Effect of point mutations on in vitro transcription from the promoter for the large ribosomal RNA gene of yeast mitochondria.

Initiation of transcription on mitochondrial DNA of Saccharomyces cerevisiae was studied in an in vitro system with a mtRNA polymerase fraction reconstituted from separately purified components and with DNA templates containing the promoter of the gene coding for large rRNA. The effect of various point mutations in this promoter region was quantitated in assays containing a wildtype promoter in equimolar amount as internal control. Despite the strong conservation around the position at which RNA initiation occurs (ATATAAGTApuTA, initiation nucleotide underlined), none of the single point mutations abolished transcription-initiation completely. Some reduce the efficiency of initiation to 10-20% compared to the wild type promoter, while others have a much less pronounced effect. A change of the A at position +4 into a G even results in a promoter up mutation. Remarkably, alteration of the A at position +1 into a G or a T affects the efficiency of initiation only slightly and initiation is maintained at the same position.