In vitro characterization of the yeast mitochondrial promoter using single-base substitution mutants

• Published in: The Journal of biological chemistry Vol. 262; no. 28; pp. 13690 - 13696
• Main Authors: Biswas, T K, Ticho, B, Getz, G S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05-10-1987; American Society for Biochemistry and Molecular Biology
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Chromosome Deletion; CLONE; CLONES; DNA, Mitochondrial - genetics; Eukaryotes; EXPERIMENTACION IN VITRO; EXPERIMENTATION IN VITRO; Fundamental and applied biological sciences. Psychology; Genes, Fungal; Genetic technics; GENOMAS; GENOME; GENOMES; IN VITRO EXPERIMENTATION; Methods. Procedures. Technologies; MITOCHONDRIA; MITOCHONDRIE; MITOCONDRIA; Molecular and cellular biology; Molecular genetics; MUTANT; Mutant screening; MUTANTES; MUTANTS; Mutation; NUCLEOTIDE; NUCLEOTIDES; NUCLEOTIDOS; Promoter Regions, Genetic; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; In vitro transcription; Enzyme; RNA polymerase; Fungi; Promoter; Mitochondria; Substitution; Ascomycetes; Oligonucleotide; Mutation; Initiation; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)76482-5

A short DNA sequence, 5'ATATAAGTA(+1)3', extending from −8 to +1 nucleotides has been shown to function as a promoter in the yeast mitochondrial genome. A complete set of single site mutations of this nonanucleotide promoter sequence has been constructed, cloned, and used to promote specific in vitro transcription using a highly purified mitochondrial RNA polymerase. Each deviation from the natural promoter sequence results in a reduction or abolition of specific transcription depending on the nucleotide substituent. The nucleotide at −8 is not considered as a component of the promoter. Any nucleotide at position +1 is compatible with correct transcriptional initiation. The consensus sequence that exists in vivo is the strongest promoter since only down mutations are seen among the substitutions. The mutant analyses indicate that a very short octanucleotide sequence comprised of 5'TAT/aAA/g/cGT/a/cN(+1)3' is the minimal sequence necessary to direct accurate initiation by mitochondrial RNA polymerase.