Transcription of Mitochondrial DNA in vitro from Neurospora crassa

Intact mitochondrial DNA from Neurospora crassa (linear molecules of contour length 25–26 μm) has been transcribed in vitro with RNA polymerase from Escherichia coli. At low concentrations (6 μg/ml) mitochondrial DNA is transcribed as efficiently as DNA from E. coli or calf thymus. The transcription product has the low G + C content characteristic of mitochondrial RNA. After formation of an initial complex between enzyme and mitochondrial DNA only a limited number of bound enzymes can start RNA synthesis in the presence of rifampicin. The sigma factor is required to confer rifampicin resistance. By comparing the initial rate of rifampicin resistant transcription of bacteriophage and mitochondrial DNA under saturating enzyme concentrations, the number of specific binding sites per mitochondrial genome (molecular weight 50 to 60 × 106) has been estimated to be 8 to 9. The transcription product synthesized in the presence of rifampicin has been characterized by competition‐hybridization and by RNAase digestion in solution. About 50% of the sequences hybridizing with mitochondrial DNA are homologous to RNA isolated from intact mitochondria; 35% of the product consists of rapidly renaturing double‐stranded regions characteristic of ribosomal RNA, and 5% of slowly renaturing double strands. 25% of the sequences synthesized in vitro form double strands only after annealing for 16 h at 62 °C but do not contain sequences complementary to stable mitochondrial RNA synthesized in vivo. From these results it is concluded that bacterial RNA polymerase recognizes mitochondrial DNA sequences resembling bacteriophage initiation sites, and that enzymes bound to binding sites conferring rifampicin resistance transcribe asymmetrically cistrons coding for mitochondrial RNA.