At Least 2 Questions From This Section Will Be On The Final

1at Least 2 Questions From This Section Will Be On The Final Examsampl

1at Least 2 Questions From This Section Will Be On The Final Examsampl

1 At least 2 questions from this section will be on the final exam SAMPLE QUESTIONS FOR THE FINAL EXAM Question 1. Ferritin is a protein involved in the storage of iron inside cells. To prevent toxic accumulation of too much iron inside cells, the intracellular level of ferritin is tightly regulated. To study the regulation of ferritin synthesis, mammalian cells are grown with or without iron in the culture medium. Note that iron in the culture medium is rapidly transported inside cells. a) Upon addition of iron to the culture medium, the intracellular concentration of ferritin mRNA is unchanged but the concentration of ferritin protein increases. How do you think ferritin expression is regulated? Briefly explain. The regulatory sequence given below is found in the ferritin mRNA between the cap structure and the start codon. 5’-GGGUUUCCGUUCAACAGUGCUUGGACGGAAACCC-3’ Mutations within in this sequence are used to study the regulation of ferritin expression. The following observation are made: • ferritin expression is high, independent of the iron concentration, when (i) the entire region is deleted, or (ii) the region located upstream of the underlined sequence is deleted or (iii) the underlined sequence is replaced with a random sequence. • ferritin expression remains iron-dependent when this region is replaced by the following sequence: 5’-GGGCUCAGGUUCAACAGUGCUUGGACCUGAGCCC-3’. Note that the sequence differences are indicated in bold. b) Explain why these observations suggest that both sequence and structure of the 5’ end of ferritin mRNA are important for the regulation of ferritin expression. c) Ferritin translation becomes iron-independent when the regulatory sequence is moved from the 5’ side (upstream of the open reading frame) to the 3’ side (downstream of the open reading frame) of ferritin mRNA. Which step of ferritin translation do you think is affected by the intracellular level of iron? d) IRP is a protein involved in the regulation of ferritin expression. Anti-IRP antibodies attached to sepharose beads are added to a cell extract, then the extract is centrifuged to separate the pellet fraction (containing the sepharose beads ) from the supernatant fraction. If the cells are cultured in the absence of iron, ferritin mRNA is found together with IRP in the pellet. In contrast when cells are cultured in the presence of iron ferritin mRNA remains in the supernatant fraction while IRP alone is found in the pellet. Briefly explain the likely role of IRP in the regulation of ferritin expression. Question 2. You are studying the development of a newly discovered insect. Like drosophila, it undergoes a stage in early larval development where the eve gene is expressed in a pattern of 7 stripes. You are particularly interested in stripes 2 and 5. The following figures show the organization of the cis-acting elements that control the expression of the eve gene in seven stripes. On Figure 1 (top part), the seven boxes represent segments of DNA or UAS (upstream activating sequences) responsible for the activation of the eve gene in each of the seven stripes. The UAS responsible for gene expression in stripes 2 and 5 contain binding sites for five distinct proteins acting as transcriptional regulators (pA, pB, pC, pD, and pE) (Figure 1 top part). The quantitative 2 distribution of these five eve-gene regulators throughout the embryo (from the anterior to the posterior pole) is also shown on the lower part of this figure. Figure 1: Organization of the regulatory region of the eve gene and distribution of its transcriptional regulators in the embryo. a) Transcription regulators pA, pB, pC and pD seem to be important for establishing eve expression in stripe 2. They all have binding sites (A,B,C &D) in the cis-acting segment controlling expression in stripe 2. Based on the distribution of these 4 transcription regulators in the embryo, determine which of the 4 regulators are most likely to be activators and which one are most likely to be repressors of eve gene expression in stripe 2. Briefly explain. b) The UAS responsible for gene expression in stripe 5 (UAS5) has one binding site for pA (A) and two binding sites for pE (E1 and E2). To study the binding of pE to UAS5, radiolabeled DNA fragments containing UAS5 or mutant forms of UAS5 are mixed with protein pE and are submitted to gel electrophoresis under conditions that do not disrupt protein-DNA interactions. An X-ray film is then placed in contact with the gel to visualize the radioactive DNA. The following figures show the results of these experiments. 3 Figure 2: DNA-protein binding assay. Protein was added at low (+) or high concentration (+++) to radiolabeled DNA segments containing both wild type E-binding sites (E1E2), or wild type E1 and mutant E2 (E1E2), or mutant E1 and wild type E2 (E1E2). The mutations in E1 and E2 are known to abolish protein-DNA interactions. In the first lane, 0 indicates that no protein was added to the radiolabeled DNA. E1 and E2 have each a single protein E binding site. Propose a model explaining the differences observed between the addition of a low and a high amount of protein pE and the difference between the protein-binding properties of E1 and E2. c) Briefly explain how proteins pA and pE lead to eve expression in stripe 5. Be sure to account for how the posterior (right) side of stripe 5 is formed. (hint: part b should help you answering this question). Question 3: Scientists have introduced in bacteria a wild type gene coding for GFP (green fluorescent protein) or two engineered versions of this gene containing an additional cis-regulatory element (crRL or crR12). The transcription of the wild-type GFP gene leads to the production of an mRNA containing a cis-acting element labeled X. Transcription of the engineered GFP genes leads to the production of mRNAs containing the cis- acting X and either crRL or crR12 in the 5’ portion of the mRNA. The synthesized mRNA molecules are diagrammed below. 1) To identify the function of X, scientists performed the following assay: purified small ribosomal subunits, large ribosomal subunits, 16S rRNA (ribosomal RNA), or Alanyl-tRNA were bound to a filter and the filter was then incubated with radioactive wild type GFP-mRNA molecules (with or without the cis-acting element X). The radioactivity associated with the filter was measured after removal of the unbound radioactive material. The results are given in the following table. Radioactive GFP mRNA Small ribosomal subunit Large ribosomal subunit 16s rRNA Ala-tRNA X deleted 18 cpm 22 cpm 7 cpm 13 cpm X present 1500 cpm 20 cpm 2300 cpm 18 cpm 4 Based on these data and your knowledge of translation in prokarytotes determine the function of X. Briefly explain. 2) The sequences of the 5’ end of crRL, crR12and WT-GFP mRNAs are: A filter assay, similar to the one described above, was performed by adding radioactive crRL mRNA and crR12 mRNA to a filter with 16SrRNA bound to it. A second assay was performed to test the sensitivity of X to a RNAse that only cleaves single-stranded RNA. The results of both experiments are presented in the following table. Radioactive mRNA Filter bound 16S rRNA RNAse sensitivity of sequence X GFP mRNA 2300 cpm High crRL mRNA 22 cpm Low crR12 mRNA 9 cpm Low None 15 cpm Note: The RNase assay is performed in solution and in the absence of 16S rRNA Based on the sequence information and the experimental data, suggest a role for the cis-acting regulatory elements crRL and crR12. Briefly explain. 3) The level of GFP synthesis was measured in bacteria expressing GFP mRNA, crRL mRNA or crR12 mRNA. The following histogram shows the results of this experiment. Level of GFP synthesis WT GFP crRL crR12 Expressed mRNA G FP s yn th e si s Based on the results of this experiment and the sequence information (see part 2), explain why the scientists describe crRL and crR12 as translation terminators. 4) The diagram below is a reminder of the sequence of the 5’-end of the crRL mRNA. 5 Bacteria expressing a gene where the sequence of the crRL element was replaced by its complementary sequence had a normal level of GFP synthesis, whereas the level of GFP synthesis remained low when mutations were introduced in the underlined sequence only. Based on these information, draw the secondary structure adopted by the 5’ end of the crRL mRNA. Question 4 You have decided to study the role of SWI1 and SWI2 proteins in transcriptional enhancement via steroid receptors. You know that when a cell is exposed to glucocorticoids (steroid hormones) the intracellular binding of this hormone to its cognate receptor results in the transcriptional activation of a specific sub-set of genes. A promoter called the glucocorticoid response element characterizes these genes. Yeast cells are co-transformed with the following constructs: - A yeast expression plasmid, pE, responsible for the constitutive expression of the gene encoding the glucocorticoid receptor. - A yeast reporter plasmid, pR, containing the gene encoding β-galactosidase under the control of the glucocorticoid response element. a) Describe how the synthesis of β-galactosidase in the yeast cells is affected by the addition of glucocorticoid. Include in your description the variation in the level of transcriptional activity of the glucocorticoid receptor gene in both the absence and presence of hormone. b) The activity of β-galactosidase before and after addition of glucocorticoid was measured using three different strains of yeast each co-transformed with pE and pR. The strains are the following: - SWI+, is a wild-type strain producing active SWI1 and SWI2 proteins - swi1- and swi2- are two mutant strains producing inactive SWI1 and SWI2 proteins respectively. The results of the β-galactosidase activity measurements were the following: Strain Glucocorticoid Activity (arbitrary units) SWI+ No 100 SWI+ Yes 1000 swi1- No 97 swi1- Yes 105 swi2- No 92 swi2- Yes 102 Based on the fact that proteins SWI1 and SWI2 are found in a multimolecular transcriptional complex, explain these results. c) If strains SWI+, swi1- and swi2- are only transformed by the yeast expression vector pE in which the β- galactosidase gene has replaced the glucocorticoid receptor gene the level of β-galactosidase activity is similar in each strain. Why is this control experiment critical to explaining the results presented in part (b)? Question 5: HP1 is a DNA binding protein that interacts with a specific sequence (TGCTTATTC). You want to analyze, by a Dnase I footprinting assay, the effect on DNA binding of the interaction between HP1 and its 6 binding partner PA. In each assay, you combine a radiolabeled fragment of DNA that binds to HP1 and a specific combination of proteins. After incubation with DNase I, each reaction mixture was resolved by gel electrophoresis, and then exposed to film. The autoradiogram showing the results for each combination of proteins is diagrammed below. Lane 1: DNA alone, no protein added in the nuclease assay. Lane 2: DNA + 5 ng (nanograms) of HP1 Lane 3: DNA + 5 ng of HP1 + 2ng of OCT1 (a protein known to interact with HP1) Lane 4: DNA + 5 ng of HP1 + 10 ng of OCT1 Lane 5: DNA + 5 ng HP1 + 5ng PA Lane 6: DNA + 5 ng PA 1) For each lane (1 to 6) give a brief interpretation of the results. 2) When DNA is mixed with PA and OCT1, the band pattern observed on the autoradiogram is identical to one of the pattern shown above. Which one? Explain. 3) Explain how DNA binding proteins can recognize a specific sequence without opening the DNA double helix. Question 6 IPTG is a structural analog of lactose commonly used to activate genes placed under the control of the Lac promoter. When added to a bacterial culture, IPTG diffuses freely inside the bacteria. 1) Bacteria, containing a functional lactose operon, were transformed by a plasmid that contains - LacI: the gene coding for the lac repressor under the control of a constitutive promoter - gfp: the gene coding for the green fluorescent protein GFP under the control of the lac promoter. The lac promoter is the promoter that normally controls the expression of the lac operon. a) Could bacteria synthesize GFP in the absence of lactose in the culture medium? Briefly explain. b) Lactose is then added to the culture medium. Briefly explain the consequences (at the molecular level) of the addition of lactose on the synthesis of GFP when the culture medium contains glucose and when the culture medium does not contain glucose. ) The following diagram represents the amount of GFP synthesized per milligrams of bacteria (y-axis) over time when bacteria are grown in a medium without glucose and supplemented with either IPTG or with lactose. a) Based on your knowledge of the lac operon, explain the difference in GFP synthesis when IPTG and lactose are used as inducers? b) What is the main factor limiting the level of GFP synthesis when lactose is used as an inducer?

Paper For Above instruction

The regulation of ferritin expression in mammalian cells exemplifies the intricate control mechanisms that cells employ to maintain iron homeostasis. Ferritin, a critical iron-storage protein, is tightly regulated at both transcriptional and translational levels to prevent iron toxicity. This regulation predominantly occurs via Iron Regulatory Proteins (IRPs) binding to Iron-Responsive Elements (IREs) located in the mRNA of ferritin. The complex interplay between IRPs and IREs ensures that ferritin synthesis is appropriately responsive to intracellular iron levels. In conditions of low iron, IRPs bind to the IREs in the 5’ untranslated region (UTR) of ferritin mRNA, blocking translation and thereby conserving iron. Conversely, when iron is abundant, IRPs lose their affinity for the IREs, permitting translation and leading to increased ferritin protein synthesis. This dynamic regulation highlights the importance of both the sequence and the secondary structure of the IRE in the 5’ UTR, which are essential for IRP binding and proper regulation (Mason, 2004; Hentze et al., 2010).

The specific sequence and secondary structure of the IRE are critical because they constitute the recognition motif for IRP binding. Mutations or deletions within this region can abolish IRP interaction, resulting in membranous or unregulated ferritin expression that is insensitive to iron levels. Structural integrity, especially the conserved hairpin loop motif, is necessary for IRPs to identify and bind effectively, thereby modulating translation as a response to iron fluctuations. These observations are consistent with the findings where deletion or replacement of specific sequences within the IRE resulted in iron-independent ferritin expression, indicating the functional necessity of the sequence-structure conformation.

The regulation occurs primarily at the translation initiation step, whereby IRPs hinder the assembly of the ribosomal complex at the 5’ cap structure of ferritin mRNA. Iron levels modulate this interaction, with IRPs acting as repressors in low iron conditions. When iron levels rise, IRPs disassociate from the IRE, allowing initiation complexes to assemble and trigger ferritin translation (Rouault & Cooperman, 2010). This regulatory mechanism ensures rapid cellular adaptation to iron availability, safeguarding cellular health.

IRP's role involves binding to the IRE in the ferritin mRNA, preventing translation under iron-deficient conditions. In iron-replete states, IRPs undergo conformational changes or disengagement, freeing the IRE to facilitate translation. The experimental evidence that ferritin mRNA is associated with IRP in iron-deficient conditions and released when iron is abundant underscores this regulatory role. IRP thus functions as a key molecular switch, integrating iron status with the post-transcriptional control of ferritin synthesis.

In the context of the insect development pattern resembling Drosophila’s segmentation, the regulation of the embryonic expression of the eve gene involves complex interactions of multiple transcription factors binding to cis-acting regulatory elements like UASs. The formation of stripes 2 and 5 of eve expression depends on specific combinations of activators and repressors. For stripe 2, the distribution data indicate that proteins pA, pB, pC, and pD are likely to include activators that promote gene expression, given their spatial expression patterns aligning with the stripe. Conversely, certain proteins acting as repressors would show an inverse distribution pattern, inhibiting expression where they are highly expressed. For instance, if pB’s expression peaks outside the stripe region, it may function as a repressor suppressing eve in those domains, whereas pA, pC, and pD, co-expressed within the stripe, could serve as activators (Farrell & Gergen, 2013).

In the assay analyzing pE binding to UAS5, the increased binding affinity at higher protein concentrations suggests a dose-dependent interaction characteristic of a specific and high-affinity binding site. The mutant forms lacking the binding sites E1 or E2 likely exhibit diminished or abolished binding, supporting the model that pE interacts directly with these sequences. Furthermore, differences in the binding patterns of E1 and E2 may indicate distinct roles or affinities, with E1 possibly showing stronger or more specific binding under low conditions, and E2 facilitating additional regulation at higher concentrations.

Proteins pA and pE are instrumental in activating eve expression in stripe 5 through their binding to specific enhancer elements. pA likely acts as an activator, promoting transcription where it binds, while pE may enhance or modulate this activity through cooperative binding or stabilization of