Abstract 9406550 Much of chloroplast gene regulation occurs at the post- transcriptional level. The aim of this project is to undertake a systematic study of chloroplast translation initiation using genetic and biochemical approaches. We will focus on the pet genes, whose products form the cytochrome b6/f complex, which transports electrons between photosystem II and photosystem I. Mutations predicted to interfere with translation initiation will be introduced into pet genes and second-site suppressors of these mutations will be analyzed. In a parallel approach, efforts will be made to generate null mutations in nuclear translational activator genes by insertional inactivation. Non-photosyntheic transformants will be generated and screened for defects in chloroplast protein accumulation and translation. The inserted DNA tags the gene, which facilitates cloning the mutated gene and subsequently the wild-type version. In addition to these genetic approaches, in vitro biochemical methods will be used to identify and characterize proteins that interact with the 5' UTR. Protein extracts from suppressor and insertion mutants will be assayed for any differences in protein binding factors. The results of the project will lead to a greater understanding of the cis-acting sequences controlling translation, and the proteins that interact with these sequences. %%% Although chloroplasts contain their own genetic apparatus, most of the regulatory signals are transmitted to the chloroplast by the action of nuclear gene products. The isolation and analysis of mutants in both higher plants and algae that are defective in this signaling process have revealed that in most cases, chloroplast gene regulation occurs at the post-transcriptional level. Examples of such mutations include those that affect messenger RNA lifetimes or RNA maturation, protein synthesis or stability, and the assembly of macromolecular complexes. The first of two general approaches in this projec t is to introduce mutations that interfere with protein synthesis into chloroplast genes and then to identify compensating suppressor mutations in nuclear genes. At the same time, biochemical methods will be used to detect the protein factors directly. In a second general approach, efforts will be made to generate mutations that disrupt the functions of nuclear translational activator genes, by introducing transforming DNA into the nucleus. Once such mutants have been identified, the "tagged" gene can be isolated. The results of the proposed research will lead to a greater understanding of the sequences that control translation of chloroplast RNAs, and of the proteins that interact with these sequences. ***
