The induction of gene expression involves the interaction of sequence-specific DNA binding proteins with DNA elements termed upstream regulatory or activator sequences. In many cases, this interaction is in response to changes in the concentration of specific effector molecules present in the environment. Subsequent to this sequence-specific interaction, these molecules are thought to interact with the general transcriptional machinery, such as RNA polymerase. To study the interactions of the type described above, the yeast Saccharomyces cerevisiae presents an attractive model. The yeast secreted protein acid phosphatase is produced de novo under conditions of limiting phosphate levels and the copper metallothionein is produced in response to the presence of copper ion in the medium. This type of regulation suggests that there are changes in the specific protein-DNA interactions within the upstream elements of each gene system. Specific experiments are described to investigate: (A) The mechanism of action of the negative transcription factor PH080, a gene product required to repress the synthesis of acid phosphatase under conditions of excess phosphate. (B) Expression of the genes encoding several transcription factors in E. coli to study the protein-DNA and protein-protein interactions involved in repression/derepression of acid phosphatase synthesis. (C) The role of transcriptional factors absolutely required for acid phosphatase expression in the expression of other yeast genes. (D) Genetic analysis of mutants involved in acid phosphatase synthesis. (E) Purification and cloning of factors involved in the transcriptional response of the copper metallothionein gene. These studies will provide key insight into the molecular mechanisms of the transcriptional activation of specific genes in response to changes in the environment.
