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Molecular Biology 2021 Final Exam 1 Molecular Biology Final Exam AS.020.304 December 16, 2021 Exam Instructions 1. This exam is open-book/note/internet with no collaboration/communication with other people. The exam questions can be answered based on class content and you should not need to consult any outside materials. Outside materials may introduce unnecessary complexity or contradictory information- please base your answers on the class material. 2. Plagiarism of any kind (copying or using similar wording or images from other sources including class materials) is prohibited- please make sure you use your own words and images. Note that this includes self-plagiarism and copying from materials you may have created with others. Please compose your answers to these questions yourself during the exam. 3. Download the PDF to your computer before working on it and re-open it from the downloaded location. You can type or write your answers in the form version. Please add your name, save the file, then open it again to confirm that your answers are saved. The completed exam should be submitted to Gradescope by Thursday December 16 at 5pm . If possible, please upload the PDF provided with typed or hand-written answers. If this is not working for you, the best thing to do would be to put your answers on a blank document with each answer clearly labeled with the question number and on the same page number of the document as the original question. If you have any technological challenges, email Dr. Fisher. 4. The deadline to submit the exam to Dr. Fisher is 5pm on Thursday December 16. 5. If you have any questions related to this exam, contact Dr. Fisher by email. I will be available as much as possible. The learning assistants are not available to answer any questions about this exam. 6. There is space on the last page of the exam to add any clarifications or longer explanations for your answers. These can be used in grading and in re-grade requests. 7. The codon table and wobble base pairing rules are on page 15 Ethics Pledge: “I have not and will not communicate with anyone in any way about the content of this exam until after 5pm on Thursday December 16th. All answers will be in my own words and I will not plagiarize from any class materials or other sources.” Sign or Type your name here indicating your adherence to the instructions and ethics pledge above: ______________________________

Molecular Biology 2021 Final Exam 2 1. Quality control. 20 points total. Consider the three parts of the central dogma and the quality-control steps or each. List one way that the product (DNA, RNA, or protein) is checked or corrected during synthesis and one way that the product is checked or corrected after synthesis is complete. If such a step does not exist, write “none”. Please base your answers only on material covered in this class. Recommended word limit: 15 words per section. DNA Replication 6 points During synthesis: After synthesis: Transcription—please also include what type of RNA is being checked/fixed. 8 points During synthesis: After synthesis: Translation 6 points During synthesis: After synthesis:

Molecular Biology 2021 Final Exam 3 2. Investigating a mutant. 30 points total. You are studying a gene of interest and decide to induce mutations in wild-type cells to try to find mutations that affect the gene of interest so you can better understand how the gene functions. You decide to induce mutations using X-rays which will induce double-strand breaks randomly in the genome. A. What type of DNA repair will be used to fix the double-strand breaks and induce mutations? Select the best answer. 3 points. a. Mismatch repair (MMR) b. Base Excision repair (BER) c. Nucleotide Excision repair (NER) d. Non-homologous End Joining (NHEJ) e. Homology-directed repair (HDR) Answer: ________________________ You irradiate the cells and identify some that have the same phenotype as cells with the gene of interest deleted. All other cell functions appear wild-type. You decide to investigate that mutant in hopes of finding out more about how this gene and protein function. First, you look for mutations in the gene of interest by amplifying the gene using PCR and then sequencing the PCR product. The PCR design is shown below. B. Why are DNA primers required for PCR to amplify this sequence? Select the best answer. 3 points a. DNA polymerase cannot extend the 3’ end of an RNA b. DNA polymerase can extend the 3’ end of a primer c. DNA polymerase can extend the 5’ end of a primer d. DNA polymerase begins DNA synthesis with two dNTPs e. none of these Answer: _____________________________

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Molecular Biology 2021 Final Exam 4 2 continued. The PCR result shows that the gene is present in the mutant cells and the sequence matches the wild-type sequence exactly. C. What other hypothesis could explain the phenotype observed in these mutant cells? Select all that apply. 3 points. a. an miRNA is active in the mutant cells that is not expressed in the wild-type cells b. there are chromatin differences between mutant and wild-type cells c. there are DNA methylation differences between the mutant and wild-type cells d. the protein encoded by the gene of interest is post-translationally modified in the mutant and not the wild-type e. the protein encoded by the gene of interest is post-translationally modified in the wild-type and not in the mutant cells f. a mutation outside the PCR-amplified region affects transcription of the gene of interest g. all of the above Answer: ________________________ D. Pick one of the choices you selected above (a-f) and explain using the guides below. 4 points. a. is the miRNA expressed or not in the mutant? What happens to the gene of interest in the mutant? b. in what way is the chromatin different? How does that impact the gene of interest? c. in what way is the methylation different? How does that impact the gene of interest? d. or e. suggest a post-translational modification and explain the connection to gene/protein function. f. where is this other mutation and how does it impact the gene of interest? Choice __________ Explanation:

Molecular Biology 2021 Final Exam 5 2 continued. Next, you decide to investigate the mRNA encoded by the gene of interest. E. What technique would you use to compare the abundance of the relevant mRNA in mutant vs. wild-type cells? Select the best answer. 3 points a. SILAC b. ChIP c. RNA-Seq d. RIP-Seq e. Ribo-Seq f. Northern blot Answer: ________________________ Your experiments show that the transcript encoded by the gene of interest is not detectable in the mutant cells. You decide next to sequence the genome of the mutants. You find that the gene of interest is in a different chromosomal location in the mutants: it has moved from chromosome 1 to chromosome 4. F. How can the change in location, and no changes in the sequence of the gene, change the expression? Select all that apply. 4 points a. the gene is separated from is enhancer in the mutant location b. the gene is regulated by a stronger enhancer in the mutant location c. the gene is in a hyper-methylated region in the mutant location d. the gene is in a hypo-methylated region in the mutant location e. none of these Answer: ____________________

Molecular Biology 2021 Final Exam 6 2 continued. G. A labmate suggests that the new chromosome location is a region of heterochromatin. Which of the following histone modifications would you expect to find associated with the gene of interest if the labmate is correct? Select the best answer. 3 points. a. H3K9me3 b. H3K27me3 c. H3K4me3 d. all of the above e. none of the above Answer: ____________________ You don’t have the appropriate antibodies to investigate the histone modifications, so you decide to use ATAC-Seq to investigate chromatin in the wild-type and mutants. The results are shown below. Two parallel pink lines represent the genomic region around the gene of interest on chromosome 1. The two parallel blue lines represent the genomic region around the gene of interest on chromosome 4 H. Is this consistent with the hypothesis that the gene of interest is repressed by heterochromatin in the mutant? Select the best answer. 3 points. a. yes because peaks represent open chromatin around enhancers and promoters b. yes because peaks represent open chromatin spanning the entire gene c. yes because peaks represent closed chromatin around enhancers and promoters d. no because peaks represent open chromatin around enhancers and promoters e. no because peaks represent closed chromatin around enhancers and promoters f. none of these is true Answer: _____________________________

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Molecular Biology 2021 Final Exam 7 I. On chromosome 4 in the mutants, would you expect to find an insulator between a heterochromatin region and the gene of interest? 4 points. Answer yes or no: __________________

Molecular Biology 2021 Final Exam 8 3. Translation inhibitors. 30 points total. Many different small molecules and protein toxins have been shown to affect translation, often leading to cell death. Answer the following questions based on your understanding of ribosome structure and function. A. Toxin A causes depurination of a specific site in a rRNA in the 60S subunit. What aspects of translation function are likely affected by Toxin A? Select the best answer. 3 points. a. mRNA circularization b. identification of the start codon by the pre-initiation complex c. aminoacylation of tRNAs d. delivery of tRNAs to the ribosome e. peptidyl transferase function during elongation f. ribosome translocation toward the 3’ end of the mRNA g. ribosome disassembly at stop codons Answer: ________________________ Toxin B cuts a specific tRNA with a UUU anticodon in the anticodon sequence. This is diagrammed below with Toxin B represented by a red shape. B. what amino acid is attached to the tRNA with the UUU anticodon? Note that the codon table and wobble rules are on the last page of this exam. Select all that apply. 3 points a. Leu b. Lys c. Phe d. Pro e. none of these Answer: _____________________

Molecular Biology 2021 Final Exam 9 3 continued. Treating cells with Toxin B causes ribosomes to stall at AAA codons. C. What other codons (if any) would also be likely places for ribosome stalling in eukaryotes in the presence of Toxin B, according to the wobble base pairing rules? 3 points a. AAU b. AAC c. AAG d. AAI e. UAA f. CAA g. GAA h. none of these Answer: _____________________ D. You want to test whether the ribosome will stall at any other codons, so you create a reporter construct that encodes green fluorescent protein (GFP) with a C-terminal tag comprised of every amino acid-encoding codon (including AAA as a positive control). Which of the following constructs diagrammed below will create GFP followed by all aa-encoding amino acids? Select the best answer. 3 points Answer: _________________________ . E. How many codons will you add to the GFP for this test? Select the best answer. 3 points a. 20 b. 45 c. 61 d. 64 e. other—please include the number in your answer if selecting e Answer: _________________

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Molecular Biology 2021 Final Exam 10 3 continued F. What technique would you use on this reporter construct to see where the ribosome stalls? Select the best answer. 3 points. a. mass spectroscopy b. density gradient centrifugation followed by centrifugation c. ribosome profiling d. RNA-Sequencing Answer: __________________ G. Toxin G binds to the A site of ribosomes to inhibit translation. Which of the locations marked with shaded rectangles labeled 1-3 is the A site on the diagram below? Answer: __________________ H. What is the function of the A site during translation? Select the best answer. 3 points. a. the site where the initiator tRNA binds b. the site where amino acyl-tRNAs bind c. the site where tRNAs are bound to the growing peptide chain d. the site where tRNAs exit the ribosome e. the site where incorrect amino acids are removed from tRNAs f. the site where incorrectly-added amino acids are removed from the growing peptide chain Answer: __________________________

Molecular Biology 2021 Final Exam 11 3 continued I. Toxin I prevents ribosome translocation. How might it work? Select all that apply. 3 points. a. blocks eIF function b. blocks EF1 (EF-Tu) function c. blocks EF2 (EF-G) function d. blocks RF function e. all of these f. none of these Answer: _______________________ J. A different class of molecules is toxic to cells because they allow decoding of stop codons. In the presence of these molecules, an aa-tRNA binds at the stop codons and the ribosome continues translating past the normal stop codon. Briefly, why would this additional translation be deadly to the cell? Suggest one way this would harm cells. Recommended word limit, 40 words .

Molecular Biology 2021 Final Exam 12 4. RNA. 20 points total. A. You find a new gene in the genome. You hypothesize that the transcript is either 1. An RNA that codes for a protein 2. a lncRNA, or 3. a pri-miRNA. For each type of RNA, match the sequencing feature(s) that would best correspond to the type of transcript. If none of the features fits the type of RNA, please write none. Some features will not correspond to any of the RNA types. 6 points total. Features: a. sequence that forms an imperfect hairpin b. sequence with no start codon c. sequence with a start codon followed by a long ( > 60-nucleotide) open reading frame d. sequence with a start codon and 22-nucleotide open reading frame RNA that codes for a protein: ___________________ lncRNA: ___________________ pri-miRNA: ___________________ B. You figure out that the gene generates a pri-miRNA. Select all the key proteins that are required to process the pri-miRNA to the final miRNA and enable its function in translational repression: 4 points. a. Drosha b. U2 snRNP c. Dicer d. Argonaute e. PIWI f. Exportin5 g. RITS h. Guanyl transferase i. All of the above j. None of these Answer: ______________________

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Molecular Biology 2021 Final Exam 13 4 continued. You next want to identify targets that are repressed by this miRNA. Assuming that this miRNA works only via repressing translation, answer the following questions (C and D). C. Using SILAC, what would you expect to observe? Select all that apply. 2 points a. more target protein in cells that express the miRNA b. less target protein in cells that express the miRNA c. no difference in the amount of target protein in cells that express the miRNA d. more non-target protein in cells that express the miRNA e. less non-target protein in cells that express the miRNA f. no difference in the amount of non-target protein in cells that express the miRNA Answer: __________________________ D. Using RNA-Seq, what would you expect to observe? Select all that apply. 2 points a. more target RNA in cells that express the miRNA b. less target RNA in cells that express the miRNA c. no difference in the amount of target RNA in cells that express the miRNA d. more non-target RNA in cells that express the miRNA e. less non-target RNA in cells that express the miRNA f. no difference in the amount of non-target RNA in cells that express the miRNA Answer: __________________________ You generate an enhancer reporter in which GFP has the 3’UTR of a target gene. E. Would you expect to see GFP fluorescence in cells that contain this reporter and express the miRNA? 2 points. Yes/no: _____________________ F. What would you change about the reporter construct to test the function of the 3’UTR? Recommended word limit: 40 words . 2 points. G. Would you expect to see GFP fluorescence in cells that contain the version you described above (part F) and express the miRNA? 2 points. Yes/no: _____________________

Molecular Biology 2021 Final Exam 14 If there is anything you would like to add or clarify about any of your previous answers, please do so in the space below:

MB2021 final exam_BLANK

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