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DNA'Protein Interactions: Principles and Protocols (Methods - download pdf or read online

By Tom Moss

ISBN-10: 0896036251

ISBN-13: 9780896036253

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Additional info for DNA'Protein Interactions: Principles and Protocols (Methods in Molecular Biology)

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Metzger and Heumann the optimal salt conditions optimal for complex. Add 6 mM Mg2+, if this is not already present. Add Exo III and incubate at 37oC. In order to establish the optimum conditions, perform a series of experiments using concentrations Exo III between 1 and 200 U per reaction (20 µL) and incubation times varying between 1 and 45 min. Exo III seems to be rather stable over a wide range of ionic strengths, and no large changes in activity were observed in the range between 0 and 100 mM NaCl (or KCl) in the incubation mixture (see Note 2).

The technique can be used to determine the site of interaction of most sequence-specific DNA-binding proteins but has been most extensively applied to the study of transcription factors. Because the DNase I molecule is relatively large as compared to other footprinting agents (see Chapters 5 and 6 on the use of hydroxy radicals and diethylpyrocarbonate), its attack on the DNA is relatively easily sterically hindered. Thus, DNase I footprinting is the most likely of all the footprinting techniques to detect a specific DNA–protein interaction.

Temperature-dependent footprinting studies showed that the transcription initiation complex undergoes three different conformations characterized by a specific “footprint” until a transcription competent complex is formed. These conformations could be attributed to the so-called closed, intermediate, and open complex. A bent conformation of DNA in complex with DNA was concluded from the OH radical probing of the lambda PR promoter complex (5). 2. Site-specific cleavage of the DNA of a RNA polymerase binary complex by both free and EDTA-chelated Fe 2+ was detected in absence of Mg2+ ions (6,7).

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DNA'Protein Interactions: Principles and Protocols (Methods in Molecular Biology) by Tom Moss


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