Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids
| Autor: | Sandipan Roy Chowdhury, Rumit Maini, Sidney M. Hecht, Rakesh Paul, Shengxi Chen, Basab Roy, Sasha M. Daskalova, Larisa M. Dedkova |
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| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Peptidyl transferase Stereochemistry Protein Conformation Stereoisomerism Biology Molecular Dynamics Simulation RNA Transfer Amino Acyl 010402 general chemistry 01 natural sciences Biochemistry Ribosome Article Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Protein structure Heterogeneous-Nuclear Ribonucleoprotein L Dihydrofolate reductase Protein biosynthesis Escherichia coli Humans Nucleotide Motifs 030304 developmental biology chemistry.chemical_classification 0303 health sciences Alanine Protein Stability Escherichia coli Proteins Hydrogen Bonding Recombinant Proteins 0104 chemical sciences Amino acid RNA Bacterial RNA Ribosomal 23S Tetrahydrofolate Dehydrogenase chemistry Puromycin RNA Ribosomal Peptidyl Transferases biology.protein Mutant Proteins Ribosomes |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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