Combinatorial interactions between transcription factors are a common theme, yet in most cases the mechanisms governing these interactions are not known. At glycolytic enzyme gene upstream activating sequence (UAS) elements of Saccharomyces cerevisiae, repressor activator protein 1 (Rap1p) and glycolysis regulatory protein 1 (Gcr1p) come together to form some of the strongest promoter-binding complexes known. The properties and mechanisms responsible for combinatorial interactions between Rap1p and Gcr1p will be elucidated. Rap1p serves as a model for multi-functional transcriptional factors that are capable of carrying out seemingly opposite roles in transcription, functioning as both an activator or repressor depending on the sequence context of their binding sites. Gcr1p serves as a model for a class of sequence-specific transcription factors that display high affinity but low specificity for their binding sites in vitro. With regard to Rap1p and Gcr1p, there are indications that they both may participate in combinatorial interactions with other binding partners as well. At glycolytic enzyme gene UAS elements, Rap1p facilitates the binding of Gcr1p. To determine the mechanism by which Rap1p facilitates Gcr1p's binding, alternate models for binding cooperativity will be tested. A protein-protein interaction model and a DNA-bending model will be investigated. A genomic approach will be used to identify other proteins that participate in combinatorial interactions with Gcr1p. Yeast genomic arrays will be used to identify every gene within the cell that is dependent on Gcr1p for full expression. The regulatory regions of Gcr1p-dependent genes will be searched for common sequence motifs. Common sequence motifs adjacent to Gcr1p-binding sites will be investigated to test the hypothesis that they are the binding sites for binding partners of Gcr1p. Both in vivo footprinting and site-directed mutagensis will be undertaken to confirm the importance of the putative binding sites. Once the sequence motifs are established as binding sites, they will be used to identify clones from a phage lambda-gt11 yeast expression library expressing proteins that bind to the sequence motif. Identification of the phage clones encoding the DNA-binding activity will lead directly to the identification of yeast genes encoding other binding partners of Gcr1p. These experiments will define the precise nature of the DNA binding interactions that occur between Rap1p and Gcr1p and will lay the ground work for structural studies on the DNA-binding domain of Gcr1p. This work will also lay a foundation for elucidating the network of combinatorial interactions within cells.
A major control point of gene expression is the regulation of transcription, the process by which the information in DNA is copied into mRNA. The goal of this project is to define the interactions between two proteins that regulate the transcription of yeast genes that encode proteins that break down glucose. The yeast Saccharomyces cerevisiae has proved indispensable as a model system for studies of gene expression. The experiments will determine the molecular mechanisms by which these two proteins interact with each other and with DNA to regulate gene expression. Furthermore, additional genes that may be regulated by similar mechanisms will be identified. This work will lay a foundation for elucidating the network of interactions between transcriptional regulatory proteins that occur within cells.
