Supercoiling is essential for the formation and stability of the initiation complex at the divergent malEp and malKp promoters.
malEp and malKp are divergent and partially overlapping promoters of the Escherichia coli maltose regulon, whose activity depends on the presence of two transcriptional activators. MalT and CRP (cAMP receptor protein). Their activation involves a common 210 base-pair regulatory region encompassing multiple binding sites for both activators. Using a supercoiled plasmid containing malEp and malKp as template, purified proteins and a single-round transcription assay, we developed an in vitro system in which both promoters behave as in vivo. In this system, malEp and malKp are active only in the presence of both MalT and CRP, and various mutations in the MalT or CRP binding sites affect the promoters in the same way as they do in vivo. We showed that supercoiling plays a crucial role not only for the formation of the initiation complex at malEp and malKp but also for its stability. In addition, dimethylsulphate protection experiments provide evidence that the nucleoprotein complexes formed by CRP and MalT bound to malEp and malKp on supercoiled and relaxed DNA are different. We speculate that one of the roles of supercoiling might be to assist the assembly of a preinitiation complex involving the regulatory region DNA and several molecules of MalT and CRP.
Richet
E
E
Unité de Génétique Moléculaire, URA 1149 du CNRS, Institut Pasteur, Paris, France.
Raibaud
O
O
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002474:Cell-Free System; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008320:Maltose / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D011953:Receptors, Cyclic AMP; D014157:Transcription Factors; D008320:Maltose