Footprinting technique

All footprinting techniques are designed to characterize binding interactions by determining the accessibility of the backbone of macromolecules to chemical/enzymatic cleavage or modification reactions. Synchrotron X-ray footprinting uses synchrotron radiation to generate reactive chemical species (such as hydroxide radical -OH) to detect and quantitate backbone accessibility. 

Use of the Footprinting Technique to Determine the Binding Site of a DNA-binding Protein... 

Protein binding sites can be studied by two different methods using RNA modifications the footprinting technique and the modification-selection experiment. The latter is described in Section 4.6. 

Fig. 4.13. Flow chart of the protein footprinting technique. Purified recombinant protein, which has been radioactively labelled at the N- or C-terminal, is cleaved by proteinases or chemicals with single-hit kinetics. The substrate is kept at native conditions in uncomplexed and complexed form. The cleavage products are analysed in high resolution SDS gels.

Other studies have attempted to utilise the properties of MIPs as selective catalytic materials for the hydrolysis of esters [116-118], whilst others [119-124] have used a footprint technique to successfully imprint catalytic sites onto inorganic surfaces. 

The DNAase-hypersensitive sites are small and dispersed throughout the chromatin they are interpreted as being due to the absence of histones or nucleosomes. Often, the binding of other proteins can be detected in the hypersensitive sites, using more sophisticated "footprinting" techniques. There is growing evidence for the existence of multi-subunit complexes of proteins bound at hypersensitive sites which may control the initiation of transcription. 

Protein-DNA interactions can be characterized experimentally by the DNA footprinting technique and the electrophoretic mobility shift assay (EMSA). (a) In vitro DNAse I footprinting (b) EMSA. Reactions contained an end-labeled radioactive fragment (a) or oligonucleotide (b) incubated with the zinc-finger DNA binding domain of the... 

Other "footprinting" techniques, e.g., observing cleavage of the DNA by hydroxyl radicals generated by reduction of IT2O2 by Fe(II) (Fig. 5-50), have also been employed. If was shovm that RNA polymerase binds to both the -10 and -35 sequences and also to sequences further upstream. The subunit associates with the DNA, principally the transcribed strand, along a region from about the -25 to the +12 position relative to the transcription start site. The a subunits bind to an UP element from —40 to -60 via their C-terminal domains (CTDs). See Fig. 28-4B. ... 

The ability to discern changes in the regulation of protein expression, not only by the use of antibodies, but also by means of Northern blotting techniques to monitor changes in mRNA levels, and footprinting techniques or gel retardation/ mobility shift assays to determine interaction of regulatory proteins with nucleic acids. 

Mirkovitch, and Darnell, J. E., Jr. (1991) Rapid in vivo footprinting technique identifies protein bound to the TTR gene in the mouse liver. Genes Dev. 5, 83-93. 

I cleavage are affected by both the width of minor groove and the flexibility of the DNA. Furthermore, these parameters can be affected by protein binding. DNase I footprinting data should thus be complemented by other footprinting techniques such as those based on micrococcal nuclease, hydroxyl radical (Fe -EDTA and H2O2), and/or methylation by dimethyl sulfate (64). 

Since the initial description and application of the DNase I footprinting techniques to the study of the lac repressor-binding site (43), various technical refinements have been made (44,45). Perhaps more importantly, a deeper understanding of the structural requirements for DNase I to cleave DNA has provided a solid basis for interpreting the footprint data. 

---