Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites

Abstract

To identify transcription initiation sites in wheat mitochondria, the nascent 5'-ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [alpha-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed.