Initiation of transcription from each of the two human mitochondrial promoters requires unique nucleotides at the transcriptional start sites

Abstract

Promoters for transcriptional initiation on each strand of human mtDNA lie in close proximity in the displacement-loop region. Transcription initiates within these promoter elements, providing an opportunity to analyze the effects of nucleotide sequence changes on the efficiency and fidelity of this event within two delimited regions. Each promoter was individually altered by a site-directed mutagenesis approach and assayed in vitro for the ability to support transcription. The data clearly confirm the previous assignment of promoter functions for these elements and show that single nucleotide substitutions immediately upstream of the transcriptional start sites can abolish transcription. In all cases of decreased transcription, the correct site of initiation is maintained. Although the heavy-strand promoter and light-strand promoter are similar in primary sequence, they exhibit remarkably different sensitivities to base substitutions.