Question 6 (3pts). A DNA fragment contains two operator sites for the Lac Repres
ID: 144274 • Letter: Q
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Question 6 (3pts). A DNA fragment contains two operator sites for the Lac Repressor, one between positions +1 and +20, and another one between positions-1 dependent Polymerase does 00-80. It has a 070. promoter which has been modified such that binding of RNA s not require the presence of CAP/CRP in lane 1 the DNA fragment is incubated for 20 minutes with DNase . and the reaction on products are fractionated by electrophoresis. Indicate on the blank lanes the band patterns predicted for the following conditions. For each lane the reaction contains ONLY THE COMPONENTS LISTED and all components have been added before DNase I cleavage. Explanations provided were not required Lane 2: Incubation with the Lac Repressor assume that looping between operator sequences does not result in a footprint) Footprinting between -100 and-80 and +1+20. Lane 3: Incubation with 70-Holoenzyme Footprinting between~ -40/-35 to +20. ne 4: Incubation with the Lac Repressor and with the 70-Holoenzyme Same as lane 2 - repressor prevents binding of holoenzyme. Lane 5: Incubation with the Lac Repressor and with the 70-Holoenzyme but the DNA fragment has been mutated such that the Operator site at position -100-80 has been inactivated and does not bind the Lac Repressor. Same as lane 3-1Operator is not sufficient to allow binding of repressor and repress RNA polymerase binding. In lanes 6 and 7 the DNA fragment is not treated with DNase I, but instead with KMn04 after incubation in the following conditions: Lane 6: Incubation with 70-Holoenzyme Cleavage between -10-+1 Lane 7: Incubation with 70-Holoenzyme, but 70 contains a mutation of the aromatic residues of region 2 to alanine. -100 -80 -60 -40 -20 No cleavage - No open complex formation +20 +40Explanation / Answer
The observations made in these 7 lanes can be described as below:
Lane 1: Since the DNA is digested with DNase I, it cleaves the whole strand and opens up the helix, hence, all bands can be visualized as a ladder. This lane serves as control.
Lane 2: The pattern of electrophoresis demonstrates the two operator sites at the respective upstream and downstream positions.
Lane 3: The incubation with holoenzyme suggests binding of the protein to the promoter, hence protection from cleavage.
Lane 4: The incubation with lac repressor causes binding of repressor at its respective sites however, the binding of repressor fails to protect the start site and it gets degraded by DNase.
Lane 5: Since the upstream site has been genetically mutated causing failure of binding, it is no more protected by RNA polymerase and hence gets cleaved, thus the bands appear.
Lane 6: Incubation of DNA with KMNO4 causes its clumping and prevents digestion by the enzymes. Only prolonged digestions result in the appearance of primary sites, as the start site and immediate upstream regulator.
Lane 7: Since the holoenzyme is mutated, it is no longer biologically active to perform its proteinaceous function, hance fails to bind to DNA. This is why whole DNA is precipitated by KMNO4 and hence no band is observed.
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