DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in nondenaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between CJ and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5: 1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and CJ factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the CJ factor.The eubacterial DNA-dependent RNA polymerase (subunit composition: Pj?aza) in complex with a promoter adopts a number of sequential conformational states until an active transcription complex is formed. Four different transient complexes have been proposed to describe the transition from a non-specific RNA-polymerase DNA complex to a specific initiation complex [I -31: (a) a non-specific RNA-polymerase-DNA complex; (b) a specific closed complex; (c) an isomerisation complex as a result of a conformational change of the RNA polymerase; (d) a second isomerisation complex (open complex) as a result of the melting of the DNA.Adding only the starting nucleoside triphosphates, an initiation complex is formed, which catalyzes the synthesis of RNA molecules abortively to a length of approximately 8 nucleotides. After addition of more triphosphates and thus formation of longer transcripts, the initiation factor c is released from the ternary complex [4 -61.The different intermediary transcription complexes have been postulated from experiments using the radioactive incorporation test [7], kinetic experiments using the filter-binding method [8,9] and the abortive initiation assay [lo]. This paper characterizes some of the intermediary transcription complexes on the basis of their electrophoretic mobility on non-denaturing gels. Previously non-denaturing gels were used for the study of the interaction of complexes with long DNA fragments could not be characterized because the protein-DNA complexes did not enter the gel matrix. Smaller DNA fragments were used by Fried and Crothers [12] to discriminate different occupational states of the repressor/operator system. We applied this gel method on the investigation of the interaction of DNA-dependent RNA polymerase with a promoter-carrying DNA fragment. MATERIALS AND METHODSRNA polymera...

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Pretreatment with recombinant human granulocyte CSF (G-CSF) protected mice in two different models of septic shock. Intravenous injection of 250 micrograms/kg G-CSF to mice prevented lethality induced by 5 mg/kg LPS. Injection of 50 micrograms/kg G-CSF protected galactosamine-sensitized mice against LPS-induced hepatitis. In either case, this protection was accompanied by a suppression of LPS-induced serum TNF activity. In contrast, when galactosamine-sensitized mice were pretreated with 50 micrograms/kg murine recombinant granulocyte/macrophage CSF instead of G-CSF and subsequently challenged with LPS, serum TNF activity was significantly enhanced and mortality was increased. The suppressive effect of G-CSF on LPS-induced TNF production was also demonstrated in rats. In vivo, no TNF was detectable in the blood of LPS-treated rats, which had been pretreated with G-CSF. Ex vivo, alveolar macrophages, bone marrow macrophages, Kupffer cells, or peritoneal macrophages prepared from G-CSF-treated rats produced significantly less TNF upon stimulation with LPS than corresponding populations from control rats. However, when these macrophage populations were incubated with G-CSF in vitro, LPS-induced TNF production was unaffected. These data suggest that the G-CSF-mediated suppression of TNF production is not a direct effect of G-CSF on macrophages. To examine whether, independent of the protection against LPS, G-CSF treatment still activated neutrophils, it was demonstrated that granulocytes from G-CSF-treated rats were primed for PMA-induced oxidative burst and for ionophore/arachidonic acid-stimulated lipoxygenase product formation. The experiments of this study support the notion that G-CSF is a negative feedback signal for macrophage-derived TNF-alpha production during Gram-negative sepsis.