RESUMO
Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the -10 region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the -10 region in the gyrA promoter. On the basis of analysis of various mutants in the -10 region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA anmd gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling.
Assuntos
Algoritmos , Sequência de Bases , Sequência Consenso , DNA Girase/genética , Análise Mutacional de DNA , DNA Super-Helicoidal/química , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Homeostase , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Moldes Genéticos , Ativação TranscricionalRESUMO
EtBr binding to DNA has been used to determine the superhelical density of Goat mtDNA. The differential binding of closed circular and nicked circular DNA is estimated by simple fluorescence measurements, which is a function of state of DNA supercoiling. The method is simple and quick over the cesium chloride density gradient centrifugation studies.
Assuntos
Animais , DNA Mitocondrial/química , DNA Super-Helicoidal/química , Fluorometria , Cabras/genéticaRESUMO
Supercoiled DNA on treatment with NaOH followed by neutralization produces a condensed structure (form Id). This structure does not split into topoisomers when run on long gel in presence of intercalating agents and the migration of this form does not change appreciably in presence or absence of ethidium bromide. Relaxation of form Id by topoisomerase I from pea chloroplast is facilitated more than form I. Single-stranded binding (SSB) protein binds more to form Id as evidenced from gel retardation study. Hydroxyl radical nicking is facilitated in this form. Compared to form I, this form produces half the number of transformants, but adsorption and penetration remain almost same in both the forms. Post-transformational growth using 32P labelled form I and form Id showed greater amount of degradation in form Id.