Suppose you want to study the transcription in vitro of one particular gene in a DNA molecule that contains several genes and promoters. Without adding specific regulatory proteins, how might you stimulate transcription from the gene of interest relative to the transcription of the other genes on your DNA template? To make all of the complexes identical, you would like to arrest all transcriptional events at the same position on the DNA template before isolating the complex. How might you do this?
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- Cells go to great length to correctmistakes in the processes of DNAreplication, transcription, splicing,and translation. Are there analogousstrategies to correct mistakes in theselection of which genes are to beexpressed in a given cell type? Couldthe great complexity of transcriptioninitiation in animals and plants reflectsuch a strategy?Some transcription regulators bind to DNA andcause the double helix to bend at a sharp angle. Such“bending proteins” can affect the initiation of transcrip-tion without directly contacting any other protein. Can youdevise a plausible explanation for how such proteins mightwork to modulate transcription? Draw a diagram that illus-trates your explanation.A cloned gene fragment contains a regulatory element that isrecognized by a regulatory transcription factor. Previousexperiments have shown that the presence of a hormone resultsin transcriptional activation by this transcription factor. To studythis effect, you conduct an electrophoretic mobility shift assayand obtain the following results: Explain the action of the hormone.
- A single UASG regulates the expression of threegenes, all of which are adjacent: GAL7 and GAL10 asdescribed in Problem 8, and also GAL1.a. Would you expect these genes to be transcribedinto individual transcripts, or to be cotranscribed asone mRNA? Explain.b. How could you determine experimentallywhether each gene is transcribed separately orinstead that the three are cotranscribed into asingle mRNA?c. GAL1 and GAL10 are not only adjacent to eachother, but also are transcribed divergently with asingle UASG between them. Describe experimentsusing GFP and RFP transgenes that would allowyou to determine which of the four GAL4 bindingsites in this UASG element is (are) important for thetranscription of GAL1 and/or GAL10.You have isolated a protein that binds to DNA in theregion upstream of the promoter sequence of the sysgene. If this protein is a positive regulator, which ofthe following would be true?a. Loss-of-function mutations in the gene encodingthe DNA-binding protein would cause constitutiveexpression of sys.b. Loss-of-function mutations in the gene encodingthe DNA-binding protein would result in little orno expression of sys.In eukaryotes, transcription initiation involves enhancersequences located far from the promoter. In addition, thechromatin of eukaryotic chromosomes must be unwoundto allow access by _________?
- A. Identify the mutation(s) that lead to the most loss in transcriptional activity, and discusswhether those match expectations based on the consensus sequence for the Initiator.B. Hypothesize a molecular mechanism to explain how the mutations identified in Part Aresult in loss of transcription activity.C. Identify whether any mutations cause transcription to initiate from a different positionthan wild-type, and provide a brief explanation as to why that occurs for those mutantcore promoters. THIS IS A PRIMER EXTENSION ASSAY. Using a cDNA library, you isolated two differentcDNA clones that have sequences indicating that theyboth correspond to mRNAs transcribed from the samenerve growth factor gene. The beginning and endingsequences of the clones are the same, but the middlesequence is different. How can you explain the different cDNAs?Why transcription initiation requires the assembly of transcription regulatory proteins on DNA sites known as enhancers ?
- Transcription factors function in the nucleus. However, like (almost) all eukaryotic proteins,they are translated in the cytosol. Can you draw a visual to explain how transcription factor proteinsenter the nucleus from the cytoplasm? Can you also include a representation of relevant proteins and proteindomains to explain how these proteins reach their destination. Thank youSome prion-related diseases, such as familial fatal insomnia, areinherited. How would you expect the mutation has altered the PrPgene in this case? Would it have affected the promoter, the regulatorysequences, or the coding sequence of the PrP gene?Enhancers can influence the transcription of genes far away on the same chromosome. How are the effects of enhancers restricted so that they do not exert inappropriate transcriptional activation of non-target genes?