The cyanobacteria provide an excellent experimental system for studying the control of gene expression in photoautotrophs. We have been applying the powerful tools of molecular genetics and recombinant DNA technology to the study of the regulation of gene expression in these organisms as well as to the study of protein-protein interactions which are required for the in vivo assembly of the phycobilisome. We have cloned and characterized the genes encoding nine of the twelve structural components of the Synechococcus sp. PCC 7002 phycobilisome; efforts to clone the remaining three genes are currently in progress. Mutations have been constructed in most of these genes. Analyses of these mutant is being performed to define the requirements for assembly of this multiprotein complex as well as to define the pathway of excitation energy migration through the phycobilisome. Two genes, cpcE and cpcF, which apparently encode proteins required for the post-translational chromophorylation of apophycocyanin, and whose products are required for stable accumulation of phycocyanin, have also been cloned, sequenced, and inactivated. Studies to define the role of these proteins in phycocyanin biosynthesis are proposed. a number of genes encoding phycobilisome components have also been isolated and characterized from Cyanophora paradoxa and Pseudanabaena sp. PCC 7409. The expression of some of these genes, cpeBA and cpcB2A2, has been shown to be regulated at the level of mRNA abundance by light wavelength. The proposed experiments are intended to probe two different but related aspects: 1. What specific structural requirements are necessary for phycobilisome assembly and energy transfer? 2. What mechanisms regulate the expression of the various phycobiliprotein genes? The techniques of site-directed mutagenesis will be employed to analyze the function of the L-CM 97 polypeptide. Translational fusions of various phycobiliprotein promoters tot he lacZ gene will be constructed; deletion analyses will be performed in an attempt to define more precisely which sequences are important in controlling the transcription of the phycobiliprotein genes. The constructs generated will be used to examine the effects of light wavelength on phycobiliprotein gene expression. We will initiate attempts to purify the RNA plymerases of Synechococcus sp. PCC 7002 and Pseudanabaena sp. PCC 7409 in an attempt to generate at in vitro system for the analysis of transcriptional regulation in cyanobacteria. The studies proposed should greatly extend out knowledge of mechanisms regulating gene expression in photoautotrophs.
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