Mapping protease susceptibility sites on the Escherichia coli transcription factor sigma70.
N-terminally and C-terminally histidine-tagged versions of Escherichia coli RNA polymerase initiation factor sigma70 were subjected to limited proteolysis and electrophoretic separation. The protein fragments were transferred to nitrocellulose, and biotinylated nitrilotriacetic acid was used to detect the His-tagged ladder that resulted. Using size markers of known lengths derived from chemical cleavage of the same His-tagged sigma70, we were able to map the sites of proteolysis for sigma70 free in solution, bound to core RNA polymerase, and in the Mg2+-dependent open complex with lambdaPR promoter DNA. Numerous sites of changed susceptibility were mapped. Most of these sites mapped near residues 100 and 500. In addition, the highly acidic region around residue 190 became susceptible to cleavage in the open promoter complex. These results suggest that sigma70 undergoes significant conformational changes upon binding to core RNA polymerase and upon open promoter complex formation.
McMahan
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SA
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706, USA.
Burgess
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D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000235:genetics; D020035:Holoenzymes / Q000235:genetics / Q000302:isolation & purification; D006868:Hydrolysis; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D010957:Plasmids / Q000138:chemical synthesis; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004269:DNA, Bacterial; D020035:Holoenzymes; D010446:Peptide Fragments; D012808:Sigma Factor; D006639:Histidine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D010450:Endopeptidases