Problem set 4

Problem set 4 - BIMM 100, Fall 2010, Assignment #4...

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Unformatted text preview: BIMM 100, Fall 2010, Assignment #4 (Oct 28, 2010) 1.) What is the difference between a proximal promoter element and a distal enhancer? a.) T/F enhancers are position and orientation dependent. Explain why this is true or false. b.) T/F Promoter ­proximal control elements can be moved 50 or more bases with no effect on gene expression. 2.) You are a scientist studying the FUN gene in mice, and you decide you would like to experimentally determine the DNA Control ­elements in promoter proximal regions of the gene. a. Design an experiment to determine which sequences are required for transcription of the FUN gene. b. You run two experiments. Below are shown your results. Which sequences are important for transcription? Circle the important regions? Luciferase activity (% wild type) 5’ 3’ Bases  ­1 to  ­200 89% 5’ 3’ Bases  ­1 to  ­150 90% 5’ 3’ Bases  ­1 to  ­100 20% 5' 3’ Bases  ­1 to  ­50 48% 5’ Bases  ­1 to  ­20 2% LINKER SCANNING ASSAY  ­ NORTHERN BLOT Can you explain this result? No deletion  ­20/ ­35 mutant  ­35/ ­50 mutant  ­50/ ­75 mutant  ­125/ ­150 mutant  ­150/ ­175 mutant Why more? C.) You have a sneaking suspicion that the either the transcription factor MOLFUN or MOLBAD binds to the proximal promoter region of the FUN gene. Design an experiment would allow you to test this hypothesis and at the same time see where the transcription factor binds on the proximal DNA d.) Below are your results using the  ­1 to  ­200 DNA used in 2b. What is the transcription factor that binds? The DNA was 32P ­labeled at the  ­1 position of the template strand. Approximately where on the proximal region does your protein bind? Explain how you can tell. DNA Ladder DNA only DNA +MOLBAD DNA + MOLFUN 90bp 65bp 43bp 35bp 29 bp 25bp 16bp 5bp 3bp 3.) You are studying the transcription factor NF ­κB in tumor cells, and you have already determined the DNA sequence it binds to. But you decide it’s important to determine what regions of the transcription factor are important for binding DNA (DNA binding domain). So you systematically delete regions of the cDNA encoding NF ­kB and then express it in bacteria to get purified NF ­kB protein fragments. You next isolate the different protein and combine with your known DNA promoter and run an EMSA. Below are your results. What regions of the protein are important for binding DNA? Explain the differences in DNA mobility shifts between lanes. Bonus: Explain the shift in lane six? (NFkB cDNA CLONE) b.) Does this experiment tell you anything about the activating domain of the transcription factor? Why or why not? Can you tell whether it is an activator or repressor? 5. Initiation factors which assist polymerase binding at transcription start sites and melting of double stranded DNA template strands are called general __________. The factor that binds the TATA ­box is _________, which consists of a protein that binds directly to the TATA ­box, called ________________ and multiple associated factors called _________. Another important general ______________, TFIIH has ATP dependent ___________ and __________ activities transcription initiation by ________________ and phosphorylating the _____________, respectively. 6.) What is the CTD, and what are the roles of the CTD? 7.) You are studying the DNA binding Transcription factor TICK and you believe that it is a specific transcription factor for the gene TOCK. You decide to test this hypothesis through an in vitro transcription reaction. a.) What components will you need for this in vitro transcription reaction? b.) On the Northern below, lane 1 shows the result of transcription without adding the TICK transcription factor . Show the result in lane 2 if you add TICK and it is an activator, and in lane 3 if TICK is a repressor. (1) (2) ( 3) (4) c.) It turns out that TICK is a repressor of TOCK; show in lane 4 the result if you added a fusion protein with the TICK DNA binding domain fused with the GAL4 activating domain (assume that you are using yeast components). 8.) T/F Transcription of a single gene may be regulated by binding multiple transcription factors to alternative control elements, directing expression of the same gene in different types of cells and at different times during development. How could you test this? 9.) You are a scientist studying Sasquatch transcription. You have isolated some Sasquatch cells and separated the proteins into different fractions, and then assayed the fractions for RNA synthesis with and without α ­amanitin. Knowing that α ­amanitin strongly inhibits RNA polymerase II and weakly inhibits RNA polymerase III, which peaks likely correspond to RNA pol I, II and III, respectively? 10.) What are the 3 human RNA polymerases and which types of RNAs does each one synthesize? Which one transcribes protein ­coding genes? What is the function of each type of RNA? I. Which one of the following is true (can be more than one a. Each one of the RNA polymerases requires the same polymerase specific general transcription factors and recognizes different DNA control elements. b. Each one of the RNA polymerases requires its own polymerase specific general transcription factors and recognizes same DNA control elements. c. Each one of the RNA polymerases requires its own polymerase specific general transcription factors and recognizes different DNA control elements d. TF ­I Transcription Factors recruit Pol I to rRNA promoters e. TF ­III Transcription Factors recruit Pol I to rRNA promotors. 11.) What are some common eukaryotic Pol ­II promoter elements? 12.) T/F DNA binding motifs of transcription factors recognize specific DNA sequences by hydrogen bonding with the surface of the bases normally engaged in base pairing. Explain why or why not. a.) What are some DNA binding protein motifs? What is the advantage of having a transcription factor that exists as a heterodimer? 13.) How do transcription factors activate or repress transcription? (give two general molecular mechanisms). 14.) You are conducting a linker scanning analysis of the 5’ upstream region of a tRNA gene. You clone the 5’ upstream region in front of a reporter gene, and assay for transcription. Can you explain this puzzling result? tRNA 5’ upstream region (yellow) ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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