Unusual Usage of Noncomplementary Dinucleotide Primers by the Yeast Mitochondrial RNA Polymerase

The mitochondrial RNase P RNA gene in yeastSaccharomyces cerevisiaeis transcribed from a variant mitochondrial promoter (SP). The sequence of this SP promoter [TATAAGAAG (+2)] differs from the conserved mitochondrial promoter sequence [TATAAGTAA (+2)] by −1T → A and +2A → G nucleotide substitutions. To determine the effect of these nucleotide alterations in mitochondrial promoter function, anin vitrotranscription analysis was carried out. In the presence of high concentrations of rNTPs (i.e., 125 μm), transcription initiation on the wild-type or variant promoter occurred at the conventional 3′ adenine nucleotide. However, at low rNTP concentrations (i.e., 5 μm) and in the presence of a complementary dinucleotide primer corresponding to positions −1 + 1, the mitochondrial RNA polymerase started transcription one nucleotide upstream of the conventional start site. Surprisingly, in the presence of some noncomplementary dinucleotides (i.e., GpA or CpA), which do not have perfect Watson–Crick base pairing with the initiator sequence, transcriptional initiation also occurred with the SP promoter but not with the conserved promoter sequence. This finding is the first example of utilization of noncomplementary dinucleotide primer by an RNA polymerase. Further analysis of mitochondrial promoter function by site-directed mutagenesis determined that the guanine nucleotide at position +2 is mainly responsible for this unusual function of the SP promoter.