Regulation of Photosynthetic Electron Transport Genes in Chlamydomonas Chloroplasts

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9723274 Stern Photosynthetic electron transport is largely carried out by multi-subunit complexes, whose constituents are encoded by nuclear and chloroplast genes. The synthesis of these complexes relies on the coordinate gene expression in these two organelles. A particularly intriguing aspect of this interdependence is that many of these nuclear genes regulate the synthesis of a specific chloroplast gene product. This regulation is carried out largely at the post-transcriptional level, mediating processes such as RNA processing, RNA stability, translation, and macromolecular complex assembly. This research will focus on nuclear regulatory genes and their RNA targets in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii, particularly those that encode subunits of the cytochrome b6/f complex of the electron transport chain. The cytochrome b6/f complex mediates electron transfer between the plastoquinone pool and plastocyanin, and is subject to developmental and environmental regulation. The specific goals of this project are first, to analyze two mutations that impair the stability of the chloroplast petA and petD mRNAs, which encode cytochrome f and subunit IV, respectively. Second, chloroplast ribosome recognition elements in mRNA will be functionally defined. The analysis of nuclear gene products and mechanisms that control mRNA stability will be carried out simultaneously using molecular genetics and biochemistry. Molecular approaches will be used to investigate the mechanism by which degradation occurs in mRNA stability mutants, and to clone the gene identified by one such mutation. Biochemical approaches will take advantage of in vitro systems to analyze the details of RNA-protein interactions, focusing on the ability of these stability factors to recognize specific RNA sequences, and perhaps to carry out other functions, such as RNA processing. The analysis of the subunit IV mRNA stability factor, which we hypothesize to encode an mRNA 5' untranslated reg ion-binding protein, will be facilitated by using a molecularly tagged allele of this locus to clone the nuclear gene encoding this protein. To analyze ribosome recognition elements, two regions of mRNA close to the translation initiation codon will be tested for a possible role in ribosomal RNA recognition. One of these regions is a Shine-Dalgarno element whose existence in chloroplast mRNAs is not yet firmly established. The other region consists of the three nucleotides immediately upstream of the initiation codon. Mutagenesis of these elements, and potential pairing partners in ribosomal RNA, .will be used to test the proposed interaction of these two RNA molecules. The overall goal of this research is to contribute to our understanding of chloroplast gene regulation, in a way that may be broadly applicable to photosynthetic eukaryotes. Some of the results may illuminate evolutionary strategies used by the nucleus to seize control of this cytoplasmic organelle. %%% Photosynthetic electron transport is largely carried out by multi-subunit complexes, whose constituents are encoded by nuclear and chloroplast genes. The synthesis of these complexes relies on the coordinate gene expression in these two organelles. A particularly intriguing aspect of this interdependence is that many of these nuclear genes are regulatory and control the synthesis of a specific chloroplast gene product. Therefore, genes once expressed autonomously in the prokaryotic ancestor of this organelle now require specific nuclear gene products for their activity. This research focuses on nuclear regulatory genes and their RNA targets in the chloroplast of the unicellular green alga Chamydomonas reinhardtii. The research will contribute to our understanding of chloroplast gene regulation, in a way that may be broadly applicable to phosynthetic eukaryotes. Some of the results may illuminate evolutionary strategies used by the nucleus to seize control of this cytoplasmic organelle. ***