Journal of Microbiology

Amino acid substitutions conferring cold-sensitive phenotype on the yeast MTF1 gene: Jang , Sei Heon; J. Microbiol. 1997;35(3):228-233.

Abstract: The MTF1 gene of Saccharomyces cerevisiae encodes a 43 kDa MITOCHONDRIAL RNA polymerase specificity factor which recognizes mitochondrial promoters to initiate correct transcription. To better understand structure-function of the MTF1 gene as well as the transcription mechanism of mitochondrial RNA polymerase, two cold-sensitive alleles of the MTF1 mutation were isolated by plasmid shuffling method after PCR-based random mutagenesis of the MTF1 gene. The mutation sites were analyzed by nucleotide sequencing. These cs phenotype mtf1 mutants were respiration competent on the nonfermentible glycerol medium at the permissive temperature, but incompetent at 13℃. The cs phenotype allele of the MTF1, yJH147, encoded an L146P replacement. The other cs allele, yJH148, contained K179E and K214M double replacements. Mutations in both alleles were in a region of Mtflp which is located between domains with amino acid sequence similarities to conserved regions 2 and 3 of bacterial s factors.

Identification of Critical Amino Acids in the Core RNA Polynerase Binding Region of Yease Mtflp: Yang, Jae Sub , Jang, Sei Heon; J. Microbiol. 1998;36(3):208-213.

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.

Search