromycin
Ribosomes
Alanine
Tetrahydrofolate Dehydrogenase
Amino Acids
Clone Cells
Proteins
Isomers
Heterogeneous-Nuclear Ribonucleoproteins
Stereochemistry
Structural Models
Computer Simulation
Escherichia coli
Nucleic Acids
Tyrosine
Demonstrations
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids. / Maini, Rumit; Chowdhury, Sandipan Roy; Dedkova, Larisa; Roy, Basab; Daskalova, Sasha M.; Paul, Rakesh; Chen, Shengxi; Hecht, Sidney.

In: Biochemistry, Vol. 54, No. 23, 16.06.2015, p. 3694-3706.

Research output: Contribution to journalArticle

Maini, Rumit ; Chowdhury, Sandipan Roy ; Dedkova, Larisa ; Roy, Basab ; Daskalova, Sasha M. ; Paul, Rakesh ; Chen, Shengxi ; Hecht, Sidney. / Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids. In: Biochemistry. 2015 ; Vol. 54, No. 23. pp. 3694-3706.
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