title: Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids.
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 tRNA(CUA) 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.
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Rumit
authors.initials: R
authors.lastname: Maini
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Sandipan Roy
authors.initials: SR
authors.lastname: Chowdhury
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Larisa M
authors.initials: LM
authors.lastname: Dedkova
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Basab
authors.initials: B
authors.lastname: Roy
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Sasha M
authors.initials: SM
authors.lastname: Daskalova
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Paul
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Shengxi
authors.initials: S
authors.lastname: Chen
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry; D034664:Heterogeneous-Nuclear Ribonucleoprotein L / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D059372:Nucleotide Motifs; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011691:Puromycin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D050505:Mutant Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D011691:Puromycin; C479919:FolM protein, E coli; D013762:Tetrahydrofolate Dehydrogenase; D010458:Peptidyl Transferases; D000409:Alanine