Many of the transfer RNA genes of chloroplast genomes have been found to be encoded in polycistronic operons. These operons can contain genes for other tRNAs, rRNAs, constitutively expressed proteins, or proteins whose expression is developmentally regulated. Regulation of chloroplast gene expression for most genes occurs posttranscriptionally. The regulatory mechanisms which could involve RNA processing events and/or translation initiation/elongation steps are not known. The focus of this proposal is to determine the RNA maturation pathways for developmentally regulated and constitutively expressed chloroplast polycistronic operons, and to determine the role of RNa processing steps, in any, in regulating organelle development. Some emphasis will be placed on mixed tRNA-mRNA operons, since precursor RNAs accumulate and tRNA processing must proceed mRNA translation. The main objectives of the proposed research are: (1) to determine the primary structure of precursor transcripts from polycistronic operons; (2) to determine if precursor transcripts and non- translated mRNAs that accumulate in proplastids are localized on polysomes or in ribonucleoprotein particles; (3) to characterize the RNa processing pathways for tRNA-mRNA mixed chloroplast operons containing constitutively expressed genes and photosynthetic membrane genes; (4) to determine if there are developmental specific RNA processing event during organelle biogenesis; (5) to purify selected RNA processing enzymes, and determine if the RNa developmentally regulated; (6) to characterize a novel class of chloroplast introns found only in constitutively expressed genes. In order to characterize organelle RNAs, we are developing a procedure for preparing cDNA clones of chloroplast transcripts. The procedure will be applicable to mature RNA species, primary transcripts, and RNa processing intermediates. The procedure will be general for transcripts from any source, not restricted to chloroplast RNAs, and will be applicable to polycistronic transcripts, including mixed tRNA-mRNA operons. These cDNAs will be subsequently expressed in vitro with T3 or T7 viral regulatory sequences and promoters as a means to generate substrates for in vitro characterization of RNa processing reactions.
