Purification of ArcA and analysis of its specific interaction with the pfl promoter-regulatory region.
ArcA is one of several transcription factors required for optimal anaerobic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl promoter-regulatory region we developed a procedure for the isolation of ArcA. The purification of ArcA involved chromatography in heparin agarose, hydroxylapatite and Mono-Q matrices and delivered a protein that was > 95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA-DNA complexes could be resolved depending on the ArcA concentration used. This finding suggested that either multiple ArcA-binding sites are present in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA-binding activity of ArcA could be increased as estimated 10-fold by prior incubation of the protein with carbamoyl phosphate, suggesting that phosphorylation activates DNA binding or oligomerisation. DNase I footprint analyses identified four sites that were protected by ArcA from cleavage. Two of these sites spanned the transcription start site and -10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration host factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of promoter 7. These results are congruent with the hypothesis that a higher-order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoters of the pfl operon.
Drapal
N
N
Lehrstuhl für Mikrobiologie, Universität München, Germany.
Sawers
G
G
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000494:pharmacology; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D034982:Integration Host Factors; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D034982:Integration Host Factors; D007506:Iron-Sulfur Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C041610:arcA protein, E coli; D002221:Carbamyl Phosphate; D000123:Acetyltransferases; C022560:formate C-acetyltransferase