Purification of the Escherichia coli purine regulon repressor and identification of corepressors.
The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
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RJ
Rolfes
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Zalkin
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight; D009876:Operon; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C065825:PurR protein, Bacteria; C489317:PurR protein, E coli; D012097:Repressor Proteins