In Vitro and in Vivo Processing of Cyanelle tmRNA by RNase P

Abstract

Abstract Ribonuclease P, the ubiquitous endonuclease required for generating mature tRNA 5 ends, is a ribonucleoprotein in most organisms and organelles, with the exception of mitochondria and chloroplasts of multicellular organisms. The cyanelle of the primitive alga Cyanophora paradoxa is the only photosynthetic organelle where the ribonucleoprotein nature of this enzyme has been functionally proven. tmRNA is another highly structured RNA: it can be aminoacylated with alanine, which is then incorporated into a tag peptide encoded on the same RNA molecule. This dualfunction RNA has been found in bacteria, and its gene is also present in mitochondria and plastids from primitive organisms. Since nothing is known about the expression of this RNA in organelles, we have performed processing studies and determined the promoter of cyanelle pretmRNA. This RNA is transcribed as a precursor molecule in vivo. Synthetic transcripts of cyanelle pretmRNA, including or lacking the mature 3 CCAend, are efficiently and correctly processed in vitro by bacterial RNase P ribo and holoenzymes and by the homologous cyanelle RNase P. In addition to these experimental data, we propose a novel secondary structure model for this organellar tmRNA, which renders it more similar to its bacterial counterpart.