The main objectives of the proposed research are (1) to determine the relationship between the rpoA, rpoB, rpoC DNA-dependent RNA polymerase subunit genes encoded in chloroplast DNA of Euglena gracilis and higher plants and the RNA polymerase activities of chloroplasts, (2) to define how the complex rpoB- rpoC transcription units are transcribed, and the resulting pre- mRNAs are processed and translated to yield polypeptide subunits, (3) to identify, isolate and characterize any additional chloroplast or nuclear localized genes for chloroplast RNA polymerases, and (4) to determine how the expression of the chloroplast RNA polymerase subunit genes is regulated during light induced organelle development. Emphasis will be placed on the importance of transcriptional vs post-transcriptional events in regulating the transcriptional vs post-transcriptional events in regulating the transcriptional status of the developig plastid. Chloroplast contain two types of transcriptional activity. In order to determine if the rpoA, B, and C genes encode subunits of either or both polymerase activities, polypeptide coding domains of the chloroplast rpoA, B, and C genes will being expressed in E. coli to yield fusion polypeptides for use in generating monospecific antibodies directed against the putatative RNA polymerase subunits. Antibodies will be used to immunopurify the enzymes, to identify subunits by Western blotting, and to specifically inhibit the different transcriptional reactions. Homogenous preparations of these enzymes should be obtained via immunoaffinity chromatography and FPLC, in addition to currently available purification procedures. Genes for RNA polymerase subunits that are not encoded by the rpoA, B, and C loci will also be studied. In order to determine how rpoC transcripts are processed, and if there are unusual features to this process such as alternat splice sites, cDNA clones of rpoC mRNA will be prepared and sequenced. Through the types of experiments described above and studies on the regulation of expression of the RNA polymerase subunit genes, and of the resulting enzyme activities, it will be possible to define many important fundamental aspects of RNA synthesis in cell organelles.
Showing the most recent 10 out of 16 publications
