Anin VitroTranscription Assay for Yeast Mitochondria Using Organellar Lysates

Abstract

The nuclear geneNUC1encodes the major mitochondrial (mt) ribonuclease in the yeastSaccharomyces cerevisiae.We describe anin vitromt transcription assay system based on lysates of purified mitochondria from a petite (ρ−, mt deletion mutant) yeast strain in whichNUC1has been insertionally inactivated. Controlin vitrorun-on transcription assays using intact mitochondria demonstrate that the rate of incorporation of labeled precursor into mt RNA is identical in organelles from thenuc1ρ−mutant and its otherwise isochromosomalNUC1parent strain. Brij-35 lysates of mitochondria from thenuc1strain incorporate precursor into mt RNA at nearly the same rate as do intact organelles from that strain, while similar mt lysates fromNUC1cells show no such incorporation. Other control studies show that mt lysates from thenuc1strain retain functional mt cAMP-dependent protein kinase and other critical activities. When the cloned template DNA encoding the yeast mt 21S rRNA gene, which is not retained in thenuc1ρ−strain, is added to mt lysates from that strain, transcripts are produced from the template under standard assay conditions.