Ethylation interference footprinting of DNA-protein complexes.
Structural studies of DNA-protein complexes reveal networks of contacts between proteins and the phosphates, sugars and bases of DNA. A range of biochemical methods, termed chemical footprinting, aim to determine the functional groups on DNA which are protected in solution by bound protein against modification or where chemical pre-modification interferes with subsequent protein binding. One of these approaches, termed ethylation interference footprinting, reveals which backbone phosphate groups are contacted by protein and the positions where the DNA-protein interface is so tight that the modification cannot be accommodated. This chapter describes the steps necessary to perform an ethylation interference experiment, including modification of DNA using ethylnitrosourea, fractionation of the products based on their affinities for a DNA-binding protein and analysis of the "bound" and "free" fractions to reveal sites critical for complex formation. This is illustrated using results from our experiments with the Escherichia coli methionine repressor, MetJ.
Manfield
Iain W
IW
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
Stockley
Peter G
PG
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D005591:Chemical Fractionation; D018983:DNA Footprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000378:metabolism*; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011868:Radioisotopes; D012097:Repressor Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011868:Radioisotopes; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; D005038:Ethylnitrosourea