DNA sampling: a method for probing protein binding at specific loci on bacterial chromosomes.
We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter.
Butala
Matej
M
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Busby
Stephen J W
SJ
Lee
David J
DJ
D001426:Bacterial Proteins / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000737:chemistry*; D003087:Colicins / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases; D048168:Escherichia coli K12 / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification; D005821:Genetic Techniques; D056968:Lac Repressors; D013058:Mass Spectrometry; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics
D001426:Bacterial Proteins; D003087:Colicins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D004706:Endodeoxyribonucleases