Development of an isolation and purification scheme for chloroplast RNA polymerase in Pelargonium

Abstract

The rpo genes encoding the RNA polymerase (RNAP) in the chloroplasts of the common geranium, Pelargonium, are evolving at high rates. RNAPs in other organisms, however, are more evolutionarily conservative, presumably because of the enzyme's complexity and key role in production of photosynthetic machinery. Therefore, the rapid evolution of genes encoding such a vital enzyme in Pelargonium deserves deeper investigation. We report progress towards isolation of Pelargonium chloroplast RNAP in order to conduct detailed functional studies and to confirm that the peptide sequence of the functioning enzyme reflects the rapidly evolving gene sequences. Spinach chloroplast RNAP served as a model for development of a procedure for the isolation of previously uncharacterized Pelargonium RNAP. Chloroplasts have been isolated and lysed to expose soluble RNAP, which has been partially purified using heparin-sepharose chromatography and DEAE-sepharose chromatography. A fluorescence-based assay that utilizes the nucleic acid binding dye, RiboGreen, was used in tandem with the isolation and purification procedures to measure RNAP activity. This research was supported by the J. Reid and Polly Anderson Endowment.