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MCB Seminar 4B, 2015 : Translation Assignments: GR1: Q1, Q2, Q11; GR2: Q3, Q4, Q8; GR3: Q5, Q6; GR4: Q7, Q9, Q10; GR5:Q12, Q13, Q17; GR6+8: Q14, Q15, Q16; GR7: Q18, Q19 1. There are 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. 2. A mutation in MNK1 kinase leads to its inactivation. The cell acquired, however, a compensatory mutation in a phosphotase gene resulting in the reduced levels of the phosphotase that dephosphorylates 4E-BP proteins. Explain the mechanism of such a compensation. 3. 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. 4. A researcher injects a double stranded (ds) RNA into the cell cytoplasm. She finds that the translation of certain mRNAs becomes inhibited because of the destruction of these mRNAs in the cytoplasm. Describe the mechanism of the observed effect. What should be the relation between the mRNA sequences and the sequences in injected ds RNAs? 5. Male type Tra mRNA has a stop (UAG) codon in exon 2 which is followed by other exons in this mRNA. This mRNA is transported into the cytoplasm but rapidly degraded there. Describe the mechanism of the Nonsense Mediated Decay (NMD) that operates on this type of mRNAs. 6. When glucosyl-transferase activity of UDP-glucose:glucoprotein Glucosyl Transferase (UD-GT) in the ER was blocked by the addition of specific drug the protein synthesis in the cell gradually stopped. A subsequent analysis showed that this was due to the accumulation of unfolded glucoproteins in the ER.(a) Explain in details how the blockage of glucosyl-transferase activity of UD-GT would affect the calnexin/calreticulin promoted folding in the ER. (b) Describe the unfolded protein response (UPR) at the translation level. 7. All tRNAs have a universal CCA sequence at their 3’ends. What is the role of this sequence in tRNA binding to the ribosome and in the peptidyl-transferase reaction? Which site on the ribosome (A or P) should have higher affinity to tRNA? 1 8. All cell have a specialized initiator tRNAiMet as well as elongator tRNAMet. What are the major differences between these tRNAs. What is the main reason for using the specialized tRNA to initiate translation? What is the main disadvantage in using this specialized tRNA for initiation? 9. Ubiquitin (Ub) is activated in an ATP driven reaction catalyzed by Ub activating enzyme E1 and becomes attach to E1 through a thio-ester bond. What are the similarities and differences between the mechanism of Ub activation and tRNA charging by aminoacyl-tRNA synthetases? 10. Describe general organization of the 26S proteosome. How are the ubiquitinylated proteins recognized by the 19S cap? What happens with Ub-s attached to the degraded protein? What is the role of the ATPase subunits of the 19S cap? What is the proteolitic specificity of the active sites of the proteosomal 20S core? 11. Why must the ribosome attached to the translocon maintain a tight seal between the ER and cytoplasmic compartments? How is the tight seal maintained in the ribosome absence? 12. Some proteins are delivered into the ER post-translationally, through the Sec63 modified translocon. This mechanism uses Bip chaperon in the ER. Describe the Brownian ratchet model of the translocation into the ER for this case. 13. 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. 14. 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. 15. 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. 16. Lumen proteins often have KDEL sequence at their C-terminus. What is the roll of this sequence ? 17. tmRNA is used both for the ribosome rescue and for the tagging of incomplete proteins for degradation. A researcher modifies the ORF of tmRNA so that instead of the C-terminal ALAA sequence it codes for LALL. How would such a replacement affect bacterial physiology? How are ALAA containing peptides normally degraded? Why does the ribosome stalled on truncated mRNA need a rescue? 2 18. Translation of transferrin receptor (TfR) mRNA is repressed at high Fe and derepressed at low Fe in the cell by the mechanism that uses the IRE: IRP1 interactions. Explain how the translation repression works in this case. 19. E.coli strain contains two genes for initiator tRNA. A researcher mutates one of the gene changing the original anticodon 5’-CAU of tRNAfMeti into CUG that reads Gln. What will happen with the initiation of protein syntheses in the cell? Which aminoacid will be found at the N-terminus of dysfunctional proteins? 3