Key for Exam 3,  Spring 2007

1.    Describe what role the following play in the translation process. (6 pts each)

A.     eIF2

A G protein which when GTP bound interacts with the initiator tRNA and the small ribosomal subunit and participates in finding the Cap and scanning to the AUG start codon.

B.     eEF1alpha

A G protein that when GTP bound interacts with the incoming tRNA at the A site, preventing the amino acid end from stably associating with the A site until the codon-anticodon interaction is deemed correct. Then GTP hydrolyzes to GDP and the factor is released, allowing the energy released to help the stable association to the A site.

C.     eEF2

A G protein that when GTP bound is involved with the translocation of the ribosome one codon along the mRNA following peptide bond formation. GTP hydrolysis provides the energy.

2.                  Describe one method by which translation can be regulated. (6 pts)

Stem-loop structures in the 5’ untranslated region can be bound by a factor, for example the IRE Binding Protein, and prevent initiation of translation by preventing ribosome scanning when a protein, such as ferritin, is not needed.  OR

A global decrease in translation could happen in response to low energy or other reasons by having phosphorylation of eIF2 or eIF2beta prevent their disassociation when GDP is being forced out of eIF2. If they cannot disassociate, the GTP will not replace GDP and no further initiation complexes can form.

3.                  If a cell requires an increase in ATP production, what signaling pathway that we studied would be used? Describe this pathway thoroughly from the initial signaling ligand arriving at the cell up to the increase in ATP production. (10 pts) 

Hormone would bind a G linked receptor and activate it to bind to a G protein. This causes the alpha subunit to replace GDP with GTP and to disassociate with the beta/gamma subunits. The alpha subunit then activates adenyl cyclase which catalyzes formation of cAMP. The cAMP binds protein kinase A, causing the release of the catalytic subunits from the regulatory subunits. These then cause phosphorylation of glycogen synthase which becomes inactive and cannot sequester glucose into glycogen. They also cause phosphorylation of phosphorylase kinase which activates it. It then causes phosphorylation of glycogen phosphorylase, which activates it to cause breakdown of glycogen into glucose monomers. This overall cycle causes increased amounts of glucose in the cell for use in ATP production.

4.                  How are the immediate early genes induced in response to the presence of a growth factor? (8 pts)

A growth factor binds and causes the dimerization or a receptor tyrosine kinase and activation of its tyrosine kinase activity. The two dimers autophosphorylate one another’s cytoplasmic domains at tyrosines. Greb binds the phosphotyrosine regions of the receptor through its SH2 domain. Through its SH3 domain it binds to sos (a GEF). Sos binds to ras and causes the GDP to be replaced by GTP thus activating ras. Ras recruits raf to the membrane and causes its activation. Raf phosphorylates MEK, activating it. MEK phosphorylates ERK, activating it. ERK moves to the nucleus and phosphorylates Elk-1. Elk-1 then can bind to the SRE upstream promoter element along with SR factors. This activates the early response genes.

5.                  Describe two ways that a cdk can be activated in a cell and two ways that it can be inactivated?  (4 pts)

Activated by phosphorylation of thr 160; by binding a cyclin.

Inactivated by phosphorylation of thr 14 and 15; by binding of a cyclin/cdk inhibitor.

6.                  What role does cdc25 play in the DNA damage checkpoints that induce cell-cycle arrest?  (8 pts)

Cdc25, a phosphatase, usually helps cell-cycle progression by dephosphorylating a cdk at the inhibitory amino acids (see above), thus helping activate them. At DNA damage checkpoints, ATM or ATR activate chk 2 or chk1 kinases, respectively. They then phosphorylate cdc 25A or C, inhibiting their ability to act as a phosphatase. This helps cause cell cycle arrest.

7.                  What is a caspase? (6 pts)

A cysteine protease that cleaves proteins after aspartic acids. They target either themselves, other caspases, or cellular proteins for degredation in response to apoptosis inducing signals and events in the cells.

8.                  Outline the molecular events that induce apoptosis in response to mitochondrial damage in mammalian cells. (8 pts)

A single domain proapoptotic protein moves to the mitochondrial membrane and binds an antiapoptotic protein, preventing it from interacting with and inhibiting a multi-domain proapoptotic protein. The pro-apoptotic protein then can form multimers with other active pro-apoptotic multi-domain proteins and form channels that allow the efflux of cytochrome c and anti AIP proteins from the inner mitochondrial space out to the cytoplasm. The cytochrome c binds to sites on Apaf-1. This activates the apoptosome which also contains caspase 9. Caspase 9 is activated and cleaves other procaspases and activates them. Apoptosis is thus initiated.

9.                  p53 is mutated in a large number of human cancers. Explain how p53 is activated and why its activation plays such an important role in preventing the formation of cancers. (6 pts)

p53 is stabilized by phosphorylation  by either ATM directly or through the activation of chk1 kinase. This keeps mdm-2 from binding to it and targeting it for degredation. The stable p53 then activates the transcription of p21, an inhibitor of several cdk/cyclin complexes. This causes cell cycle arrest.

P53 can also, when stabilized, activate the transcription of several singl domain proapoptotic proteins which will then initiate the apoptosis pathway.

Both the cell-cycle arrest function and the apoptosis function protect the cell from replicating damaged DNA or from proceeding with cell division in undesirable circumstances involving the chromosomal material. If this protective function is lost, the cell moves towards malignancy.

10.              Why is the retinoblastoma gene (Rb) considered to be a tumor-suppressor gene and how does its role in the cell explain that? (6 pts)

Because its normal role in the cell is to prevent movement through the restriction point into S phase. It does this by binding to E2F, a transcription factor that activates the transcription of several cyclins and other genes for DNA synthesis. Only if phosphorylated can Rb be prevented from binding E2F. As long as it is bound, the cell cannot pass through the restriction point. Therefore, if Rb has lost both function alleles of its gene, no functional Rb can form and E2F is left unrestricted in its activity to cause movement into S phase.

11.              How is a chromosomal translocation involved in the formation of Burkitt’s lymphoma? (6 pts)

c-myc usually resides on chromosome 8. The genes for the heavy chain of the immunoglobulins resides on chromosome 14. When a reciprocal translocation moves c-myc to the vicinity of the strong promoter/enhancers regulating the heavy chain gene, c-myc gets activated for transcription every time the B cell is activated to produce antibody molecules. Therefore, c-myc is expressed in an aberrant fashion. Since it is a transcriptional activator involved in activating expression of cell division and other growth regulating genes, its overexpression leads to cell to malignancy and the formation of Burkitt’s lymphoma.

12.              Give one example of an oncogenic virus that has informed our understanding of cancer.  Explain what we learned from it. (6 pts)

Many possibilities: Any of the retroviruses that picked up an oncogene that we discussed; any of the DNA tumor viruses we discussed like WV40, Adenovirus, HPV. The answer should discuss what the retroviral oncogene is and how it causes cancer or how the CAN tumor virus inactivates what tumor suppressor proteins.

13.              What were the four topics that we debated during the semester? (2 pts each)

Should girls be required to have HPV vaccinations?

Should viral genomes be published online to foster scientific inquiry even if this risks access by terrorists?

Should the human genome sequences be used to develop mandatory genetic screening of infants?

Should pharmaceutical companies give up patent protection to all drugs for poor, third world countries to be more affordable?