ProblemSet5key.pdf - Problem Sets-153B Summer 2017 Problem...

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Problem Sets-153B- Summer 2017 2nt 3nt 4nt 6nt 10nt 18nt 80nt 120n 1 2 3 4 5 Problem Set 5 – Transcription 1--The prokaryotic holoenzyme RNA polymerase is incubated with following DNA sequence under various conditions. “-“ indicates the promoter region, +1 the transcription initiation site. The polymerase and/or DNA are incubated in the following conditions. Whenever rATP is used, it is radiolabeled on the a group with 32 P. After incubation, radiolabeled molecules are loaded on a sequencing gel, and the gel exposed for autoradiography. The sizes of the molecules are indicated on the right of the gel in nucleotides (nt). Lane1 RNA Polymerase+rATP-No DNA Lane2 RNA Polymerase+rATP+DNA Lane3 RNA Polymerase+rATP+rUTP+DNA Lane4 RNA Polymerase+rATP+rUTP+rGTP+DNA Lane5 RNA Polymerase+rATP+rUTP+rGTP+rCTP+DNA A- What type of molecules are the scientists visualizing on this gel ? B- What is the significance of the result observed in lane 1 ? C- Describe and explain the differences observed between lanes 3 and 4. D - what is the significance of the short species observed in lanes 4 and 5 ?
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Problem Sets-153B- Summer 2017 1 2 3 4 5 6 +1 -40 -60 -80 -100 -120 +40 2- A DNA fragment carrying a CAP/CRP binding site, the lac promoter and two operator sequences is used in this experiment. +1 corresponds to the transcription initiation site. Negative numbers correspond to the promoter and regulatory sequences. In lane1, the DNA is treated with DNAse I. In lane 2, the DNA is incubated with CAP/CRP-cAMP, and then treated with DNAse I. In lane 3, the DNA is incubated with CAP/CRP-cAMP, RNA polymerase and then with DNAse I. In lane 4, the DNA is first incubated with CAP/CRP-cAMP, RNA polymerase; the lac repressor is then added, and the complexes are treated with DNAse I. In lane 5, the DNA is first incubated with the Lac repressor in the presence of allolactose, then with CAP/CRP- cAMP, and RNA polymerase and then treated with DNAse I. In lane 6, the DNA is incubated with RNA polymerase, and then with DNAse I. A- what is the significance of the result observed in lane 1 ? what is the purpose of using these conditions ? B- Indicate approximately next to the gel the location of the CAP/CRP binding site, or give a numbered approximation of the location of this region. C- Compare and explain the results observed in lanes 3 and 6. D – Explain the results observed in lanes 4 and 5
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Problem Sets-153B- Summer 2017 3 - Shown below is the structure of the Lac Operon and of the upstream region A - Indicate the name of the products of the I, LacZ, LacY, and LacA structural genes, and indicate briefly their function(s) in lactose metabolism. I: LacZ: LacY: LacA: B-What are the predicted effects of the following mutations on the production of the polycistronic LacZ-LacY-LacA mRNA ? In each case, explain briefly your answer - Mutations that disrupt the –10 and 35 regions of P I - A mutation that disrupts the structure of the Helix-Turn-Helix motif of the Lac repressor - A mutation that disrupts the cAMP binding site of CAP/CRP - A mutation in the ribosome binding site of the Lac Z gene (10pts)
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  • Summer '15
  • paul tarr

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