This project will define the biochemical steps by which a cell senses a change in its environment and relays the information to genes to achiever appropriate modification of cell function. The psbA and psbD genes of the cyanobacterium Synechococcus sp. strain PCC 7942 are three- and two- member multigene families, respectively, which encoded the D1 and D2 reaction center proteins of photosystem II (PSII). Each family's members respond differentially to changes in light intensity and light quality. The psbA genes respond to an increase in intensity of white light through transcriptional induction of psbAII and psbAIII and accelerated degradation of psbAI and psbAIII messages. The psbDII gene is also light-responsive, and appears to be coregulated with psbAII and psbAIII. The psbA family encodes two different forms of the D1 protein, and the D1 composition of the PSII reaction center reflects the light-responsive regulation of the genes. Three distinct cis elements are present in the regulatory regions of psbAII and psbAIII: basal promoters, negative elements upstream of the promoters, and enhancer-like sequences downstream of the promoters that are required for induction by high light. The genes also exhibit a blue/red photoreversible response which is almost indistinguishable from the high-light response.
The aims of this project are 1) To determine whether the same signal transduction pathway is able to sense and respond to changes in both light intensity and light quality. Transcriptional activation of reporter genes whose defined cis elements respond to high light, and post-transcriptional changes in message degradation rates, will be examined following exposure to blue light; 2) To obtain mutants that fail to respond normally to changes in light quality or intensity. Mutants will be identified by monitoring bioluminescence from individual colonies of strains that carry fusions between psbA genes and the Vibrio harveyi luxAB genes. Complementation by libraries created in a recombinational vector that delivers DNA to the cyanobacterium at very high efficiency will identify important loci of the signal transduction pathway. 3) To define the functions of enhancer-like and negative cis elements that control psbA and psbD expression. Elements will be tested for their ability to regulate a heterologous and chimeric control regions comprising elements from different psbA genes will be constructed to assess their combinatorial activities. 4) To identify the trans-acting factors that bind to the gene' control regions. A genetic selection in E. coli will be used which promotes antibiotic resistance when a binding site and the gene encoding its binding protein are present in the same cell. 5 & 6) To identify sequences that target specific psbA transcripts and their protein products for regulated turnover. Chimeric psbA genes will be constructed to mix segments of genes whose transcripts are or are not subject to accelerated degradation at high light, and to produce proteins that have domains of the two forms of D1, whose half-lives in the membrane differ.
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