DNase I footprinting.
ABSTRACT Footprinting is a method for determining the sequence selectivity of DNA-binding compounds in which ligands protect DNA from cleavage at their binding sites. Footprinting templates are typically 50-200 base pairs long, and DNase I is the most commonly used nuclease for these experiments. This chapter describes the preparation and labelling of suitable DNA footprinting substrates, the footprinting experiment itself, and the way in which these data can be used to estimate the dissociation constant of the interaction.
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ABSTRACT: We have previously shown that iron regulates the transcription of inducible nitric oxide synthase (iNOS). To elucidate the underlying mechanisms we performed a series of transient transfections of murine fibroblast (NIH-3T3) and macrophage-like cells (J774.A1) with reporter plasmids containing the iNOS promoter and deletions thereof. By means of this and subsequent DNase I footprinting analysis we identified a regulatory region between -153 and -142 bp upstream of the transcriptional start site of the iNOS promoter that was sensitive to regulation by iron perturbation. Gel shift and supershift assays revealed that the responsible protein for this observation is NF-IL6, a member of the CCAAT/enhancer binding protein family of transcription factors. Binding of NF-IL6 to its consensus motif within the iNOS promoter was inducible by IFN-gamma and/or LPS, was reduced by iron, and was enhanced by the iron chelator desferrioxamine. Introduction of a double mutation into the NF-IL6 binding site (-153/-142) of an iNOS promoter construct resulted in a reduction of IFN-gamma/LPS inducibility by >90% and also impaired iron mediated regulation of the iNOS promoter. Our results provide evidence that this NF-IL6 binding site is of central importance for maintaining a high transcriptional rate of the iNOS gene after IFN-gamma/LPS stimulation, and that NF-IL6 may cooperate with hypoxia inducible factor-1 in the orchestration of iron-mediated regulation of iNOS.The Journal of Immunology 05/1999; 162(10):6171-7. · 5.52 Impact Factor
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ABSTRACT: To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.Molecular and Cellular Biology 07/1988; 8(6):2628-37. · 5.37 Impact Factor
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ABSTRACT: TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.Journal of bacteriology 02/2012; 194(8):1897-911. · 3.94 Impact Factor