Looping looping weaver 2002 molecular biology ed 2 ara

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Unformatted text preview: A binding domains CAP (purple) (purple) • DNA CAP binding sites (red) • DNase hypersensitive sites DNase (blue) (blue) Effect of the DNA looping on DNase susceptibility ara OPERON REGULATION BY “LOOPING” “LOOPING” Weaver 2002 Molecular Biology Ed. 2 • • • • ara operon: encodes genes for arabinose metabolism ara regulation: negative (araC repressor) & positive (CAP-cAMP) ara repressed in absence of arabinose inducer (all this similar to lac lac operon) operon) expression of araC controlled by the operator araO1 expression araC araO ara OPERON REGULATION BY “LOOPING” “LOOPING” Weaver 2002 Molecular Biology Ed. 2 • • • operator (araO2) that controls expression of ara metabolic genes that (araBAD) is located far upstream of the promoter ( PBAD) (araBAD) likely way for the operator to control transcription from the distant likely promoter is by DNA looping promoter ara operon transcript. controlled by DNA looping; araC represses by araC looping DNA between O2 and I1, blocking RNAP access looping LOOPING OUT DNA: PROTEINS BIND SAME FACE OF DOUBLE HELIX SAME Weaver 2002 Molecular Biology Ed. 2 • • insertions (or dels) of 10, 20, 30 bp – integral units of 1 helical turn, insertions minimal interference; insert or del of 5, 15, 25 bp – units of half helical turns, looping disruption helical DNA looping brings 2 regulatory sites together if sites on same face DNA of the double helix - if proteins bind helix on opposite faces, then helix must twist 180˚ for proteins to face one another (very helix energetically unfavorable) energetically unfavorable) ara OPERON REGULATION BY “LOOPING” “LOOPING” Weaver 2002 Molecular Biology Ed. 2 • • • in absence of arabinose, araBAD products not needed and AraC in exerts negative control by binding to araO2 and araI1, looping DNA araO araI when arabinose present, it binds to and changes conformation of when AraC, such that AraC now binds araI1 and araI2; at this location, araI araI at together with CAP-cAMP, AraC acts as positive regulator of transcription transcription Experimental evidence for this model? araC LOOPING OF DNA LOOPING Weaver 2002 Molecular Biology Ed. 2 • • • • • Labeled minicircle DNA with araC binding sites Labeled araC AraC added, then unlabeled araI competitor DNA AraC araI Looped DNA migrates faster (more supercoiled) Mutant binding...
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