Cyanelle Ribonuclease P: Isolation and Structure–Function Studies of an Organellar Ribonucleoprotein Enzyme

Publisher Summary Although all photosynthetic organelles are phylogenetically related to cyanobacteria, RNase P from different types of plastids shows striking diversity: whereas no RNA subunit can be detected in the chloroplast enzyme from green algae or higher plants, the plastids of some primitive algae encode an RNase P RNA of the cyanobacterial type. The photosynthetic organelle (cyanelle) of the glaucocystophyte Cyanophora paradoxa combines properties of free-living cyanobacteria and true chloroplasts, which makes it an ideal model system to study the evolution, structure, and function of RNase P from plastids. This chapter describes the methods used to grow this organism in amounts sufficient for enzyme preparation; the isolation of the organellar RNase P holoenzyme, detection of its activity, and analysis of the RNase P RNA structure; and the preparation of RNA and protein components from this and related organisms for use in homologous and heterologous reconstitution experiments. The unusual variability of RNase P in the phylogenefic lineage leading to and including modern chloroplasts necessitates the analysis of different members of this group of organisms and requires different approaches and methodologies.