In vitro characterization of the yeast mitochondrial promoter using single-base substitution mutants

A short DNA sequence, 5'ATATAAGTA(+1)3', extending from -8 to +1 nucleotides has been shown to function as a promoter in the yeast mitochondrial genome. A complete set of single site mutations of this nonanucleotide promoter sequence has been constructed, cloned, and used to promote specific in vitro transcription using a highly purified mitochondrial RNA polymerase. Each deviation from the natural promoter sequence results in a reduction or abolition of specific transcription depending on the nucleotide substituent. The nucleotide at -8 is not considered as a component of the promoter. Any nucleotide at position +1 is compatible with correct transcriptional initiation. The consensus sequence that exists in vivo is the strongest promoter since only down mutations are seen among the substitutions. The mutant analyses indicate that a very short octanucleotide sequence comprised of 5'TAT/aAA/g/cGT/a/cN(+1)3' is the minimal sequence necessary to direct accurate initiation by mitochondrial RNA polymerase.