Identification of a single transcriptional initiation site for the glutamic tRNA and COB genes in yeast mitochondria

Abstract

We have identified a single transcriptional initiation site for the glutamic tRNA and COB (cytochrome b) genes by using the complementary techniques of in vitro capping of RNA and in vitro transcription. In the capping reaction, mitochondrial RNA is labeled with [alpha-32P]GTP by vaccinia virus guanylyltransferase. This reaction is specific for the 5' ends of RNA retaining the terminal triphosphate of transcriptional initiation. Exploiting the extremely low G+C content (18%) of yeast mitochondrial DNA, we digested in vitro capped transcripts from various petite deletion mutants with the G-specific RNase T1. By petite deletion mapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNA gene. When the sequence of this oligonucleotide was determined, it perfectly matched the DNA sequence 391 base upstream of the glutamic tRNA. Purified yeast mitochondrial RNA polymerase initiated transcription in vitro at the same site as shown by the sequence of the 33-base oligonucleotide product of the reaction performed in the absence of CTP. Initiation starts at a nonanucleotide sequence previously implicated in yeast mitochondrial transcriptional initiation. Because there is no evidence of an initiation site in the 1,050 bases between the glutamic tRNA and COB genes, the two genes are likely to be transcribed together. Further evidence of a long common transcript was provided by RNA blot hybridization.