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Transcription/translation Seminar 2012 Questions. 1. At which stage of mRNA synthesis does the cap attachment to pre-mRNA occur? What is the connection between the CTD phosphorylation and Cap attachment? 2. What is the signal of nuclear mRNA polyadenylation? What role does CTD play in mRNA polyadenylation? Describe how the polyadenylation of mRNA is carried out in the nucleus. 3. Why does CTD become phosphorylated first at Ser 5 and then at Ser2? How is the Ser2 phosphorylation connected with the de-acetylation of histones in the nucleosomes of transcribed DNA? Describe the role of Set2 in this process. Name kinases that run the Ser 5 CTD phosphorylation (some of them are important for the cell cycle). 4. What is the role of histone acetylation by HATs? Name at least one protein complex that runs the acetylation. How is the acetylation related to the TF binding to promoter proximal regions? 5. Histone 3 is often methylated in chromatin regions containing non-transcribed genes. What does this methylation do? Describe the effects of K9 methylation in gene silencing. 6. What do ISWI, SWI and SWR1 chromosome re-modeling machines do and what is the difference between them? Why do SWI machines have bromo-domains? 7. What is the role of EJCs (exon junction complexes) in mRNA transport into the cytoplasm. Describe the normal transport of mRNA from the nucleus that relies on the use of Tap/p15 transporter. What is the role of Dbp5 helicase here? How does the Brownian ratchet work? 8. Rev protein of HIV participates in the transport of HIV mRNAs into the cytoplasm. HIV virus with a mutant Rev does not form infectious virus particles. Explain why. Describe also how Rev normally works. 9. Transformer protein (Tra) in Drosophila determines the alternative splicing of Dsx genes. You find a mutation in the transformer gene that results in the female phenotype even in the mutants with a disrupted Sxl gene. What kind of mutations in the Tra gene could have occurred? 10. A researcher introduces a mutation into the AAUAAA sequence in the end of a gene. She finds that a AAGAAA mutation leads to the production of a much longer mRNA that is poorly poly-adenylated. Explain this observation. 11. You study transcription initiation in vitro using 'naked' DNA (without nucleosomes) that contains a reporter gene under control of promoter containing CRE proximal element. Would the phosphorylation of Ser133 in CREB stimulate the transcription ? Would the addition of dephosphorylated CREB stimulate the transcription? Explain why. Assume that all general transcription factors (including TAFs of TFIID) and CBP/p300 are present. 12. There is much less eIF2B molecules than eIF2 molecules in the eukaryotic cell. In a particular cell line there is 1 eIF2B per 10 eIF2. What would you expect to happen with the protein synthesis if the level of eIF2 phosphorylation by PKR is (a) 5% ; (b) 20% ? Explain why. 1 13. Oocytes contain large amounts of un-translated mRNA with short polyA sequences. Translation of these mRNAs can be activated by hormones and other signals. Explain the mechanism of translation inhibition by Maskin and how this inhibition is relieved in response to a hormonal signal. Describe the role of mRNA poly-adenylation in activation mRNA translation. 14. A researcher injects a double stranded (ds) RNA into the cell cytoplasm. She finds that the translation of several mRNAs becomes inhibited because of their destruction in the cytoplasm. Describe in details the mechanism of the observed effect. What should be the relation between the mRNA sequences and the sequences in the injected ds RNA? 15. A cell line is used to over-express a heterologous protein X containing a signal sequence to promote its secretion. Upon induction of protein X synthesis there is a short burst in the synthesis of protein X after which the translation slows down considerably. The protein is known to fold very slowly and it employs BiP chaperon extensively. Come with a good explanation of why and how the protein synthesis stops. A researcher also measures the level of BiP chaperon in the cell and finds that it increases dramatically after some time. How was the BiP synthesis induced? 16. FMDV virus invades proliferating cell. Describe the way it shuts down the translation of cellular mRNAs but maintains the translation of its own mRNAs. Would a simultaneous growth factor stimulation of translation through Ras/Raf signaling pathway make any difference for translation of cellular mRNAs? Explain why. 17. A researcher studies a Sar1 protein with a temperature sensitive mutation which is unable to hydrolyze GTP at non-permissive temperature. He notes that the formation of COPII vesicles continues for some time after the shift to non-permissive temperature but the formed vesicles are unable to fuse to the cis Golgi. In addition, after some time the formation of COPII vesicles also stops. Explain these observations. 18. A mammalian cell has a mutation in Ire1a protein that drastically increases its affinity to a BiP chaperon. The cell however responds almost normally to elevated levels of unfolded protein in the lumen of the ER by increasing the synthesis of ER chaperons. However, an additional mutation in a DNA binding domain of transcription factor ATF6 results in a cell death upon unfolded protein accumulation. Explain these observations. 19. A researcher introduces several mutation in exon 3 of a long transcription factor (90 kD) which gene contains 9 exons of approximately equal length. She finds that some of these mutations lead to inhibition of transcription for genes usually activated by this factor. She also finds that the same mutations result in a much shorter version of the factor (approximately 40 kD). The truncated factor is expressed but in a much smaller quantities than expected. Besides, much smaller fraction of ribosomes was involved in translation than that observed for the control with the wild type factor. Discuss the following reasons for the observed effects: (a) Exon 3 is a part of activation domain. (b) Nonsense mutations in exon 3. (c) Problems with splicing at exon 3 and their probable consequences. (d) Whatever reason you can invent. Give an explanation that takes into account all the experimental observations. Suggest complimentary experiments to confirm or disprove your explanation. 2