Understanding the mechanisms by which eukaryotic gene expression is regulated is a central problem in molecular biology. Many proteins involved in this process bind to DNA sequences in the promoter region of the target gene. In many cases, a variety of different proteins bind to a single promoter, allowing complex combinatorial control of mRNA synthesis. These proteins often interact with one another as well as the general transcription apparatus. The experiments in this proposal are designed to probe the chemistry of transcription factor-promoter interactions, in particular how direct and indirect protein-protein interactions influence promoter occupancy inside the cell. For example, we have recently developed a novel in vivo method to quantitatively probe binding of the yeast activator GAL4 to its genomic target sites. Using this """"""""plasmid titration"""""""" assay, we have shown that GAL4 and one or more factors bound to the TATA box occupy promoters cooperatively in vivo. We will further probe this point and develop methods to elucidate which of the general transcription factors is targeted by GAL4. We will also test current models for the action of the SWI/SWF complex and the MOT1 protein, global enhancers and suppressors of gene expression, respectively. Each is thought to modulate transcription by affecting transcription factor-promoter interactions. We will also examine the effect of so-called """"""""architectural"""""""" activators, which are thought to modulate gene expression by bending promoter DNA, thus influencing the spatial relationship between the binding sites for other regulatory proteins. While there are many laboratories interested in protein-DNA interactions relevant to transcriptional regulation, we will pursue a novel approach that focuses largely on quantitative in vivo experiments. These will complement qualitative genetic probes and quantitative in vitro binding experiments that are commonly carried out by molecular biologists and bio-organic chemists, respectively.
