The Role of the Nuclear-Encoded Plastid RNA Polymerase in Plastid Function and Development

Project Details

Description

MCB-9905043

PI: Pal Maliga

ABSTRACT

The plastid genes of higher plants are transcribed by two RNA polymerases: a multisubunit, E. coli-like enzyme encoded by the plastid genome (plastid-encoded plastid RNA polymerase or PEP) and a phage-type RNA polymerase related to the mitochondrial RNA polymerase (the nuclear-encoded plastid RNA polymerase or NEP). Since the NEP catalytic subunit is related to the mitochondrial enzyme, it is assumed that NEP contains a catalytic core and a specificity factor. Funds are requested to complete characterization of two NEP promoters. The first is a Type I promoter, PatpB-289, which has a novel, as yet uncharacterized, promoter element (Box II). The second is the only known Type II promoter, PclpP-53. Promoters first will be characterized in vitro using extracts from a unique biomaterial, tobacco plants lacking PEP due to targeted deletion of the rpoA gene. These plants are non-photosynthetic, but can be grown in the greenhouse by grafting mutant shoots onto wild-type (photosynthetically competent) rootstocks. Conclusions from the in vitro studies will be verified in vivo via transgenic approaches. In vivo promoter activity will be followed by accumulation of the jellyfish Aequorea victoria green fluorescence protein (GFP). The project will define NEP promoter architecture in higher plants, and determine tissue-specific and developmental expression of the two plastid promoters. NEP composition will be determined by purifying the complex from transgenic plants which carry a gene for a His-tagged catalytic subunit in their nuclear genome. His-affinity resins will be used to facilitate affinity purification of the native NEP. Fractions containing NEP holo-enzyme will be identified by specific transcription in in vitro transcription assays. NEP proteins will be separated by SDS-PAGE, isolated and sequenced. The microsequence data will facilitate the design of suitable primers for cloning and will serve in EST database searches for expressed cDNAs. The immediate target for cloning will be the NEP specificity factor.

This project will further our understanding of the nuclear control of plastid gene expression and facilitate the engineering of plastid NEP promoters for biotechnological applications.

StatusFinished
Effective start/end date8/1/997/31/03

Funding

  • National Science Foundation: $335,000.00

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