RESUMO
OBJECTIVE: To increase the reporter repertoire of the yeast three-hybrid system and introduce the option of negative selection. RESULTS: Two new versions of the yeast three-hybrid system were made by modifying the MS2 coat RNA-binding protein and fusing it to the Gal4 DNA-binding protein. This allows the use of Gal4 inducible reporters to measure RNA-protein interactions. We introduced two mutations, V29I and N55K into the tandem MS2 dimer and an 11 amino acid deletion to increase RNA-protein affinity and inhibit capsid formation. Introduction of these constructs into the yeast strains MaV204K and PJ69-2A (which contain more reporters than the conventional yeast three-hybrid strains L40-coat and YBZ-1) allows RNA-protein binding interactions with a wide range of affinities to be detected using histidine auxotrophy, and negative selection with 5-fluoroorotic acid. CONCLUSION: This yeast three-hybrid system has advantages over previous versions as demonstrated by the increased dynamic range of detectable binding interactions using yeast survival assays and colony forming assays with multiple reporters using known RNA-protein interactions.
Assuntos
Ligação Proteica/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Leveduras/metabolismoRESUMO
Many conventional, modern genome engineering tools cannot be used to study mitochondrial genetics due to the unusual structure and physiology of the mitochondrial genome. Here, we review a number of newly developed, synthetic biology-based approaches for altering levels of mutant mammalian mitochondrial DNA and mitochondrial RNAs, including transcription activator-like effector nucleases, zinc finger nucleases and engineered RNA-binding proteins. These approaches allow researchers to manipulate and visualize mitochondrial processes and may provide future therapeutics. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.
Assuntos
DNA Mitocondrial/genética , Expressão Gênica , Genes Mitocondriais/genética , Engenharia de Proteínas , RNA Mitocondrial/genética , Animais , DNA Mitocondrial/metabolismo , Humanos , Mamíferos/genética , Mutação , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Biologia Sintética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismoRESUMO
It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.