Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11MCB502 Final ExamName:_________________________Each problem is worth 10 points.Problem 1. Upon joining your thesis laboratory you decide to follow-up on how the yeast TWT8 gene is regulated. During your rotation project the senior graduate student you were working with mentioned her preliminary studies showing 3 sites (she called the sites “a, b, c”) around the transcription start of the TWT8 open reading frame that contribute to the production of the Twt8 RNA. Unfortunately, the mechanism(s) by which the 3 sites control TWT8 was not resolved. Prior to leaving the laboratory the senior student made a series of constructs with DNA insertions or deletions between the 3 sites The data she collected is below.Question A (8 points) Based on the data above, what mechanisms of regulation are used by the two different pairs of response elements to control TWT8 expression?Insert sizeDeletion Size Activity50025010050010005002501000--++++++Activity--+++++++Between Sites A & BInsert sizeDeletion Size Activity1000500250100010005002501000-++++++++++Activity++++++++++++++Between Sites B & CQuestion A (2 points) Draw a model of the 3 response elements when all 3 are bound by transcription factors and being used for TWT8 regulation. DNA bending is occurring between sites A & BDNA looping is occurring between sites B & CABC1. 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, gene regulation by DNA bending requires a <250 bp spacer between the operator and transcription start site.4. The non-canonical histone protein H2A.Z marks the boundaries of nucleosome depleted regions.5. As the concentration of a partial antagonist is increased the compound switches from inducing transcription to repressing. True or False (circle one) Problem 2 (2 pts each question)Question A (2 points) Why did the original ChIP-Seq/bioinformatic analysis not reveal the HNF47 consensus site?Problem 3Your advisor has spent the last 2 decades investigating how the tissue specific transcription factor Hepatic Nuclear Factor 47 (HNF47) contributes to liver development. Recently he had his best postdoctoral fellow perform a ChIP-Seq experiment using highly-specific antibodies directed against HNF47. Pleasingly they found that HNF47 was bound to 12484 different sites along the genome and that many of the nearby genes were known to contribute to liver function. Confusingly, a bioinformatic analysis of the bound DNA sequences did not reveal the known consensus site of HNF47 but rather it showed seemingly random sequence. You perform additional in silico work suggesting that the bound sites might contain 7 different consensus sequences with one of the motifs actually matching the known HNF47 consensus of AGTGGGACT. Question C (5 points) What genome wide experiment could you perform to test your explanation provided in Question B.Question B (3 points) Provide an explanation as to why your in silico analysis suggests the data set contains 7 different consensus sequences.HNF47 primarily works at tethering response elementsHNF47 can tether to 6 different transcription factors in addition to bind to its own consensus siteSequential ChIP-Seq targeting each of the transcription factors known to bind to the identified 6 other consensus sites along with targeting HNF47Problem 4 (2 points each)1. Cholesterol is the backbone molecule for all steroid hormones. 2. Nuclear Run-On is the only assay that provides a direct measure of transcription rates. 3. Composite element is a response element that contains DNA binding sites for multiple transcription factors.4. Quorum sensing is the process prokaryotes use for cell-to-cell communication.5. Circle the name(s) of the assay(s) used to detect DNA binding activities.A. Electrophoretic-mobility shift assayB. Northern blotC. Run-off assayD. Quantitative RT-PCRE. Run-on assayProblem 5The MULTi promoter is known to be regulated by 5 different factors called A, B, C, D, and E. To resolve how these 5 factors influence MULTi expression you use the ChIP assay with sequential precipitations steps along with antibodies specific for each factor from samples taken at different time points after activation of the MULTi locus. The data is shown below.Question A (5 points)Draw a model of the complexes formed at the MULTi promoter at the different time points. Provide a label for each complex that succinctly describes how that structure might contribute to the activation of the MULTi gene.Question B (5 points)With the limited presented data the complexes formed at the 2 and 5 minute time points can have more than 1 structural organization. Relative to what you drew above, draw an alternative arrangement for each complex that uses a different type of response element. A B C D EABCDEA B C D EABCDEfirstfirstsecondsecondInputsABCDE2 Minute 5 MinuteA B C D EABCDEfirstsecond1 Minute1 2 5A AADAEB COR1 minTranscription factor-Simple response elementComplex2 minLysine Acetylase Complex5 minChromatin RemodelingComplexesAADA EBCORCircle each of the following statements as true (T) or false (F)1. T or F Nucleosomes can be part of a response element.2. T or F Bacteria compact their genomes using highly abundant, relatively small proteins.3. T or F Micrococcal nuclease is used to map protein “footprints” bound to DNA.4. T or F The structure of green fluorescent protein is comprised of a 12 a-helix bundle.5. T or F Histone methylation always 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 factors only slide histones along the 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 Histone octamers prefer to bind to curved DNA. Problem 6 (1 point per question)Problem 7You are working on how the model organism Dictyostelium towerium transitions from swarming cells (individual cells) to a fruiting body (adhered, grouped cells). You know the transition occurs under conditions of limited nutrients. Importantly, you have identified a gene, APL13, that
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