Abstract

Yeast mitochondral RNA polymerase specificity factor encoded by the nuclear MTF1 gene is required for a selective transcription on nonanucleotide mitochondral promoter by core RNA polymerase. Although there is a little amino acid sequence similarity of Mtf1p with bacterial sigma factors, the mode of transcriptional initiation of mitochondrial RNA polymerase is identical to that of E. coli RNA polymerase. To study the interaction of mtf1p with core polymerase, we carried out region-directed random mutagenesis of the core binding domain with the pool of mutant oligonucleotide. Out of 4,000 transformants screened for petite phenotype on glycerol media by plasmid shuffling, six alleles of the MTF1 gene were isolated. The positions of amino acid replacements that resulted in mtf1 mutants were limited to amino acids 53-54 and 65-67. Among mutant forms of Mtf1p overproduced in E. coli, Mtf1p with either L53H or Y65Dmutation was unable to produce a selective transcript in run-off transcription reaction, suggesting that amino acids L53 and Y65 are crucial for promoter recognition and/or contact with core polymerase.

• Keywords: Mitochondrial transcription; MTF1; RNA polymerase