RESUMO
The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.
Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is readily transmitted from person to person. We evaluated the emerging landscape of SARS-CoV-2 variants in Bangladesh from a retrospective study of nasopharyngeal swabs collected from 130 SARS-CoV-2-positive cases randomly selected over 6 months. Mutation analysis of whole-genome sequencing of 130 SARS-CoV-2 variants revealed 528 unique coding mutations, of which 102 were deletions, 6 were premature stop codons, and the remaining were substitutions. The most common mutation in the cohort was ORF1b:P314L, with a frequency of 98.5%. A total of 132 unique coding mutations were observed in the spike protein gene. Fourteen mutations were mapped to the spike protein receptor binding domain (RBD). These mutations increase the affinity between the spike protein and its human receptor, angiotensin converting enzyme 2 (ACE2), thereby increasing SARS-CoV-2 transmissibility. This study will help understand the SARS-CoV-2 virus and ultimately aid in monitoring and combatting the COVID-19 pandemic by furthering research on appropriate therapies. Analysis of age revealed closer association of the Delta variant with older populations and of the Omicron variant with younger populations. This may have important implications on how we monitor infections, distribute vaccines, and treat patients based on their ages.