RESUMO
Since its development about two decades ago, the yeast one-hybrid (Y1H) assay has become an important technique for detecting physical interactions between sequence-specific regulatory transcription factor proteins (TFs) and their DNA target sites. Multiple versions of the Y1H methodology have been developed, each with technical differences and unique advantages. We will discuss several of these technical variations in detail, and also provide some ideas for how Y1H assays can be further improved.
Assuntos
Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Animais , Clonagem Molecular , Genes Reporter , História do Século XX , Humanos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/fisiologia , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido/históriaRESUMO
In 1996, the Wickens and the Kuhl labs developed the yeast three-hybrid system independently. By expressing two chimeric proteins and one chimeric RNA molecule in Saccharomyces cerevisiae, this method allows in vivo monitoring of RNA-protein interactions by measuring the expression levels of HIS3 and LacZ reporter genes. Specific RNA targets have been used to characterize unknown RNA binding proteins. Previously described RNA binding proteins have also been used as bait to select new RNA targets. Finally, this method has been widely used to investigate or confirm previously suspected RNA-protein interactions. However, this method falls short in some aspects, such as RNA display and selection of false positive molecules. This review will summarize the results obtained with this method from the past 15years, as well as on recent efforts to improve its specificity.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Técnicas do Sistema de Duplo-Híbrido/normas , Animais , Biblioteca Gênica , História do Século XX , História do Século XXI , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas/história , Mapeamento de Interação de Proteínas/métodos , Mapeamento de Interação de Proteínas/normas , Saccharomyces cerevisiae , Sensibilidade e Especificidade , Técnicas do Sistema de Duplo-Híbrido/históriaRESUMO
The initial yeast two-hybrid experiment - published in 1989 - described an approach to detecting protein-protein interactions that has flourished over the last two decades, leading to the assembly of large-scale data sets of these interactions. Yet the yeast assay originated because of the laboratory's interests in technology development, not because of its need to identify partners of any protein then under study. In addition to such motivating forces, other features of the process of originating a technology can be revealed by considering the lessons of the two-hybrid approach. These include the value of timeliness in a method's development, the willingness of an investigator to try experimental approaches that prove fruitless, the ability of biological macromolecules to display surprising attributes, the benefits of a community expending efforts to expand the uses of a technology platform, and the role of scientific training of those who work in technology.
