RESUMO
Mass spectrometry matrix-assisted laser desorption ionization (MALDI) analysis and N-terminus sequencing as well as immunoblotting experiments using human and mouse antibodies have allowed us to identify the 25 kDa protein, previously isolated from rat liver using magnetic beads coated with a rat liver mitochondrial (mt) DNA region upstream of the Ori-L, as the homologue of human mt transcription factor A (mtTFA). We can therefore identify this DNA binding protein as the rat mtTFA. Furthermore, since we previously showed that the 25 kDa protein purified from rat liver was able to bind the curved mtDNA region upstream of the Ori-L as well as the curved mtDNA in the D-loop region, the results here reported lead us to state, for the first time, that mtTFA binds both the curved regions of mtDNA upstream of the two replication origins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Origem de Replicação , Transativadores/metabolismo , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Humanos , Fígado/química , Camundongos , Dados de Sequência Molecular , Peso Molecular , Ratos , Alinhamento de Sequência/métodos , Análise de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transativadores/genéticaRESUMO
An in organello footprinting approach has been used to probe a protein-DNA interaction of a nuclear coded 25 kDa protein, previously isolated in our laboratory, that binds "in vitro" a region within the ND2 gene, located upstream of the Ori-L. Footprinting studies with the purine-modifying reagent dimethyl sulfate and the pirimidine-modifying reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting has allowed the detection of a protein-DNA interaction within the curved ND2 region with contact sites located in both the strands. Potassium permanganate footprinting allowed detection of an adjacent permanganate-reactive region. We hypothesize that the permanganate-reactive region is a single stranded DNA due to a profound helix distortion induced by a 25 kDa protein binding to the nearest region.
Assuntos
Pegada de DNA , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/química , Mitocôndrias Hepáticas/ultraestrutura , Animais , Sequência de Bases , Replicação do DNA , DNA Mitocondrial/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleoproteínas/química , Mitocôndrias Hepáticas/química , Peso Molecular , Conformação de Ácido Nucleico , Permanganato de Potássio/química , Ligação Proteica , Ratos , Ésteres do Ácido Sulfúrico/químicaRESUMO
The presence of a curved DNA sequence in the gene for the NADH-dehydrogenase subunit 2 of rat mitochondrial genome, upstream from the origin of the light strand replication have been demonstrated through theoretical analysis and experimental approaches. Gel retardation assays showed that this structure makes a complex with a protein component extracted from the mitochondrial matrix. The isolation and purification of this protein is reported. With a Sepharose CL-6B and magnetic DNA affinity chromatography a polypeptide was purified to homogeneity having 25-kDa mass as shown by gel electrophoresis. To functionally characterize this protein, its capability to bind to other sequences of the homologous or heterologous DNA and to specific riboprobes was also investigated. A role for this protein as a trans-acting agent required for the expression of the mammalian mitochondrial genome is suggested.