1. EIF4 is often a target of regulation when it is desirable to influence
the rate of translation of a certain mRNA. Which activities of EIF4 explain
why it is a good target? (8 pts)
1. It serves as the binding site for the small ribosomal subunit/initiator
tRNA eIF1-GTP initiation complex to align with the mRNA to be translated.
2. It contains the helicase activity that melts stem-loops in the
5’ UTR allowing for ribosome scanning.
3. It binds to poly A binding protein to circularize the mRNA to
facilitate more efficient translation reinitiation.
2.
A. What is wobble? (4 pts)
The phenomenon whereby non-Watson-Crick basepairs can stably occur
between tRNA anticodon base 1 and mRNA codon base 3.
B. What steps in the translation process are impacted by this phenomenon
and have led to a degenerate genetic code? (8 pts)
During translation elongation the incoming tRNA, bound to eEF1alpha
first interacts with the mRNA codon at the A site by base pairing with
its anticodon. Only if the interaction is sufficiently stable can the tRNA
remain there long enough for eEFI alpha to hydrolyze GTP to GDP, releasing
the tRNA and allowing stable association of the tRNA to the A site and
the subsequent formation of a peptide bond. This “proofreading” step prevents
a tRNA that is not correct to be knocked out of the A site before it can
be erroneously used. Therefore, since wobble rules allow non-Watson Crick
stable bonds that would allow for the tRNA to remain at the A site and
be used in peptide bond formation, it is necessary that the amino acids
carried by such tRNAs carry amino acids that have degenerate codons. The
correct amino acid will then be on the tRNA and the presence of a wobble
will not cause an incorrect amino acid to be used.
3. Illustrate how the Iron-response binding protein regulates the iron
concentration in the cell when iron concentrations are low. (8 pts)
Ferritin mRNA has the stem loops in the 5’UTR. When iron is low, no ferritin is needed. So the IREBP is active and binds the IREs in the stem loops, preventing their melting during translation initiation. This stops translation.
Transferin receptor mRNA has stem loops in the 3’ UTR. When iron
is low, more transferin receptor is needed to help bring more iron into
the cell. Since the stem-loops in the 3’UTR are a signal for nucleases
to degrade the mRNA, when the IREBP is bound there under low iron conditions,
it prevents the degredation and the mRNA is available to be translated
to make more transferin receptor.
4. What role do each of the following have in the regulation of calcium concentration in the cytoplasm? (4 pts each)
A. IP3
Binds to calcium channels in the endoplasmic reticulum membrane,
causing release of calcium into the cytoplasm and migration of the channel
to the cell membrane to induce influx of calcium through another membrane
channel. The calcium causes protein kinase C to move to the membrane also.
B. diacylglycerol
Binds to protein kinase C at the membrane and activates its kinase
activity. It then phosphorylates its substrates, continuing signal transduction.
C. phospholipase C
It is the enzyme that cleaves PIP2 into IP3 and diacylglycerol.
D. protein kinase C
Activated by diacylglyerol, it phosphorylates its substrates and
continues signal transduction.
5. The ras protein is frequently found mutated in cancer cells. Describe three possible interactions or activities of ras that could be altered by such mutations. (12 pts)
1. It could cause ras to lose its inherent GTPase activity and it would always be active in transducing signals that cause cell division to occur.
2. It could cause ras to be unable to associate with a GEF, therefore preventing the necessary increase in GTPase activity needed to effectively turn ras off.
3. It could cause raf to be unable to release raf, therefore causing the Map Kinase pathway to be constantly turned on, leading to unregulated cell division.
4. It could cause ras to be unable to remain stably associated with
GDP such that GTP would constantly replace it, keeping ras active in signal
transduction in an unregulated way.
6. Progression through the cell-cycle requires the following factors. Briefly describe their role. You may use a specific example or answer generically. (4 pts each)
A. cyclin/cdk complexes
Phosphorylate target proteins that allow for events to move forward,
promoting movement through the cell-cycle.
B. ubiquitin ligases
Target proteins for ubiquitination and degredation in a proteosome.
The targeted proteins could either be an inhibitor of cell cycle progression
that when removed allow the cell cycle to move forward or perhaps be a
protein no longer needed. Another example is securin, a protein that helps
connect sister chromatids together during mitosis. When degraded, chromatids
can separate during anaphase.
C. phosphatases
Removes phosphates from target proteins that must be removed before
the protein can function in its role in cell cycle progression. An example
are proteins that are part of the replication complexes at the ori regions.
These are phosphorylated during S phase. However, the phosphates must be
removed to allow the complexes to reassemble at the oris to promote the
next round of DNA replication in a subsequent cell cycle.
7. Rb and p53 are both considered to be tumor-suppressor proteins. Discuss
this in relationship to their role in cell-cycle regulation. (8 pts)
Rb sequesters the transcription factor E2F by binding to it and keeping it inactive. E2F must be active to move the cell cycle out of G1 into S phase. When Rb is phosphorylated by cyclin/cdk complexes during G1, it releases E2F and no longer inhibits the cell cycle progression. Rb is therefore a tumor-suppressor protein because it inhibits cell cycle progression as described. If both alleles of Rb are lost or mutated, E2F cannot be inhibited and unregulated cell cycle progression occurs.
P53 is usually bound to mdm-2 and thus targeted for degredation.
This keeps its levels very low. However, in response to DNA damage signals,
p53 is phosphorylated at sites that prevent mdm-2 from binding to p53.
p53 is not degraded and is free to activate transcription of p21. p21 binds
cyclin/cdk complexes, preventing them from phosphorylating target proteins.
This arrests cell cycle progression. If both p53 alleles are lost or mutated,
p21 cannot be activated and the cell cycle keeps progressing despite the
DNA damage. P53 also plays a role in initiating apoptosis, usually when
DNA repair has failed.
8. Outline the sequence of events that would result in the suppression of apoptosis when a survival factor interacts with a receptor tyrosine kinase in the cell membrane of its target cell. (10 pts)
See Fig. 22-32 on page 929
9. Describe two ways that a proto-oncogene can become an oncogene. (8 pts)
1. insertional mutagenesis when a retrovirus inserts its powerful
enhancers near a protooncogene and activates its transcription.
2. chromosomal translocations when different chromosomes transfer
regions such that a protooncogene is either expressed aberrantly or becomes
mutated during the transfer and therefore is active in an unregulated manner.
(our example was c-myc activation by antibody producing cells)
3. genomic amplification when genes or parts of genes are copied
into many, many copies when they should be single copy genes. (Evidenced
by the appearance of double-minutes or HSRs in chromosome stains)
4. mutation during retroviral transduction when a retrovirus picks
up a cellular gene and incorporates it into its genome. (for example, c-src
becoming v-src)
5. spontaneous mutations of genes for a variety of reasons.
10. Answer the following questions about the three last debates. (2 pts each)
A. Answer True or False: There are no laws currently that restrict the use of genetic information by insurance carriers.
False
B. How many different disorders should be included in the genetic screening
of infants according to the March of Dimes?
29
C. What state recently passed a proposition calling for the establishment of forensic DNA databases for use in criminal investigations?
California