RESUMO
TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.
Assuntos
Empacotamento do DNA/fisiologia , DNA Mitocondrial/química , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional/fisiologia , Sequência de Bases , Cristalização , DNA/química , DNA/genética , DNA/fisiologia , DNA Mitocondrial/fisiologia , Proteínas de Ligação a DNA/fisiologia , Dimerização , Humanos , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Conformação Proteica , Análise de Sequência de DNA , Fatores de Transcrição/fisiologiaRESUMO
The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1-Fis1 complex that contains both the N-terminal extension and coiled-coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1-Fis1 complex is mediated by the antiparallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses showed that these contacts are important for mitochondrial fission activity. These results indicate that, in addition to dimerization, the unusually long Mdv1 coiled coil serves a scaffolding function to stabilize the Mdv1-Fis1 complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Mitocôndrias/química , Complexos Multiproteicos/química , Dobramento de Proteína , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cristalografia por Raios X , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Sequências Hélice-Alça-Hélice , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.