Usage of non-canonical promoter sequence by the yeast mitochondrial RNA polymerase

Prior work has demonstrated that a conserved nonanucleotide [5'-TATAAGTAA(+2)] promoter sequence is used by the mitochondrial [mt]1 RNA polymerase in Saccharomyces cerevisiae. However, the highly AT-rich yeast mt genome carries many other promoter-like sequences, but only a fraction of them are involved in gene-specific transcription. To examine the sequence variability of this nonanucleotide promoter motif, single or multiple nt substitutions were introduced into the canonical promoter sequence. The transcriptional activity of these altered promoter sequences was examined under the in-vitro reaction conditions. The results presented here determined that several variant promoter sequences (i. e. TAAAAGTAA, TATAAGAAA, TATAAGTAG, TATAAGAAG, TATAAGAGA, TATAAGGGA, TATAAGTGG, TAAAAGTAG) were efficiently used by the mtRNA polymerase. However, a single (i.e. AATAAGTAA, TTTAAGTAA, TATTAGTAA, TATAACTAA, TATAAGGAA, TATAAGTAT) or multiple (TATAGGAAA, TAAAAGGAA, TATAGGGAA, TAAAGGAAA, TAAAGGGAA) nt substitution(s) in other locations drastically reduced mt promoter function. Interestingly, some of these poorly or partially active promoter variants (i.e. TATAAGGAA, TATAAGTAT, TATAAGTCA) became fully functional in the presence of sequence-specific dinucleotide primer. Since dinucleotide primer bypasses the first phosphodiester bond formation in transcription, it is suggested that the -1T-->G, +1A-->C and +2A-->T mutations affect mt transcription at the level of initiation rather than polymerase binding.