Mutational characterization and mapping of the 70S ribosome active site
| Autor: | Antje Krüger, Tasfia Azim, Adam J. Hockenberry, Anne E. d’Aquino, Michael C. Jewett, Nikolay A. Aleksashin |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Peptidyl transferase Mutant Computational biology Biology 010402 general chemistry medicine.disease_cause 01 natural sciences Ribosome 03 medical and health sciences Synthetic biology Catalytic Domain Escherichia coli Genetics Protein biosynthesis medicine Codon 030304 developmental biology 0303 health sciences Mutation Point mutation RNA 0104 chemical sciences RNA Ribosomal Polyribosomes Protein Biosynthesis Peptidyl Transferases biology.protein Synthetic Biology and Bioengineering Ribosomes |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkaa001 |
| Popis: | The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications. |
| Databáze: | OpenAIRE |
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