Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli.
Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N-terminal His6-tagged derivative and characterised both with and without the N-terminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its alpha-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 microM. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase I footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter. This region contains a 6-base palindromic sequence CGTTAT-N12-ATAACG.
Anderson
L A
LA
Department of Biochemistry, Medical Sciences Institute, University of Dundee, UK.
Palmer
T
T
Price
N C
NC
Bornemann
S
S
Boxer
D H
DH
Pau
R N
RN
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002942:Circular Dichroism; D018983:DNA Footprinting; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism* / Q000494:pharmacology; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism; D017973:Tungsten Compounds / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C491064:ModE protein, E coli; C101050:ModE protein, bacteria; D012097:Repressor Proteins; D014157:Transcription Factors; D017973:Tungsten Compounds; D014443:Tyrosine; D008982:Molybdenum; D014364:Tryptophan; C045951:tungstate