Key For Exam 3, BISC401 Honors, Spring, 2006

1.                  Eukaryotic cells often control their protein production at the level of translation events. Translation of globin mRNAs that make hemoglobin subunits is one example. When heme levels are low in a cell, translation is slowed by modifying a GEF protein that regulates the activity of eIF2, causing the GEF to bind to eIF2 irreversibly.

A.                 Using your knowledge about the translation mechanism and GEF proteins, explain why this would inhibit the translation of globin mRNAs. (8 pts)

At the end of the previous translation initiation cycle, eIF2 is bound to GDP. It requires the GEF to bind to it reversibly, forcing out GDP so GTP can replace it. If the GEF is modified due to low heme concentration, it will bind but not release the eIF2. Therefore, the eIF2-GTP cannot function in its role. It is needed both to help form the small ribosomal subunit initiation complex and then, during ribosome scanning and recognition of the AUG start codon to prevent further movement past that codon by hydrolyzing its GTP to GDP, irreversibly stopping the scanning process.

B.                 Why is it logical that low heme levels would work as a regulator in this system? (6 pts)

Heme is required to interact with each globin subunit to form a functional hemoglobin. If  heme levels are low, there is no need to make additional globin subunits until the levels rise again. Therefore, slowing translation is appropriate.

2.                  Histone synthesis is coordinated with DNA synthesis during the cell cycle. Describe a scenario related to histone mRNA stability that would correlate to the amounts of histone protein present in the cell during G1, S and G2 phases of the cell cycle. In other words, what might be different about the mRNAs and how they are degraded or not degraded that would explain histone production during these cell cycle phases? Assume the genes for histones are constitutively active at all phases and that only the mRNA stability differs. Your answer MUST address the mechanism involved in determining mRNA stability. (8 pts)

In the 3’ UTR of mRNAs that code for proteins that are quickly degraded are several AUAAA sequences that facilitate the formation of stem-loop structures. These signal nucleases to rapidly degrade the mRNA. Such as system can explain the role of mRNA stability in the regulation of histone production. Such a situation would exist during G1 and G2 phases of the cell cycle which do not require rapid histone protein synthesis. However, during S phase, histone production is needed to form new daughter DNA molecules. Thus, a protein would be present during S phase that binds to the stem-loops, preventing them from signaling for the mRNA degredation.

3.                  How is the Cap on eukaryotic mRNAs involved in setting the correct reading frame for translation? Explain thoroughly.  (8 pts)

The Cap binds to a complex of proteins collectively known as eIF4. Components of eIF4 interact with the small ribosomal subunit initiation complex, thus bringing it to the 5’ end of the mRNA. From there, the complex scans until it finds the first AUG following the Cap and initiates the translation process there, thus setting the correct reading frame. Also, eIF4 uses the energy from ATP hydrolysis to melt the stem-loop in the 5’ UTR. This must occur if the initiation complex is to be able to move along the 5UTR to the start codon.

4.                  Glycogen synthase is an enzyme that uses glucose to make glycogen. Glycogen phosphorylase causes the breakdown of glycogen into glucose. Both are regulated by phosphorylation, catalyzed by the cyclic-AMP dependent protein kinase under the direction of the hormone epinephrine which is secreted when cells require increased ATP production by aerobic respiration. Explain the entire signaling pathway, from epinephrine secretion to the phosphorylation of the two enzymes and indicate why this makes sense when cells need to increase their ATP production (remember introductory biology!!). (10 pts)

Epinephrine binds to a receptor in the membrane of the targeted cell. The cytoplasmic domain of the receptor changes shape and interacts with a G-alpha subunit of a G protein in the membrane. This interaction causes the G-alpha subunit to release GDP, allowing GTP to replace it. This releases the alpha subunit from the beta-gamma subunits. The alpha subunit binds to adenylyl cyclase, activating it to catalyze the formation of cAMP. cAMP binds to the regulatory subunits of cyclic AMP dependent protein kinase (protein kinase A), causing them to release the catalytic subunits which then can catalyze the phosphorylation of target proteins. In this system, the two target proteins are the enzymes glycogen synthase and glycogen phosphorylase. Phosphorylation inhibits the ability of glycogen synthase to function, preventing the use of glucose monomers to make glycogen and phosphorylation activates glycogen phosphorylase which initiates the breakdown of glycogen and thus releases glucose monomers. Therefore these two results increase the amount of glucose monomers available for glycoloysis, which is needed for the formation of ATP.

5.                  The regulation of genetic activity by Map Kinase involves what is called a “kinase cascade.” Explain this by describing the activation of Map Kinase starting from the formation of an active ras protein. (8 pts)

Active ras (bound to GTP) interacts with the N-terminal of raf, causing it to release from 14-3-3 (by some mechanism the phosphoserine binding the N terminal to 14-3-3 becomes dephosphorylated). The raf now is in the vicinity of the membrane instead of completely sequestered in the cytoplasm and its kinase domain is active. It catalyzes the phosphorylation of serines and threonines on MEK. This activates MEK. MEK catalyzes the phosphorylation of both serine/threonines and also of tyrosines on MAP Kinase. This activates MAP Kinase. MAP Kinase phosphorylates p90 in the cytoplasm which moves into the nucleus. Also, MAP Kinase now forms a dimer and moves into the nucleus also. Both p90 and the dimeric MAP Kinase catalyze phosphorylation of target specific transcription factors, thus activating them and causing the transcription of early response genes.

6.                   

A.                 How do the G1 cyclin/cdks control the  Retinoblastoma protein(Rb)? (4 pts)

They catalyze the attachment of phosphate groups to amino acid side chains of  Rb, causing it to be unable to remain bound to the E2F protein.

B.                 How does Rb control the entry into S phase? ( 4 pts)

By binding to E2F, Rb prevents it from acting as a specific transcription factor. E2F can activate its own transcription and that of several genes involved in producing proteins needed for DNA synthesis. If bound to E2F, Rb prevents this and therefore cell cycle progression into S does not occur. If phosphorylated (see A above), Rb releases E2F and the cell cycle progresses.

C.                 How does p53 regulate the events occurring in A and B? (6 pts)

P53 is usually bound by mdm-2 and targeted for degredation. If appropriate, it becomes phosphorylated and mdm-2 cannot remain bound. Thus p53 is not rapidly degraded. It then can work as a specific transcription factor, activating its own transcription and that of a gene for a protein called p21. p21 binds to cyclin-cdk complexes. This inhibits the ability of these complexes to catalyze phosphorylation of target proteins such as Rb. Rb then remains bound to E2F and the cell cycle is stopped from moving into S.

7.                  When cancer biologists identified tumor-suppressor proteins, one of those so identified was the bax protein. Why does bax qualify as a tumor suppressor protein? Be specific about what it is that bax does in the cell and how this explains why it is in that category. (8 pts)

Bax forms dimmers in the outer mitochondrial membrane that work as ion channels. When ions flow into the mitochondria through these channels, cytochrome is released into the cytoplasm where it binds to Apaf-1. Apaf-1 is activated to cause procaspase-9 to cleave itself releasing caspase 9. This targets the cleavage of multiple other proteins, including procaspase 3 which cleaves to caspase 3. Caspase 3 cleaves additional target proteins. This combined effect leads to apoptosis in the cell. Since apoptosis is a way to inhibit the growth of undesirable cells, bax is considered a tumor-suppressor. Both genes of bax would need to be mutated or inactive to prevent bax from forming and working to cause apoptosis.

8.                  Describe two ways that a proto-oncogene can become an oncogene. (8 pts)

Possible answers include:

            Spontaneous mutations that deregulate the activity of proto-oncogene products.

            Mutations induced in proto-oncogene products because their genes were transduced and mutated by a retroviral integration (e.g., v-src).

            Insertional mutagenesis where a retrovirus integrates near a proto-oncogene causing the aberrant expression of the gene due to the influence of the powerful enhancers present in the LTRs of the retrovirus.

            Gene amplification due to the presence of multiple copies of a proto-oncogene that would normally be present in only one copy. This is usually detected by the presence of double-minutes or hsrs seen in chromosomal stains.

            Reciprocal chromosomal translocations that move a proto-oncogene into the region of another chromosome that causes the aberrant expression of the proto-oncogene and perhaps the formation of a mutated protein from that gene. For example, the 8, 14 translocation that causes c-myc to come under the regulation of powerful antibody promoters and causes Burkitt’s lymphoma, or 9-22 which causes the formation of a fusion protein, bcr-abl, that has unregulated kinase activity during signal transduction.

9.                  Explain how potential human oncogenes were identified using the method of transfection of human DNA into mouse cells. (8 pts).

DNA from human tumors was transfected into normal mouse cell cultures. Foci were allowed to develop. DNA from the foci was removed and transfected into more normal mouse cell cultures. A second round of foci were allowed to develop. DNA from the foci was shotgun cloned into a bacteriophage library. The library was used to infect E-coli cells and resulting plaques were probed for the presence of Alu family sequences in the phage. Alu sequences are specific to human spacer DNA and indicate that a potential human gene is nearby. Since the human DNA came from a tumor it could be an oncogene. The human DNA is sequenced and the sequence is compared to known oncogene sequences, identifying genes potentially involved in forming the tumor.

10.              By what mechanism are the DNA tumor viruses SV40, HPV, and Adenovirus similar in the method by which they cause cancerous transformation of cells? (6 pts)

SV40 makes large T antigen that binds to both p53 and to Rb, inactivating them.

HPV makes E6 and E7 proteins. E6 binds to p53 and causes its degredation. E7 binds to Rb and inactives it.

Adenovirus makes E1a and E1b. E1a binds to Rb and inactivates it. E1b binds to p53 and inactivates it.

Therefore, all three viruses inactivate the same two tumor-suppressor proteins.

11.              Why are the following drugs effective tools in the treatment of cancers (In other words, what mechanism are they using?). (8 pts)

A.                 Gleevek

Gleevek binds to deregulated protein kinases that cause uncontrolled signaling pathways in a cell. The bcr-abl protein produced by the reciprocal translocation between chromosomes 9 and 22 that causes CML was the first kinase targeted by Gleevek.

B.                 Angiostatin

Angiostatin inhibits the growth factors released by tumor cells that recruit endothelial cells to the tumors and activate mitosis of these cells. This prevents the formation of new blood vessels to feed the tumors which would otherwise die by apoptosis due to insufficient nutrients or at least be unable to continue growing.

Extra Credit: (4 pts)

What is a major argument in favor of denying states the right to maintain forensic databases?

Consult the handout.

What is a major argument in favor of mandating infant DNA testing?

Consult the handout.