1.       1.    Outline the steps by which an accurate translation initiation process occurs. (8 pts)

See figure 4-24 in the text.

2.      2. Describe the role of the following in the elongation of translation (3 pts each)

A.     a.     eEF1 alpha

Accompanies the tRNA coming into the A site of the ribosome during elongation. It is bound to GTP then. Its presence prevents the amino acid end of the tRNA from stably associating with the A site. Therefore only the codon-anticodon interaction holds the tRNA there initially. The GTP is hydrolyzed to GDP. This changes the conformation of the eEF1 alpha so that it disengages from the tRNA. This also releases energy. The tRNA can now interacts stably with the A site. If an incorrect tRNA had entered in this fashion, the delay until GTP hydrolysis occurred would be too long to allow the tRNA to remain in the A site held only by its codon-anticodon interaction which would not be correct. It would be bumped out by thermal activity. This kinetic proofreading prevents errors during translation.

B.      b.    peptidyl transferase

Enzymatic activity associated with the large ribosomal subunit that is activated once the A site is stably occupied by the incoming tRNA. It breaks the bond between the P site tRNA and the amino acid to which it is covalently linked. This releases energy which is used by the enzyme to make a peptide linkage between the just released  amino acid and the amino acid held on the tRNA at the A site.

C.      c    eEF2

A G protein that works after the new peptide bond has been made. It uses energy released by hydrolysis of its GTP to GDP to fuel the movement (translocation) of the ribosome one codon along the mRNA in the 5’-3’ direction. This results in the tRNA that was at the A site now being at the P site; the tRNA that was at the P site now is at the E site, and the A site is open and situated over the next codon awaiting the next incoming tRNA.

3.       3.    What roles do the 3’UTR and the 5’UTR play in the regulation of iron content in the cell.? (6 pts)

See figure 8-31 in our text.

4.       4.    Briefly describe one of the following mechanisms:  the mTOR mediated control system, the miRNA control system, or the siRNA control system.  (5 pts)

For m-TOR see figure 8-30. You were only required to know the role of RhebGTP in the activation of mTOR and the results of this activation.

For miRNA and siRNA see figure 8-25 and the legend explaining the figure.

5.       5.    Match the following terms to the most accurate statement below. Terms may be used more than once or not at all. (2 pts each)

Phospholipase C                              Calcium                                                Cyclic AMP

IP3                                                          PIP2                                       Protein kinase A

Adenyl cyclase                                  Calmodulin                        Protein kinase C

Diacylglycerol                                   G alpha subunit                                               

a.                   Cleaves PIP2. ­­­­­­­­­­­__phospholipase C_______________________

b.                  Activates protein kinase A. __cyclic AMP_______________

c.                   Binds to calcium. _calmodulin (protein kinase C also accepted)______________

d.                  Causes the release of calcium into the cytoplasm. _IP3___________

e.                  Activates membrane-associated protein kinase C.__Diacylglycerol (calcium accepted also)___________

f.                    Catalyzes the formation of the activator of protein kinase A. __adenyl cyclase____________

g.                   Phosphorylates enzymes that control glycogen metabolism. __protein kinase A__________

6.       6.    Answer the following questions about signal transduction:

A.      Describe the Her family of receptors and how they activate a signal transduction pathway in response to ligand binding. (6 pts)

See figure 16-18. A description of all of the possible dimers that can form; the differences between the various Her family members;  and the method by which they phosphorylate tyrosines on their cytoplasmic domains following dimerization in response to ligand binding are all required in the answer.

B.      Order the following proteins in their proper sequence (1-7) as it occurs in the signal transduction pathway that is stimulated by activation of the epidermal growth factor receptor. (7 pts)

__4_raf                 __1_GRB2                            __5_Mek                              __2_sos

__3_ras                 __7_p90                                               __6_Map Kinase

7.       7.    Answer the following cell-cycle related questions.

A.      How does the cell insure that an origin of replication will be used only once during S phase?

Only non-phosphorylated replication initiation factors can bind to the ORC. Such binding is required for subsequent phosphorylation of these factors during S-phase to initiate DNA replication. These factors remain phosphorylated after working to initiate DNA replication until late in the cell-cycle a phosphatase removes the phosphates and allows for the factors to once again associate with the ORC during early G1. Therefore, the factors cannot initiate a round of replication twice from the same ORC, insuring that it is only used once during S phase. See figure 20-30.

B.      To what is the sensor responding during the intra-S phase checkpoint? (2 pts)

The presence of DNA replication forks, indicate the process is not yet finished.

C.      What causes the movement from metaphase into anaphase? (4 pts)

Securin, which associates with the centromere region of sister-chromatids, is targeted for degredation by the ubiquitination/proteosome pathway. The releases separase enzyme to be active. It breaks up the cohesion rings holding the sister chromatids together and they can separate during anaphase.

D.      How is Rb regulated during cell cycle progression? (4 pts)

Rb binds to EF2Fwhen not phosphorylated. In mid-G1 a cyclin/cdk begins to phosphorylate Rb causing the release of some E2F transcription factor. It activates transcription of its own gene and those of additional cyclink/cdks that further phosphorylate Rb causing the complete release of E2F which activates transcription of genes involved in moving the cell-cycle into S phase and the replication of DNA. See figure 20-33

8.      8.    Outline the sequence of events that ensues when there is no survival factor present to stop the apoptosis process in a cell. (8 pts)

See figure 21-41 a and figure 21-40. I also accepted the c-elegans system, figure 21-39.

9.       9.    Define the following terms related to the activation of oncogenes:  (3 pts each)

A.      Double-minutes

Pieces of fragments of chromosomes lying near the main chromosome that stain positive in FISH assays. They represent gene amplification that has occurred. If the amplified gene is a protooncogene, this would create an oncogene.

B.      Philadelphia chromosome

Chromosome 22 that has been part of a reciprocal translocation with chromosome 9 that brings the  abl gene region on 9 next to the bcr gene region on 22 creating a bcr-abl fusion gene which when transcribed in B cells produces the bcr-abl fusion protein that is a constitutive, unregulated protein kinase that operates in signal transduction pathways causing abnormal cell division that leads to cancer. See figure 25-20.

C.      Insertional mutagenesis

Powerful enhancers and promoters, especially enhancers, in the LTRs of retroviruses exert their influence on the host cell genes when the retrovirus integrates into the host chromosomes as part of its normal replication activity. If a proto-oncogene is influenced in this way, its transcription will be abnormally activated helping cause the cell to be growth deregulated and facilitating the formation of cancer.

10.  10.    P53 is categorized as a tumor-suppressor protein. Explain why, using what you know about the mechanism/s by which it acts. (8 pts)

P53 is a transcription factor that plays three critical roles in suppressing unrestricted cell growth that can lead to cancer. First, it is a major player in the DNA damage checkpoints. It is activated by phosphorylation in response to DNA damage during G1 and also G2. When so phosphorylated, p53 is released from mdm-2, a ubiquitin ligase that targets p53 for degredation in the ubiquitin/proteosome system. This increases the half-life of p53 and it can activate transcription of p21, an inhibitor of many cyclin/cdk complexes. This arrests the further progression of the cell-cycle.

P53 also plays a role in apoptosis initiation. It activates the BH3 only domain pro-apoptotic proteins Puma and Noxa, beginning the apoptosis inducing pathway. It also restricts the translation of bcl2, preventing it from inhibiting apoptosis.

P53 also activates the transcription of many DNA-repair related genes, helping the cell to repair damaged DNA. 

All of these roles played by p53 make it a central player in keeping cells from dividing with damaged DNA that could cause the accumulation of additional mutations in other genes and push the cell into the malignant phenotype. Therefore it is a tumor-suppressor. It also acts in a recessive fashion because it is the complete loss of p53 that promotes the cancer due to the absence of its protective effects.

11.  11.    This question will be given to you in class. (6 pts)

SV40 virus makes large T antigen that binds to both p53 and Rb and inactivates them. They cannot then play their normal tumor-suppressor protein role.

The oncogenic Adenoviruses make E1A, which binds to Rb, and E1B, which binds to p53, inactivating them similarly.

The HPV (human papilloma virus) makes E7 protein which binds and inactivates Rb, and also makes #6 protein that binds to p53 and targets it for degredation in the ubiquitin/proteosome system.

Thus all three DNA tumor viruses inactivate both of these tumor-suppressor proteins as they cause malignant transformation of the cells they infect.