Translational Initiation Discussion Questions

1. What are the characteristics of eIF2 that make it a G protein?
2. Weaver describes another initiation factor that appears to act like a G protein, eIF5b on page 547. Distinguish the roles the active and inactive forms of this G protein.
3. Many mRNA’s require eIF4a for translation, but some mRNA’s can be translated in vitro in the absence of a functional eIFa. Predict how these two classes of mRNA might be different.
4. Not all AUG sequences function as start codons. In fact in most mRNA there are dozens of AUG triplets but only one of them functions as the start codon. Outline the most important factors that distinguish a function start codon from all the other AUG triplets.
5. Some mRNA’s have two functional in frame start codons. Use of the first start codon (nearest the 5’ end of the mRNA) results in a longer polypeptide. When the second start codon is used then a shorter peptide is produced. This is one mechanism by which one gene can produce two polypeptides.
1. Explain how deviation from Kozak’s rule could lead to the use of two functional start codons.
2. Some refer to this process as “leaky ribosome scanning”. What is “leaky” about this process?
6. Kozak designed a multi-start codon gene to investigate if upstream start codons were favored over more down stream start codons (Fig 17.19).
1. How does this experiment support the model of “ribosome scanning”?
2. How would her results have differed if there wasn’t a bias for the first start codon?
7. Describe two circumstances where an upstream start codon in a good Kozak contexts might not be a “barrier” to initiation at a more downstream start codon.
8. What does it mean to say that some IRES’s are eIF4g dependent and some are eIFg independent?
9. Why might you expect eIF4g dependent IRES translational initiation to be influenced by a polyA tail while eIF4g independent IRES translational initiation to be not dependent on a polyA tail?
10. Describe two circumstances where an upstream start codon in a good Kozak contexts might not be a “barrier” to initiation at a more downstream start codon.
11. How does phosphorylation of eIF2 influences translational initiation?
12. Chimeric genes were used to demonstrated that the “IRE” in the 5’ UTR of ferretin mRNA is the “cis” element responsible for translational repression of ferretin? How would the results of the experiment have differed if the cis element regulating ferretin translation was within the open reading frame of the ferretin mRNA?
13. How does the IBP prevent translation of ferritin mRNA in low iron conditions?
14. miRNA decrease gene expression either by preventing translational initiation or triggering the destruction of the mRNA. What determines which of these two pathways are utilized by the miRNA?
15. Humphrey et al conducted an experiment that showed that translational regulation by miRNA’s was dependent on both the CAP and the polyA tail. How would their results have differed if regulation was only dependent on the polyA tail?
16. Would you expect an eIF4g independent IRES to be inhibited by a miRNA? Explain your answer.
17. Would you expect insulin to regulate translational initiation of mRNA’s that use IRES for translational initiation? Explain your answer.
18. Re-initiation from minicistrons was described in the lecture as one of three alternative methods on CAP independent initiation. However this phenomenon might best be described as an alternative method of translational termination. Explain this distinction.
19. Compare and contrast IRES with the Shine/Delgarno sequence.
20. Design and experiment to test whether reinitiation from a minicistron can be blocked by a “barrier” start codon. What would you conclude if translational initiation at the second start codon was blocked by the barrier start codon? What might you conclude if it was not blocked by a barrier start codon. Based on your knowledge of translational initiation, what result would be more likely. Explain your answer.
21. Design an experiment to test whether ribosome shunting can be blocked by a barrier start codon placed between the upstream 18S complementary sequences and the downstream functional start codon. What would you conclude if translational initiation at the second start codon was blocked by a barrier start codon? What might you conclude if it was not blocked by a barrier start codon. Based on your knowledge of translational initiation, what result would be more likely. Explain your answer.