Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli

Lisa Anderson, Tracy Palmer, Nicholas C Price, Stephen Bornemann, David H Boxer, Richard N Pau

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    78 Citations (Scopus)

    Abstract

    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 His(6)-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;he 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 K-d of 0.8 mu M. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate K-d of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-depen dent 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-N-12-ATAACG.

    Original languageEnglish
    Pages (from-to)119-126
    Number of pages8
    JournalEuropean Journal of Biochemistry
    Volume246
    Issue number1
    DOIs
    Publication statusPublished - 15 May 1997

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