The overall goal of this proposal is to gain insight into the relationships among DNA structure, trans-acting factors, protein- nucleic acid interactions, and the regulation of gene transcription in plants. The experiments involve the maize alcohol dehydrogenase genes. Adhl and Adh2 exhibit differential tissue specificity, yet anaerobiosis mediates the coordinate transcriptional expression of both Adh genes. This provides a unique opportunity to evaluate the roles played by trans-acting factors and protein-nucleic acid interactions in the activation of two closely related genes, and to compare and contrast the developmental and environmental activation of gene transcription. The main focus of this rather straightforward proposal is the in vitro characterization and purification of the trans-acting protein factors that affect the regulation of the Adh genes. While this general area of research has been actively pursued in animal systems, plant trans-acting factors are only now beginning to be investigated. What makes this proposal particularly feasible and appropriate is that we already know (at least some of) the factors involved in Adh regulation. From our in vivo footprinting studies we know the binding sites of at least four regulatory factors, and we know their dimethyl sulfate footprint signatures. Therefore we know precisely the factors we are searching to purify and are not simply looking for proteins that (just) bind to Adh DNA.
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