1.      

                                        Key for exam 3, spring 2010

1.    During the elongation phase of translation, there are two “proofreadings” that occur to insure the correct amino acid is at the A site before a peptide bond is formed. Describe them. (8 pts)

First:  The incoming tRNA is bound to eEF1. This bends the tRNA so that only the anticodon loop can associate with the ribosome A site by attempting to base pair to the codon. The amino acid containing end of the tRNA does not associate. Therefore, incorrect tRNAs dissociate before the eEF1 hydrolyzes GTP to GDP and releases the amino acid end. The hydrolysis is prevented by the ribosome until it checks out each codon/anticodon interaction as correct.

Second:  The time lag between release from eEF1 and stable incorporation of the amino acid end of the tRNA with the A site causes tRNAs with mismatches to the codon to preferentially dissociate before any peptidyl transferase activity occurs.

2.     2.    eIF2 is an important target for global translational control. Describe its normal role in the translation process and also describe how it is involved in dampening overall translation under certain conditions in the cell. (10 pts)

eIF2 in its GTP-bound form complexes with the initiaing tRNA and the small ribosomal subunit to form the preinitiation complex that will bind to the cap region of the mRNA and scan to find the AUG start codon. It  hydrolyzes its GTP to GDP as the large subunit binds. Therefore it plays a very important role in translation initiation. It is controlled when overall translation needs to be repressed by being targeted by a protein kinase that attaches phosphates group to it in its GDP-bound form. When it binds to eIF2b the GDP does not get removed and it cannot be unattached to the eIF2b. Since there are more eIF2s in the cell than eIF2Bs, the eIF2bs rapidly become unavailable for further work as eIF2 GEFs and no other eIF2s can be reactivated to GTP bound forms. All translation initiation is therefore diminished.

3.       3.    What would you predict would happen to mRNA stability if the 3’ UTR of histone H2 mRNA was substituted for that of the common cellular protein actin? (8 pts)

The 3’UTR of histone mRNA contains stem-loop structures that regulate its degredation in a cell-cycle regulated manner. Degredation signals are active when histones are not needed but these are blocked during S phase when histones are needed. If this 3’UTR replaced the normal 3’UTR of actin mRNAs, the same cell-cycle stability scenario would be seen for the stability of the actin mRNAs. In other words, it would rise during S phase and fall in other phases.

4.       4.    What roles are played by the RISC complex and by the RITS complex? (8 pts)

RISC complexes are involved with both miRNAs and siRNAs. When either are seen by DICER in the cytoplasm, they are processed so that the RISC complex and Argonaute can further modify them into single stranded RNAs that can then base-pair to targeted mRNAs in the 3’UTR regions of these mRNAs. If the base pairing is extensive, the RISC will cleave the targeted mRNAs, exposing ends that increase the rate of degredation of the mRNAs. If the base-pairing is less, translation is halted and the mRNA eventually is degraded in a P-body.

RITS complexes only interact with siRNAs. Once DICER has modified the siRNA, the RITS complex and Argonaute interact with it and process it into a single stranded RNA that targets nascently growing RNA transcripts as they are being transcribed. The RITS recruits histone modifying enzymes to this region that then modify the histones in the region of the DNA being transcribed, causing it to form heterochromatin and preventing future transcription of that gene.

5.       5.    Use the two diagrams below to answer the following questions about iron concentration control in the cell.

Consult your exam for the diagram

A.      Which of the stem-loops shown above is the loop controlling the amount of ferritin a cell will make? (2 pts)

The A loop

B.      Which of the stem loops is involved in mRNA degredation signaling? (2 pts)

The B loop

C.      Which loops will be bound by aconitase if iron concentrations are high? (2 pts)

Neither loop will be bound

D.      What role in iron concentration control is played by ferritin? (3 pts)

Ferritin is an iron-storage protein that sequesters excess iron

E.       What role in iron concentration control is played by the transferin receptor? (3 pts)

The transferin receptor binds to transferin which binds to iron. The receptor then facilitates the movement of the transferin and iron across the membrane into the cell when iron is needed.

6.       6.    Outline how the concentration of the second messengers, cAMP and calcium, are increased in cells that have had a GPCR activated. (12 pts)

The GPCR that associates with the G stimulatory alpha subunit will change its shape when bound by a signaling ligand, causing the alpha subunit of the G protein to uncouple from the beta/gamma subunits and to replace the GDP with GTP. This active alpha subunit moves to the adenyl yl cyclase enzyme in the membrane and activates it to form cAMP. This increases the concentration of cAMP.

Another GPCR that associates with the Gq alpha subunit will change its shape when bound by a signaling ligand, causing the alpha subunit of the G protein to uncouple from the beta/gamma subunits and to replace the GDP with GTP.This active alpha subunit moves to phospolipase Cbeta and activates it. The phospholipase Cbeta cleaves PIP2 in the cell membrane, forming Diacylglycerol (DAG) which remains in the membrane and activates membrane-associated protein kinase C, and IP3 which is released into the cytoplasm where it binds to calcium channels on the ER membrane, opening them and allowing the release of calcium from the ER lumen into the cytoplasm, thus increasing calcium concentrations.

7.       7.    Define the following : (3 pts each)

A.      SH2 domain

Protein domain present in signaling molecules that transduce signals from activated RTK receptors by binding to the phosporylated tyrosines on the receptor and to amino acids flanking those phosphorylated tyrosines. They then will bind to other proteins in the signaling pathway.

B.       GAP

GTPase activating protein that interacts with a GTP-bound G protein and increases its ability to hydrolyze the GTP to GDP and thus helps inactivate the G protein.

C.       GEF

Guanine nucleotide exchange factor that binds to GDP bound G proteins and forces out the GDP, allowing GTP to replace it and thus activating the G protein.

D.      SH3 domain

Protein domain found in proteins that transduce signals from SH2 domain containing proteins. The SH3 domain can be in the same protein as the SH2 domain or in a separate protein that interacts with the SH2 domain containing protein. The SH3 domains are high in proline content and interact with other proline containing regions on the next protein  in the signaling cascade.

8.       8.    Answer either A or B (8 pts)

A.      Describe the signaling events that lead to the activation of the E2F transcription factor during the G1 to S phase transition.

B.      Describe how the cell prevents cell-cycle progression when DNA damage is detected.

A.             A.    A signal transduction pathway is activated and activates ras. Ras then activates the MAP Kinase cascade causing the activation of specific transcription factors that cause the transcription of the immediate early genes, such as myc. Myc. Myc protein then activates the delayed-response genes such as the cyclin that activates the cdk  in the G1 phase of the cell-cycle. This cyclin/cdk complex then phosphorylates Rb. Rb releases the E2F transcription factor which then activates the transcription of DNA synthesis related genes and also of itself and the S phase cyclin. This cyclin activates another cdk which further phosphorylates Rb and other targets that move the cell into S phase.

B.          B.    DNA damage signals the activation of the ATM/ATR kinase. It phosphorylates the Chk1/2 kinase, activating it. Chk1/2 phosphorylates p53. P53 no longer remains bound to mdm-2 which was targeting it for degredation in the ubiquitin/proteosome system. The p53 half-life increases. P53 activates the transcription of the cyclin/cdk inhibitor p21. P21 binds to the cyclin/cdk complexes preventing them from phosphorylating their targets and thus inducing cell-cycle arrest.

There are additional controls involving the chk1/2 kinases that we did not discuss in class and that you were not responsible for in this answer.

9.      9.    Outline how the activation of the caspases and the start of apoptosis during the intrinsic pathway is controlled by either pro-apoptotic proteins and/or anti-apoptotic proteins. (8 pts)

Activation of the caspases requires the formation of the apoptosome, a multimer of Apaf-1 proteins which interact through CARD domains and recruit and activate the initiator procaspases. These then activate the executioner procaspases. This apoptosome cannot form unless bound by cytochrome c. The cytochrome is sequestered in the inner mitochondrial membrane. If BH123 proteins are able to form multimers in the mitochondrial outer membrane, channels will form that allow efflux of the cytochrome  c into the cytoplasm where it can activate the formation of the apoptosome. The BH123 proteins are kept inactive by binding to bcls/x inhibitor proteins. To release this inhibition, the cell activates BH3 only proteins, allowing them to move to the mitochondrial outer membrane and bind to and inactivate the bcl2/x proteins.

10.  10.    Using what you have learned about the molecular processes in cells, identify the following proteins as either proto-oncogene proteins or tumor-suppressor proteins. (8 pts)

Bad        tumor-suppressor

Ras         proto-oncogene

P53         tumor-suppressor

Rb           tumor-suppressor

Sos         proto-oncogene

PDGF receptor  proto-oncogene

Mdm-2                 proto-oncogene

Src          proto-oncogene

11.   11.    This question will be given to you at the start of the exam. (6 pts)

Any of these mechanisms was acceptable:

Insertional mutagenesis caused by integrated non-oncogenic retrovirues and their powerful promoters and enhancers. For example, int

Transduction into oncogenic retroviruses, for example src

Reciprocal translocations of chromosomes, for example the formation of bcrabl from the 9/22 translocation or the abnormal activation of myc from the 8/14 translocation.

Gene amplification where DNA replication errors cause the formation double minutes containing multiple copies of single-copy proto-oncogenes and leading often to the formation of hsr regions representing even greater numbers of copies of these genes. Examples are myc gene amplification and mdm-2.

You were expected to provide an example of the method that you selected.

Bonus Question: (4 pts)

A  mutant fruit-fly is shown to have no photoreceptor at position 7 of the fly eye. Give one possible explanation for this using what you know about the signaling system in the fly.

A mutation that would cause the loss of normal activity in any of the following could explain this:

Boss, sev, drk, sos, ras, and all of the kinases in the map kinase cascade