Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000.
Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5' end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.
Stolt
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Bacterial Molecular Genetics Unit, Department of Clinical Sciences, London School of Hygiene & Tropical Medicine, England.
Stoker
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009170:Nontuberculous Mycobacteria / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D015534:Trans-Activators
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C048817:RepB protein, Bacteria; D015534:Trans-Activators; C045264:replication initiator protein; D003850:Deoxyribonuclease I; D004265:DNA Helicases