Regulation of transcriptional initiation in yeast mitochondria

We have investigated in vitro transcriptional initiation by purified yeast mitochondrial RNA polymerase using a variety of previously described promoter variants and dinucleotides corresponding to the first two transcript nucleotides. Regardless of the actual nucleotides that occupy the first two transcript positions, the rate of initiation increases with increasing concentrations of the first two ribonucleoside triphosphates up to 125 microM whereas elongation is carried out optimally with less than 10 microM. Under normal in vitro transcription conditions, mitochondrial RNA polymerase only employs the in vitro start site (+1 position), again without regard to the nucleotide at the position. Even with initiator dinucleotide monophosphates as primers, the polymerase is only capable of initiating transcription at this position and one other, i.e. 1 base upstream (-1). Dinucleotides enhance transcription from partially active variant promoters (mutations around the initiation sites -3, -1, +1, +2), suggesting that these mutations reduce transcription by their effects on initiation. In contrast, inactive promoters (-7C, -6G, -4A, and -2A) are not active in the presence of initiating dinucleotide. We suggest that dinucleotides may function in one of three ways: (i) bypassing the energy barrier in forming the first internucleotide bond; (ii) stabilizing the initiation complex; or (iii) accelerating promoter clearance.