The sequencing of the human genome coupled with continued advances in proteomics and genomics have led to an unprecedented understanding of the relationship between genetic content and disease. As a consequence, the development of transcription-based therapeutics that would selectively reprogram aberrant gene expression in diseased cells is an increasingly attractive goal. There remain, however, fundamental questions surrounding the regulatory mechanism of transcription as well as the requirements for specificity and activity of such non-natural transcription factors. Many of these questions have proven intractable by genetic or biochemical means alone. Described herein is an approach for the generation of unique chemical tools that will be used for addressing basic questions of transcriptional regulation. These tools will further serve as molecular targets for the long-term development of transcription-based therapeutics. Saccharomyces cerevisiae or budding yeast will serve as our model system due to the mechanistic homology with mammalian systems, the availability of genomic information, and because yeast-specific transcriptional activators and/or repressors will serve as excellent candidates for anti-fungal agents. In order to accomplish these goals, transcriptional regulators will be selected from libraries of peptides screened for binding to the Mediator complex component Gall 1. A battery of biochemical techniques will be used to characterize the binding affinity and specificity of all ligands thus selected. In vitro transcription experiments will be employed to functionally characterize the ligands as activators or inhibitors of transcription that will be used to probe mechanistic questions surrounding transcriptional regulation in experiments designed to map functionally important protein-protein interactions and to probe the origin of synergy in transcriptional activation. As Saccharomyces cerevisiae is an excellent model system for metazoans, the data thus obtained will significantly contribute to the evolving model of eukaryotic transcriptional activation. Furthermore, the chemical regulators developed throughout the project will serve as the basis for achievement of the long-term goal of the design and investigation of transcription-based therapeutics. The protein under investigation, Galil, is a yeast-specific component of the transcriptional apparatus; thus artificial regulators that exert their function via specific interactions with Gall 1 will function only in yeast, providing a mechanism for the development of anti-fungal agents.
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