Molecular analysis of Transcription Factors Regulating phyA Gene Expression

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Phytochrome is a regulatory master switch that controls gene expression in response to light. The long term goal of the proposed research is to define the molecular mechanism underlying this regulation. The rapid transcriptional repression that phytochrome imposes on its own phyA genes provides an excellent model system for this purpose. The specific objectives of this proposal are: (a) to identify and isolate recombinant clones encoding DNA-binding proteins that recognize functionally defined cis-acting elements in phyA promoters; (b) to delineate the functional domains of such cloned factors; (c) to determine the molecular mechanism by which a recently identified repressor factor, RF1, disrupts phyA transcription in response to phytochrome activation. Experimental approaches will include: (a) screening expression libraries with oligonucleotide probes representing functionally defined regulatory elements for isolation of cDNA clones encoding the cognate factors; (b) in vitro binding assays to define the DNA-recognition domains of these factors; (c) gene transfer assays to delineate nuclear localization sequences; (d) yeast transfection and in vitro transcription assays with chimeric fusion proteins to define transcriptional activation or repression domains in these factors. The significance of this research is that it will help define the mechanism by which a central regulatory molecule controls gene expression. Because phytochrome appears to have no molecular counterpart in other eukaryotes, elucidation of this mechanism will broaden our understanding of how eukaryotic cells regulate gene expression in response to environmental stimuli. %%%% Plant respond to light by turning on genes that are involved with growth, photosynthesis, and other functions. A key regulatory molecule in plant cells that is responsible for this effect is phytochrome. This molecule interacts with proteins and genes to activate genetic messages for growth, etc. To understand the molecular mechanism it is necessary to identify the regions of the gene which are affected, and the proteins which are involved in the activation process. These studies will lead to greater understanding of how to regulate plant growth.