Multiple protein-DNA and protein-protein interactions are involved in transcriptional activation by MalT.
The promoters of the Escherichia coli maltose regulon are positively regulated by the MalT protein, which recognizes a short asymmetric nucleotide sequence that is present as several copies in each promoter of the regulon. We report a detailed biochemical characterization of the interaction of MalT with the promoter of the malPQ operon. The MalT sites in malPp were precisely located and their occupation as a function of MalT concentration was quantified using DNase I and dimethyl sulphate footprinting experiments. The contribution of each site to malPp activity was assessed by introducing mutations that destroy them and measuring the residual in vivo and in vitro activity. Two main results were obtained. First, although the proximal MalT site is centred at -37.5, RNA polymerase is likely to establish a contact required for malPp activity with at least one base pair of the promoter -35 region; this close proximity between RNA polymerase and MalT bound to site 1 suggests that the two proteins interact. Second, even if the interaction of MalT with the three functional sites in malPp is a co-operative process, the MalT molecules bound to the two distal sites play a more subtle role than simply increasing the occupancy of the proximal site and may also contact RNA polymerase. We suggest that, in the nucleoprotein structure responsible for the initiation of transcription, MalT, RNA polymerase and malPp are held together by several weak interactions.
Danot
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Unité de Génétique Moléculaire (CNRS URA 1149), Institut Pasteur, Paris, France.
Raibaud
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D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical; D008320:Maltose / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D011401:Promoter Regions, Genetic; D013463:Sulfuric Acid Esters; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D013463:Sulfuric Acid Esters; D014157:Transcription Factors; D017665:Hydroxyl Radical; D008320:Maltose; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate