The phytochromes are an unique family of informational photoreceptors that regulate developmentally important gene expression in response to environmental light signals. The C-terminal domain of the molecule has sequence similarity to the transmitter histidine kinase module of the bacterial two-component sensors. However, despite considerable research effort, neither the biochemical mechanism of signal transfer from the photoreceptor, nor the identity of early signaling intermediates in the transduction pathway have been determined. The PI proposes to address these two deficiencies using phytochrome A (phyA), the best characterized and experimentally most tractable member of the family. The specific objectives of this proposal are: (a) to define sequence and structural determinants of the phyA molecule responsible for its photosensory and regulatory activities; (b) to define the biochemical mechanism of signal transfer from phyA to its initial reaction partner(s); and (c) to identify early signaling intermediates specific to the phyA pathway. The experimental approaches will include: (a) molecular, genetic, and reverse genetic analysis in Arabidopsis to map functionally active sub-domains and specific residues within the photoreceptor molecule; (b) production of mg quantities of recombinant phyA structural domains in heterologous hosts for structure determination by X-ray crystallography; (c) molecular cloning of loci encoding potential signaling intermediates specific for the phyA pathway identified in genetic screens of Arabidopsis; (d) molecular cloning of phyA-interactive proteins using in vitro interaction cloning and yeast two-hybrid screening strategies; and (e) exploitation of a cyanobacterial model system, Synechocystis 6803, recently discovered to contain a phytochrome homolog also related to the two-component sensors, to accelerate analysis of the possibility that phyA is a descendent of a prokaryotic, light-regulated histidine kinase. Understanding the spectrum of molecular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals is a central goal of current biomedical research. Much has been learned about receptor kinases and phosphorylation cascades involving Ser/Thr/Tyr-class protein kinses, as well as about G-proteins and the small molecule second messengers. By contrast, very little is known about the newly discovered class of eukaryotic proteins related to the bacterial sensory histidine kinases. The discovery of these proteins suggests that eukaryotes may have retained a sensory signaling system based on that of the bacterial two-component systems, but this remains to be directly demonstrated for a multicellular eukaryote.
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