Gene-specific and General RNA Regulators in Chloroplasts

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Chloroplast gene expression is essential for photosynthesis, and has to be coordinated with the nuclear genome. This coordination is achieved by a suite of mostly post-transcriptional regulators, some of which have recognizable prokaryotic origins, and others of which appear to have been uniquely derived following endosymbiosis. A fascinating and largely unresolved question is how these proteins of very different origins interact to effect both gene-specific and general control of chloroplast gene expression. This project uses the green alga Chlamydomonas reinhardtii as a model to explore the relationship between two regulatory proteins, MCD1 and MCD4. MCD1 encodes a large, novel protein which appears to be a member of a multimeric complex, and is specifically required for accumulation of the chloroplast petD mRNA, which it protects from ribonucleolytic decay through interaction with the petD 5' untranslated region. MCD4 was isolated in a suppressor screen of strains carrying petD 5' UTR mutations which destabilize the transcript, however the mcd4 suppressor proved to be pleiotropic, displaying numerous defects in other chloroplast transcripts. The specific aims of this research are to: (1) explore the biochemical activities of the MCD1 protein through in vitro RNA-binding assays, to determine if it has properties which can account for its specificity for petD; (2) purify the multiprotein complex containing MCD1, and determine its components; (3) identify the pleiotropic gene MCD4; and (4) study the specificity and interactions of MCD4 with other RNA metabolic factors using genetic crosses, in particular two candidate ribonucleases which it might regulate, polynucleotide phosphorylase and RNase J1.This project explores an example of how plant cells have evolved to coordinate activities in different compartments. The chloroplast, where photosynthesis occurs, was captured in ancient times as a bacterium. In this project, two genes located in the nucleus are under study, since they regulate chloroplast genes, originally inherited with the bacterium, which are required for photosynthesis. The research will offer opportunities for future scientists, either as summer interns or university undergraduates, to gain valuable experience and taste the excitement of biological research. Underrepresented groups have been prominent among these interns in past years. The leader of this project is also involved in educating the lay public about plant science, through the radio program MicrobeWorld, a Science Cabaret, and other vehicles.