UNUSUAL USAGE OF NONCOMPLEMENTARY DINUCLEOTIDE PRIMERS BY THE YEAST MITOCHONDRIAL RNA-POLYMERASE

The mitochondrial RNase P RNA gene in yeast Saccharomyces cerevisiae i s transcribed from a variant mitochondrial promoter (SP). The sequence of this SP promoter [TATAAGAAG (+2)] differs from the conserved mitoc hondrial promoter sequence [TATAAGTAA (+2)] by -1T --> A and +2A --> G nucleotide substitutions. To determine the effect of these nucleotide alterations in mitochondrial promoter function, an in vitro transcrip tion analysis was carried out. In the presence of high concentrations of rNTPs (i.e., 125 mu M), transcription initiation on the wild-type o r variant promoter occurred at the conventional 3' adenine nucleotide. However, at low rNTP concentrations (i.e., 5 mu M) and in the presenc e of a complementary dinucleotide primer corresponding to positions -1 + 1, the mitochondrial RNA polymerase started transcription one nucle otide upstream of the conventional start site. Surprisingly, in the pr esence of some noncomplementary dinucleotides (i.e., GpA or CpA), whic h do not have perfect Watson-Crick base pairing with the initiator seq uence, transcriptional initiation also occurred with the SP promoter b ut 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 p osition +2 is mainly responsible for this unusual function of the SP p romoter. (C) 1997 Academic Press.