POWRE: Optimization and Characterization of Chloroplast Translation In Vitro

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9720444 Hollingsworth Regulation at the translational level is a key contol point in the expression of many choroplast genes. Such regulation is typically mediated through RNA-protein interactions at the 5' untranslated region of an mRNA. This laboratory has previously shown that specific RNA-protein interactions occur at the 5' untranslated regions of chloroplast-encoded ATP synthase genes. Proposals to analyze the effects of these interactions on translation in vitro have been stymied by reviewers' concerns about the technical challenges present by chloroplast translation extracts. The goal of this research is to answer those concerns by analyzing and optimizing a recently reported protocol for preparing chloroplast translation extracts. The project has two primary objectives: The first objective is to optimize the translation assay to expand the repertoire of transcripts that can be efficiently translated. Two transcripts with several orders of magnitude difference in translation efficiencies in vitro will be used as substrates for these experiments. Analysis of the effects of cis-acting sequences on translation will be initiated using chimerics of the two original substrates. The second objective of this research is to expand the applicability of the extract preparation of protocol. Experiments will be performed to expand the protocol from chloroplasts of tobacco to chloroplasts from other research-intensive species, including spinach and Arabidopsis. This project will be supported through the POWRE program. It will determine the feasibility of a new line of inquiry that should expand the applicability of the in vitro chloroplast translation to inquiries examining translational regulation of chloroplast-encoded ATPase genes. %%% This project examines the regulation of protein synthesis in chloroplasts by using an experimental system in which an RNA transcript from a specific chloroplast gene is added to an extract from tobacco chloroplasts. The RNA is used as a template for protei n synthesis. This allows features of the RNA that might affect the efficiency or regulation of chloroplast protein synthesis to be studied.