9507329 Troxler RNA polymerase is a key component of the transcriptional apparatus in plastids and is responsible for expression of about 130 genes for rRNAs, tRNAs and proteins required for transcription, translation and photosynthesis. Genes for 4 RNA polymerase subunits have significant homology to genes coding for the core subunits of RNA polymerase in bacteria. Plastid genomes do not contain genes for proteins with recognizable homology to RNA polymerase sigma factors in bacteria. This is surprising because many plastid genes contain promoter-like elements that resemble the -10 (TATAAT) and -35 (TTGACA) consensus promoters in genes recognized by sigma 70 -type sigma factors I RNA polymerase holoenzymes from bacteria. Lack of knowledge of sigma factor components in plastid RNA polymerase is a major gap in understanding how plastids recognize their genes. The investigator has isolate the gene for a sigma factor from the unicellular rhodophyte, Cyanidium caldarium, by screening a genomic sublibrary with a degenerate oligonucleotide coding for a highly conserved region called the "rpoD box" in sigma 70 of Escherichia coli and sigma 43 of Bacillus subtilis. The deduced amino acid sequence of the putative sigma factor contains highly conserved domains for promoter recognition, DNA melting and core binding found in sigma factors from bacteria. The mRNa for the putative sigma factor is a 2.2 kb polyadenylated transcript expressed only in illuminated cells suggesting that this protein is encoded in a nuclear photogene. Genomic DNA digested with restriction enzymes contained 4-5 restriction fragments that hybridized to the rpoD box oligonucleotide suggesting that there is a family of plastid RNA polymerase sigma factors in C. caldarium. These studies are the first to identify a plastid RNA polymerase sigma factor in a photosynthetic eukaryote and provide the "missing link" for investigating mechanisms of plastid gene recognition. The specific aims of its project are to (1) isolate and c haracterize clones for the two principal sigma factors in plastid RNA polymerase from C. caldarium, and (2) examine the expression patterns of sigma factor mRNAs and proteins in dark and light. %%% RNA polymerase is a key component of the transcriptional apparatus in plastid intracellular organelles such as chloroplasts and is responsible for expression of about 130 genes. The investigator has recently reported the first identification of a gene for the plastid polymerase subunit which confess upon the enzyme specificity for different classes of genes. Such specificity enables cells to tailor their metabolism to changes in their environments. The investigator's study of this subunit should help fill a major gap in the understanding of plastid biology. ***
