PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11MCB502 Midterm ExamName:_________________________Each problem is worth 10 points.Problem 1: Promoter bashing results are shown below for the PRtZ gene using -galactosidase as a reporter.-galactosidase-1-1200DNA Fragment Relative Activity-1200 to -1 +++++++-900 to -1 +++-600 to -1 +++-300 to -1 +++-50 to -1 +-1200 to -50 ++++++-1200 to -300 ++-1200 to -600 ++-1200 to -900 ++-900 to -300 ++++Question A (2 pts): How many response elements are at this promoter?3 points: Response element between -1200 and -9003 points: Response element between -300 and -504 points: Spacing is required for DNA looping and synergistic activation*-1 to -50 has a very weak basal promoterQuestion B (2 pts): Where are the REs located?Question C (6 pts): Provide an explanation for the results of the internal deletion (-900 to -300)?1. Gene regulatory mechanisms are generally conserved between prokaryotes and eukaryotes. True or False (circle one) 2. The preferred DNA binding motif of a transcription factor is termed a consensus site.2. In prokaryotes, DNA that preferentially curves is bound by Histone-like proteins.4. Bromo domain containing protein bind acetyl-lysine and Chromo containing proteins bind methyl-lysine.5. As the concentration of a partial agonist is increased the compound switches from inducing transcription to repressing. True or False (circle one) Problem 2 (2 pts each question)Problem 3: You are investigating the regulation of the ASP1 gene using the nuclear run-on, chromatin immunoprecipitation (ChIP), Micrococcal nuclease digestion and DNase I digestion assays. The only antibody that you have for ChIP assays is to the Astar protein, which is the activator of ASP1 . To visualize your Micrococcal and DNase I digestion products you have a radiolabeled probe to the ASP1 gene. The ASP1 gene is activated upon shaking your cells at 500 rpm; the time courses shown below is the temporal response following shaking. Importantly, your control gene (ASP1 ), which you know is not induced under these conditions does not display any signal changes by all four assays.Nuclear Run-On AssayTime (min) 0 5 15 30 60 120 240Question A: Why is ASP1 RNA produced even in the absence of bound Astar?DNase I AssayTime (min) 0 5 15 30 60 120 240ChIP Assay (-Astar)Time (min) 0 5 15 30 60 120 240Micrococcal nuclease AssayTime (min) 0 5 15 30 60 120 2406 points: ASP1 RNA is produced in the absence of bound Astar through epigenetic affects (i.e., the chromatin state is sustained)1 point: Nuclear run-on demonstrates transcripts are continually made1 point: ChIP assays shows that Astar interaction with the promoter is transient1 point: DNase I assay indicates that the chromatin at the ASP1 gene has become decondensed and is now accessible by DNase I 1 point: Micrococcal nuclease assay shows that the nucleosome become depositioned upon activation of the geneQuestion B: Please provide an explanation for each result shown above.Problem 4 (2 points each)1. Cholesterol is the backbone molecule for all steroid hormones. 1. ChIP-Seq can be used to measure the global occupancy of a protein along a genome. 2. Tethering element is a response element that binds 2 or transcription factors using a single DNA bound protein.3. Quorum sensing is the process prokaryotes use for cell-to-cell communication.1. Circle the name(s) of the assay(s) used to monitor the actual transcription rate of a gene.A. Electrophoretic-mobility shift assayB. Northern blotC. Run-off assayD. Quantitative RT-PCRE. Run-on assayProblem 5: You are studying the bacterial promoter BINDER1 that is active in both the presence of potassium (K+) and the absence of magnesium but is inactive in the presence of both ions. Shown below are the results of Northern blot analysis of BINDER1 RNA, Electro-Mobility Shift Assay (EMSA) of a small (60 bp) fragment the BINDER1 promoter and DNA footprinting experiments of the BINDER1 promoter . As a control for expression levels you probe for ACT3 RNA, which is not altered by these ions.DNA FootprintNorthern BlotK+ + +Mg++ + -BINDER1ACT3K+ + +Mg++ + -K+ + +Mg++ + -EMSAfreeprobe-1-10-20-30-40-50Distance fromRNA start siteQuestion (10 pts): What is the mechanism used to regulate BINDER1 RNA expression? In your answer provide an explanation for each result.7 points: The BINDER1 promoter relies on the Up-element for activity. Hence, the footprint at -47 to -45 in the absence of Mg++. In the presence of Mg++ a repressor binds over the Up-element and blocks RNA polymerase association.1 point: Northern blot indicates that BINDER1 RNA is only made in the absence of Mg++. 1 point: EMSA demonstrates that a large complex (RNA polymerase + promoter fragment) forms in the absence of Mg++ but a smaller complex (repressor + + promoter fragment) is bound in presence of Mg++. 1 point: DNA footprint shows that RNA polymerase is using the -10, -35 and Up-elements in absence of Mg++ and that a protein binds over the Up-element in the presence of Mg++.Circle each of the following statements as true (T) or false (F)1. T or F Nucleosomes are the only protein-containing subunit of chromatin.2. T or F Bacteria can form multi-cellular functional communities.3. T or F DNase I is used to map protein “footprints” bound to DNA.4. T or F The green fluorescent protein cleaves ATP to emit light.5. T or F Histone acetylation correlates with repression of transcription.6. T or F The chromatin immunoprecipitation assay is used to detect proteins that cannot directly bind DNA.7. T or F Chromatin remodeling complexes slide, eject, and transfer histones from DNA. 8. T or F Competition between biological factors is a means of regulation. 9. T or F Most transcription factors have an affinity for DNA of any sequence.10.T or F Transcription factors determine chromatin structure. Problem 6 (1 point per question)Problem 7: One of the first molecular models for studying transcription events was the fly Hsp70 locus that is composed of two copies of the Hsp70 gene (see below). It was detected early since the locus forms a large chromosomal puff in salivary gland polytene chromosomes
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