The highly conserved nonanucleotide (5'-TATAAGTAA[+2]) promoter sequence dictates initiation of gene-specific transcription by the mitochondrial (mt) RNA polymerase in yeast mitochondria. However, transcriptional efficiency of the nonanucleotide promoter in different mt genes varies severalfold. To explore the regulatory role of the promoter-proximal template sequence in mt transcription, different deletion, nucleotide (nt) substitution, and tandem promoter constructs were analyzed under in vitro transcription reaction conditions. It has been found that the conserved nonanucleotide promoter plus more than 9 nt of nonconserved sequence 3' to the promoter were absolutely essential for mt gene-specific transcription. In addition, approximately 300 nt of nonspecific DNA sequence 5' to the promoter was also important for efficient transcription. Interestingly, introduction of consecutive T residues in the early transcribed sequence of the template strongly inhibited mt transcription at low nt concentrations (i.e., 5 microM UTP). In contrast, neither other nt clusters nor a bacterial terminator-like sequences at that location inhibited mt transcription. Under the nonproductive reaction conditions, the full-length transcript from the mt polyT template was drastically reduced with the formation of several short abortive oligoribonucleotides. These results suggest that the transcriptional efficacy of the yeast mt promoter is influenced by sequence 3' to the promoter.