1. eIF2 phosphorylation is one way that the cell can control overall translation rates.
A. What happens when eIF2 becomes phosphorylated? (4 pts)
When phosphorylated, eIF2 binds irreversibly to its GEF, eIF2b. The amount of eIF2b in cells is small in comparison to the eIF2 amount. Thus the phosphorylated eIF2 sequesters all the available eIF2b in an irreversible complex. Withouth eIF2b availability, other eIF2s that require it to reactivate, cannot. This has a global negative effect upon translation.
B. What is the role that eIF2 plays during the initiation of translation? (4)
eIF2-GTP is bound to the initiator tRNA and the small ribosomal subunit. This complex then finds and interacts with the Cap and its binding factors and scans to find the first AUG. Once there, eIF2-GTP hydrolyzes to GDP and this energy release participates in the attachment of the large ribosomal subunit make the functional ribosome.
2. Outline the events that occur to make a peptide bond during translation, starting from a tRNA entering the A site of the ribosome up to the translocation to the next codon. (8 pts).
The tRNA, bound to eEF1-GTP, contacts the codon at the A site of the ribosome through anticodon-codon base pairing. If the interaction is sufficiently stable, it will remain there long enough for the eEF1 to hydrolyze the GTP to GDP, detach from the tRNA, and remove the steric hindrance from the tRNA that prevented the amino acid containing end from stably attaching. The energy released also helps with the stable attachment. This triggers peptidyl transferase activity. The amino acid end of the tRNA at the A site leans towards the P site. Peptidyl transferase breaks the bond holding the tRNA at the P site to the amino acid ( growing polypeptide chain) and the released amino acid is attached by a peptide bond to that held on the tRNA at the A site. This triggers translocation of the entire ribosome one codon to the 3’ direction, placing the exiting tRNA from the P site into the E site, the tRNA that was at the A site now at the P site, and the A site open for another tRNA to enter. This translocation is fueled by eEF2-GTP which hydrolyses the GTP to GDP releasing energy.
3. Cholera toxin causes a serious disease in humans because its presence results in abnormally high cAMP levels. Using what you know about how signal transduction controls cAMP levels, give a possible explanation for this. (6 pts)
The toxin, potentially, could influence any of the following players in the signal transduction pathway. The receptor could be irreversibly bound and interact uncontrollably with the G protein. The G protein could be altered so that it constantly remains in a state that has the alpha subunit bound to GTP and the beta and gamma subunits not there (this I believe is the actual case), the toxin could cause activation of adenyl cyclase that does not respond to inhibitory alpha subunits. Any step in the normal process could be a target in this answer.
4. What role is played by the following molecules in G-protein linked signaling? (2 pts each)
A. Phospholipase
Cleaves PIP2 to form IP3 and DAG
B. cAMP
Binds protein kinase A regulatory subunits causing them to release the catalytic subunits and allow for interaction with the substrates
C. IP3
Binds and opens calcium channels in the ER membrane allowing the release of calcium to the cytoplasm
D. Calcium
Binds to calmodulin and this complex interacts with various other molecules to alter their activity. Also binds to and causes protein kinase C to move to the membrane to continue signal transduction.
E. DAG
Activates the membrane-bound form of protein kinase C to continue signaling pathways.
5. Why is sos an important protein in Receptor Tyrosine Kinase-mediated signal transduction? (6 pts)
Sos is a GEF for ras-GDP. In response to activation by a signaling ligand, the RTK self-phosphorylates at tyrosines. SH2-domain containing proteins such as GRB2 bind there and then interact with an SH3 domain containing protein or have such a domain themselves, as does GRB2. The GRB2 or analogous protein binds to sos through its SH3 domain, causing sos to bind the inactive ras-GDP, causing it to release GDP and allow for reactivation by GTP.
6. A cascade of kinase activity activates c-fos gene transcription. Outline the sequence. (8 pts)
Activated ras (see above) causes raf kinase to move to the membrane and release its regulatory domain from 14-3-3. The raf becomes activated this way and phosphorylates both itself and MEK at serines and threonines. A protease completes the release of raf from 14-3-3. The activated MEK phosphorylates Map Kinase at both serine/threonines and at tyrosine, activating it. The activated Map Kinase dimerizes. It phosphorylates p90. Both phosphorylated p90 and dimerized Map Kinase enter the nucleus. The p90 phosphorylates SRF transcription factor and Map Kinase phosphorylates TCF transcription factor. The TCF and 2 SRFs bind the SRE enhancer element and activate the transcription of the early response gene c-fos.
7. Give four categories of proteins that work towards moving the cell-cycle forward and also give at least one example of a protein in each category. (8 pts)
A. Kinases: cdks, Chk1/2, ATM/ATR, etc.
B. Phosphatases: cdc25, cdc14, etc.
C. Ubiquitin ligases: SCF, APC-cdc20, APC-Cdh1
D. Inhibitors: p21, Rb, etc.
I gave credit for some other answers that categorized things slightly differently.
8. Mutated forms of p53 are found in a majority of human tumors. Using what you have learned about the function of p53 in cells, explain why this is not surprising. (8 pts)
P53 plays a role in cell-cycle arrest, in initiation of apoptosis, and in DNA repair. When a DNA damage signal activates ATM/ATR or CHK1/2 to phosphorylate p53, the p53 is no longer targetd for ubiquitination by the mdm-2 protein. The stabilized p53 then works as a specific transcription factor to activate transcription of p21 (causes cell-cycle arrest by interference with cyclin/cdk activity in G1 or G2) or of apoptosis inititiating proteins like Noxa and Puma. Also, DNA repair enzymes and co-factors are activated as well. Since all of these processes are involved in protecting the cell from unregulated cell division or cell division in the presence of damaged DNA, cells lacking this protection are more likely to become malignant.
9. Bad protein is part of an important control mechanism in cells. Explain how mutated bad leads to disruption of this mechanism and also describe a potential consequence of this disruption for the cell. (10 pts)
The usual role for bad is anti-apoptotic. It sits on the outer mitochondrial membrane preventing the bcl2 dimer from inhibiting the formation of the bax dimer ion channel. Thus the ion channel forms and iflux of ions to the mitochondria causes a release of cytochrome c to the cytoplasm. It binds to apaf-1 which activates the caspase cascade by causing the procaspase 9 to cleave itself to caspase 9. This moves to cleave procaspase 3 to caspase3 and both then go on to cleave other substrates causing apoptosis. If bad is mutated, it could not interact with bcl2 as described and apoptosis would be inhibited by the bcl2 dimer. Damaged cells that should be signaled to enter apoptosis would not be able to. This can cause dangerous cells to be allowed to divide and contribute to malignancy.
10. Genetically, what distinguishes an oncogene from a tumor-suppressor gene? (6 pts)
An oncogene arises by a dominant, gain-of-function change in a normal proto-oncogene product that deregulates its activity and allows it to promote cell division or other growth-promoting or anti-apoptotic functions in an abnormal fashion. Only one allele need be altered genetically.
A tumor suppressor arises by a recessive, loss-of-function change in the normal protein that usually plays a protective role in controlling cell growth or allowing apoptosis. Both alleles of the gene need to be lost, or the protein product completely inactivated, for the loss of the tumor-suppressor to contribute to malignancy.
One way is genomic amplification where the myc gene, usually single-copy, is present in multiple copies, usually due to aberrant DNA replication. This is detected as a double-minute or an hsr region in chromosomal staining. The extra copies cause too much myc to be made in the cell when it is activated. This can cause unregulated cell-cycle entry.
The other method is for there to be a chromosomal translocation between chromosomes 8 and 14 that brings the myc gene to the region where the promoters and enhancers that control antibody gene expression are located. The myc gene comes under the direction of these powerful enhancer elements and gets expressed in B cells that operate in the immune response. However, myc is then expressed instead, contributing to the formation of Burkitt’s lymphoma.
Tumor-suppressor because it binds to E2F transcription factor to prevent it from moving the cell cycle into S phase aberrantly.
Proto-oncogene: ras plays a positive role in signal transduction pathways that ultimately alter genetic activity such as activation of c-fos, etc.
proto-oncogene: its kinase activity contributes to the positive movement of signal transduction mechanisms that alter growth.
proto-oncogene: Forms a dimer that prevents bax dimers from operating as ion channels thus preventing apoptosis.
The debates were on the topics of DNA databases and research into the causes of homosexuality in sheep. Two arguments per topic were required.