RESUMO
The entire human topoisomerase II alpha (hTopoII alpha) dimer was expressed in the yeast Saccaromyces cerevisiae, purified to homogeneity, and subjected to atomic force microscopy (AFM) under a tapping mode. Molecular images obtained exhibited a 'heart or donut-like' structure with a large axial hole. The main benefit of the application of AFM to study the hTopoII alpha is that clear images of the internal 'pore' have been achieved without crystallization, staining, or fixation of the sample. These images are consistent with the model in which topoisomerase II has a large internal gate for DNA strand passage.
Assuntos
DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/química , Isoenzimas/química , Microscopia de Força Atômica/métodos , Antígenos de Neoplasias , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Topoisomerases Tipo II/ultraestrutura , Proteínas de Ligação a DNA , Humanos , Isoenzimas/isolamento & purificação , Isoenzimas/ultraestrutura , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/enzimologiaAssuntos
Escherichia coli/enzimologia , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/ultraestrutura , Membrana Celular/enzimologia , Lipossomos , Substâncias Macromoleculares , Microscopia de Força Atômica/métodos , Modelos MolecularesRESUMO
Although the Na+/K(+)-ATPase alpha 1-subunit gene is ubiquitously expressed in vertebrates, its level of expression varies among tissue and cell types. In spite of similar mRNA distribution in tissues of mammals and birds, the 5'-flanking regions of alpha 1-subunit genes exhibit remarkable diversity; i.e., the core promoter activity of the TATA-less chicken alpha 1 gene strongly depends upon multiple Sp1-based regulation (six Sp1 sites), whereas the promoter activity of the TATA-like rat alpha 1-subunit gene relies on the two Sp1 and additional positive regulatory factors. Further analysis of the regulatory regions of the Na+/K(+)-ATPase alpha 1-subunit genes revealed that the vertebrate alpha 1-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation; the core promoter activities can be either enhanced or repressed depending on the availability of inhibitory factors. Two potential candidates for such inhibitory elements in both avian and mammalian Na+/K(+)-ATPase alpha 1-subunit genes are (1) a newly identified element, GCCCTC, and (2) a GCF-binding sequence, NN[G/c]CG[G/c][G/c][G/c]CN, or its reverse complement. Gel retardation assays using the inhibitory region of the chicken gene and crude nuclear extracts from tissue-cultured chicken and mouse cells showed the existence of a set of proteins that bind to this region. The amounts of individual regulatory proteins in different cell types seem to vary, resulting in differential formation of DNA/protein complexes in different cell types. Thus, the regulation of Na+/K(+)-ATPase alpha 1-subunit gene expression under different cellular environment as well as in different cell types can be achieved by a shared mechanism; modulation of the ratio of the abundance of individual inhibitory factors.
Assuntos
Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , ATPase Trocadora de Sódio-Potássio/química , Transcrição Gênica/genética , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Genes Reporter , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência , ATPase Trocadora de Sódio-Potássio/genética , Transfecção/genéticaRESUMO
Atomic force microscopy (AFM) allows to study the molecular structure of biological macromolecules with nm to A resolutions without crystallization. We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA/protein interaction: (i) Protein-binding sites can be mapped over a few kilobases of target DNA. (ii) Multimerization state of DNA-binding proteins can be determined simply by measuring the sizes of proteins bound to the DNA. These features are significant advantages over the capabilities provided by conventional techniques in biochemistry and molecular and structural biology.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Galinhas , DNA/química , Proteínas de Ligação a DNA/química , Microscopia de Força Atômica/métodos , Modelos Moleculares , Ligação Proteica , Análise de Regressão , ATPase Trocadora de Sódio-Potássio/genética , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/químicaRESUMO
The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter.
Assuntos
DNA Bacteriano/ultraestrutura , Proteínas de Choque Térmico HSP70/genética , Conformação de Ácido Nucleico , Óperon , Staphylococcus aureus/genética , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Genes Bacterianos , Microscopia de Força Atômica/métodos , Modelos Estruturais , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Bacteriano/biossíntese , Mapeamento por Restrição , Staphylococcus aureus/metabolismoRESUMO
The structure of Escherichia coli F0F1-ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping-mode operation. The majority of F0F1-ATPases were visualized as spheres with a calculated diameter of approximately 90 angstroms, and a height of approximately 100 angstroms from the membrane surface. F0 sectors were visualized as two different ring-like structures (one with a central mass and the other with a central hollow of greater than or equal to 18 angstroms depth) with a calculated outer diameter of approximately 130 angstroms. The two different images possibly represent the opposite orientations of the complex in the membranes. The ring-like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.
Assuntos
Escherichia coli/enzimologia , Proteínas de Membrana/química , ATPases Translocadoras de Prótons/química , Membrana Celular/enzimologia , Microscopia de Força AtômicaRESUMO
Ion channels and pumps in cell membranes consist of multiple transmembrane segments that are thought to be critical for transport of ions. Channel structures constituted by these transmembrane segments are characteristic of ion channels, whereas such structures have not been identified in ion pumps until now. By applying atomic force microscopy on Na+,K(+)-ATPase molecules in canine kidney membranes under tapping mode, we identified a hollow in the protein with a characteristic internal diameter of 6-20 A and an external diameter of 20-55 A depending upon treatment conditions. This hollow may be interpreted as a channel-like conformation of Na+,K(+)-ATPase. In the regions where the proteins were absent, lipid head structures with 2 A width and 6 A length were imaged in an orthorhombic lattice.