Genetic Analysis of DNA-Protein Interactions Using a Reporter Gene Assay in Yeast
Understanding the underlying structural and physico-chemical basis for the recognition of specific DNA sequences by regulatory proteins is a central goal of modern biochemical genetics. A method for the rapid identification of mutant molecules altered in the affinity and/or specificity of such interactions could be a powerful tool in the hands of those studying this difficult problem. Conventional genetic approaches for obtaining and analyzing interesting mutant forms of specific DNA-binding proteins are often infeasible because of the genetic intractability of the species being studied or as a result of difficulties in identifying relevant and specific phenotypes associated with alterations in the interaction under investigation. High-resolution genetic analysis of DNA-protein interactions is particularly problematic in metazoans. We have devised an approach that makes use of the modularity in structure and function of eukaryotic transcription factors (1), the power of the polymerase chain reaction (PCR) to generate specific DNA fragments with defined levels of mutagenesis in vitro (2,3), and the recombinogenic potential of S. cerevisiae (2,4) to carry out a high-resolution genetic analysis of the sequence-specific DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA) (5). It seems likely that this approach will be generally applicable to the study of many DNA-protein interactions.
KeywordsExpression Plasmid Activation Domain Reporter Plasmid Upstream Activator Sequence Gapped Plasmid
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