In order to identify precisely the sites of initiation of transcription of mitochondrial genes of rice, we performed in vitro capping/ribonuclease protection experiments and primer extension analyses. Using this approach, we identified the transcription initiation sites of three genes, namely, orf483, rrn18 and atp9. In previous studies, the transcription initiation sites of the rps3-rpl16-nad3-rps12 gene cluster and of two other genes, namely rrn26 and trnfM, were identified by the in vitro capping method. The alignment of sequences surrounding the initiation sites of these monocistronic and polycistronic transcripts revealed a conserved sequence motif. This conserved sequence exhibits a high degree of homology to the putative promoter sequence found in the mitochondrial DNAs (mtDNAs) of maize and wheat, an observation that suggests that this motif is recognized as a promoter in the rice mitochondria. In particular, the CRTA motif(where R is G or A) is highly conserved, as it is for example in maize, wheat, soybean and Oenothera. To ascertain the number of transcription initiation sites on the mitochondrial genome of rice, restriction fragments of rice mtDNA were allowed to hybridize with in vitro capped mitochondrial RNA and then were treated with ribonucleases T1 and A. The results of this hybridization experiment revealed that there are at least 16 sites of initiation of transcription on the mitochondrial genome of rice.
