SGER: Novel Approaches to Understanding the Mechanism of Transcriptional Activation

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Recent studies indicate that the kinetic stability of activator-DNA complexes, as opposed to equilibrium binding properties, is a major determinant of the strength of transcriptional activation. These observations will be pursued and developed. Specific mutations in a model transcriptional activator (TFIIIA-VP16) have been shown to enhance transcriptional activity in vivo in a way that correlates with kinetic parameters measured in vitro. The investigators will use a newly developed technique to measure the lifetime of activator-DNA complexes in yeast cells to determine if kinetic stabilization also occurs in vivo. The availability of mutations that uncouple equilibrium binding affinity from kinetic stability of the DNA-activator complex constitutes a unique tool that will permit the identification of kinetically limiting steps leading to transcriptional activation in vivo. Alternatively, the results may indicate that kinetically mediated hyperactivation depends upon interactions of the activator with multiple parts of the transcriptional machinery. Preliminary data suggest that kinetic stabilization of the DNA-protein complex is dependent upon the presence of portions of the protein distal to the actual mutation that is responsible for stabilization in the full-length protein-DNA complex. Additional experiments will be carried out to determine just what combination of zinc fingers is required for kinetic stabilization, and to test the hypothesis that the simultaneous binding of N- and C-terminal zinc fingers is required to induce a structural deformation in the DNA that is, in turn, required for the structural transition that leads to kinetic stabilization. X-ray crystallography will be used to determine the structure of the kinetically stable DNA-protein complex to test the hypothesis that kinetic stabilization results from intercalation of planar, aromatic side chains into the DNA helix. Thus, novel approaches to studying the mechanism of transcriptional activation in eukaryotic cells will be used to obtain new insights into this fundamental cellular process involved in gene regulation. These studies will contribute to our understanding of basic cellular function at the molecular level. The completed studies will have broader impacts in at least two ways. First, a variety of educational outreach activities at the graduate, undergraduate, and elementary school levels will be supported directly or indirectly by the scientific activities carried out by the principle investigator and his colleagues. Second, because of their relevance to the design of novel sequence-specific DNA-binding proteins through the use of the zinc finger structural platform, the results obtained will have potential applications in biotechnology and in the genetic modification of agriculturally important crop plants.