AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein.
The arabinose operon promoter, pBAD, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, araO2 and araI1. pBAD is also positively regulated by AraC-arabinose and the cyclic AMP receptor protein, CRP. We provide evidence that CRP breaks the araO2-araI1 repression loop in vitro. The ability of CRP to break the loop in vitro and to activate pBAD in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI1. An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of pBAD, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO2-mediated repression of pBAD. Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.
Lobell
R B
RB
Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Schleif
R F
RF
D051777:AraC Transcription Factor; D001089:Arabinose / Q000378:metabolism; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose