In vitro and in vivo characterization of three major dadAX promoters in Escherichia coli that are regulated by cyclic AMP-CRP and Lrp.
We have shown previously that the dadAX operon of Escherichia coli expresses multiple transcripts, which are repressed by leucine-responsive regulatory protein (Lrp). Here we used site-directed mutagenesis and in vitro and in vivo transcription assays to show that each of the three major dad transcripts requires a specific promoter. These promoters, P1-P3, overlap and are positively regulated in vivo by cyclic AMP-CRP. DNase I footprinting experiments localized two CRP binding sites in this region: CRP1, which is positioned upstream of P1-P3, and CRP2, which is located within the promoters. Site-directed mutagenesis of each site provided evidence that CRP1 is necessary for the effects of cyclic AMP-CRP on dad expression in vivo and in vitro, and that CRP2 probably plays little or no role in this process.
Zhi
J
J
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 11794, USA.
Mathew
E
E
Freundlich
M
M
D000411:Alanine Racemase / Q000235:genetics*; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000502:physiology*; D003605:D-Amino-Acid Oxidase / Q000235:genetics*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D003605:D-Amino-Acid Oxidase; D000411:Alanine Racemase