title: Identification of three regions essential for interaction between a sigma-like factor and core RNA polymerase.
abstract: The cyclic interactions that occur between the subunits of the yeast mitochondrial RNA polymerase can serve as a simple model for the more complex enzymes in prokaryotes and the eukaryotic nucleus. We have used two-hybrid and fusion protein constructs to analyze the requirements for interaction between the single subunit core polymerase (Rpo41p), and the sigma-like promoter specificity factor (Mtf1p). We were unable to define any protein truncations that retained the ability to interact, indicating that multiple regions encompassing the entire length of the proteins are involved in interactions. We found that 9 of 15 nonfunctional (petite) point mutations in Mtf1p isolated in a plasmid shuffle strategy had lost the ability to interact. Some of the noninteracting mutations are temperature-sensitive petite (ts petite); this phenotype correlates with a precipitous drop in mitochondrial transcript abundance when cells are shifted to the nonpermissive temperature. One temperature-sensitive mutant demonstrated a striking pH dependence for core binding in vitro, consistent with the physical properties of the amino acid substitution. The noninteracting mutations fall into three widely spaced clusters of amino acids. Two of the clusters are in regions with amino acid sequence similarity to conserved regions 2 and 3 of sigma factors and related proteins; these regions have been implicated in core binding by both prokaryotic and eukaryotic sigma-like factors. By modeling the location of the mutations using the partial structure of Escherichia coli sigma70, we find that two of the clusters are potentially juxtaposed in the three-dimensional structure. Our results demonstrate that interactions between sigma-like specificity factors and core RNA polymerases require multiple regions from both components of the holoenzymes.
authors.affiliation: Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: P F
authors.initials: PF
authors.lastname: Cliften
authors.forename: J Y
authors.initials: JY
authors.lastname: Park
authors.forename: B P
authors.initials: BP
authors.lastname: Davis
authors.forename: S H
authors.initials: SH
authors.lastname: Jang
authors.forename: J A
authors.initials: JA
authors.lastname: Jaehning
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
chemical_list: D005656:Fungal Proteins; C071720:MTF1 protein, S cerevisiae; D046911:Macromolecular Substances; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012808:Sigma Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases