Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator.
The mode of action of regulated promoters is largely determined by kinetic parameters which govern the interaction between promoters and proteins involved in induction and repression of transcription. To gain insight into the interplay between positively and negatively acting transcriptional regulators, in this case AraC and LacR, we have generated a panel of promoter sequences derived from P(lac), the promoter of the Escherichia coli lac operon. The function of these promoters is limited at different steps and to various extents within the pathway of RNA polymerase (RNAP)/promoter interaction. Moreover, in all promoters the cAMP receptor protein binding site was replaced by the binding motif of AraC to prevent pleiotropic effects in vivo upon activation. Analyzing the activation of these promoters by AraC in vivo under conditions of repression by LacR and derepression yielded a three step model of transcription initiation which reveals mechanisms of AraC and LacR action. Our data show three distinct rate limiting steps at which AraC can exert its function. In general, the activator accelerates the formation of the first stable complex between RNAP and promoter. At most promoter sequences, however, its main impact is on the conversion of the closed to the open complex. However, AraC is also capable of eliminating limitations at steps following open complex formation.
Lutz
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R
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Lozinski
T
T
Ellinger
T
T
Bujard
H
H
D051777:AraC Transcription Factor; D001089:Arabinose / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007700:Kinetics; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases