9630763 Maliga The plastid genome of higher plants encodes subunits which are homologous to certain bacterial DNA-dependent RNA polymerase (RNAP) subunits. The promoters utilized by this plastid RNAP are similar to E. coli (70 promoters. Promoter selection by the plastid-encoded RNAP is dependent on nuclear-encoded (-like factors. In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter. Several reports have suggested the existence of an additional plastid-localized, nuclear-encoded RNAP. To confirm the existence of a second plastid transcription machinery, the rpoB gene for the essential ( subunit of the E. coli-like enzyme was deleted from the tobacco plastid genome. Transcripts in plastids lacking the ( subunit ((rpoB plants) accumulate to relatively high levels for genes which encode components of the gene expression apparatus. Therefore, the second RNA polymerase is referred to as the genetic system (GS) RNAP. In the (rpoB plastids transcription was detected from promoter regions with no similarity to (70 consensus elements. The research objective is to identify the role of the two RNA polymerases in plastid function and development. The proposal is focused on the role of the GS enzyme. The specific objectives of the research are: 1) Identification of promoters for the GS polymerase in tobacco plastids. Genes for which mRNAs are abundant in tobacco plastids lacking the rpoA, rpoB and rpoCl and rpoC2 genes will be identified by RNA gel blot analysis. The transcription initiation sites will be mapped for these mRNAs, and a consensus sequence will be derived based on sequence comparison. 2) In vivo definition of GS promoter architecture. Functionally important elements of GS promoters will be identified by testing expression of reporter genes from the mutant promoters in vivo in tobacco plastids. 3) Determination of tissue and cell-type specific transcription from GS pr omoters. This will be accomplished by testing tissue-specific accumulation of mRNAs and immunolocalization of reporter gene products. 4) Testing the role of GS and E. coli-like RNAPs in the expression of the plastid rRNA operon. The rRNA operon has both GS and E. coli-like (70 promoters. Expression of the operon from only one promoter will test role of each of these promoters in plastid function and development. 6) Developing an in vitro system for the analysis of the GS transcription machinery. An in vitro assay will be established using tobacco extracts in which GS RNAP transcriptional properties and GS promoter functional elements will be identified. Purification of the GS RNAP will facilitate the identification of subunit composition, the prerequisite of cloning GS subunit genes. %%% Plants contain specialized organelles called chloroplasts which convert light energy to carbohydrates. These chloroplasts have an unusual gene expression apparatus which compares with certain bacterial systems. When this apparatus is blocked another gene expression system is seen that is unlike the bacterial system. This system will be studied to determine what contribution it makes to plant gene expression in leaf and root tissues. ***
