Transcriptional commitment of mitochondrial RNA polymerase from Saccharomyces cerevisiae

Abstract

The transcriptional commitment of mitochondrial RNA (mtRNA) polymerase and the conditions required for the formation of a stable ternary complex have been determined by in vitro transcription study. Four different transcription complexes were made in vitro by incubating purified mtRNA polymerase, cloned synthetic mitochondrial promoters and selective ribonucleotides. The responses of these complexes to heparin, an inhibitor of unbound mtRNA polymerase, have been examined to determine their involvement in transcription. This study leads to the following observations. (1) Under normal reaction conditions, 40 n m-heparin completely inhibited mitochondrial transcription. (2) A preinitiation mitochondrial DNA-RNA polymerase complex (complex 0) showed partial resistance to heparin (~25% resistant to 40 n m-heparin) when heparin and ribonucleoside triphosphates (rNTPs) were added together to the preformed complex. This complex was rapidly inactivated when preincubated with heparin before the addition of rNTPs. (3) The early initiation (complexes 2 and 4) containing DNA template, RNA polymerase and a short RNA product showed more resistance (approx. 40 to 50%) to 40 n m-heparin but destabilized upon further incubation with heparin before addition of the rest of the rNTPs. (4) After generation of ten or more phosphodiester bonds (complex 11), the early transcription complex is converted into a stable initiation complex, leading to the polymerase consignment to elongation. On the basis of stability and heparin sensitivity, three initial steps of mitochondrial transcription have been defined: polymerase-promoter interaction, initiation, and the transition from initiation to elongation. The formation of preinitiation complex is the rate-limiting step ( t 1 2 approx. 50 s), whereas the initiation and elongation reactions are very fast processes ( t 1 2 > 5 s ) in mitochondrial transcription.