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Molecular Cell Biology course 1BL320
Spring 2015
Exam, part I, on 2015-02-15
(receptors and signal transduction;
gene expression, protein synthesis and targeting)
Total: 50 points
For passing, 25 points = 50% are required.
Please, write in legible handwriting!
Answers to the questions may be in Swedish or English.
Language dictionary is allowed (no biological literature).
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Questions 1-3
Johan Lennartsson
(18 p)
Receptors & signal pathways
1. Provide a short explanation of the catalytic activities of the following enzymes. If an
enzyme adds a post-translational modification include the target amino acid(s) in your
answer.
a) Kinase
(0.5p)
b) Phosphatase
(0.5p)
c) Phospholipase
(0.5p)
d) Caspase
(0.5p)
e) GTPase
(0.5p)
f) Ubiquitin-ligase
(0.5p)
2. Ion channels can show remarkable ion selectivity.
a) Provide an explanation for how Na+ channels show selectivity for Na+ over K+ ions.
(1.5p)
b) Provide an explanation for how K+ channels show selectivity for K+ over Na+ ions.
(1.5p)
3. Receptor tyrosine kinases (RTKs) are a group of cell surface receptor critical in
receiving and transducing signals leading to cell division.
a) In the absence of growth factor (ligand) it is important that activity of the RTK is
suppressed. Provide three mechanisms that suppress basal activity and explain how these
are relieved upon growth factor-induced RTK activation.
(3p)
b) Which post-translational modification is essential for the activation and signaling from
RTKs? In what ways does this modification affect the RTKs ability to signal?
(1 p)
c) One important signaling pathway activated many RTKs is the Erk MAP kinase
cascade. Please make a schematic drawing of this pathway, including the names,
approximate intracellular localization and enzymatic function of the protein components.
(4p)
d) In some cells only a sustained activation of Erk MAP kinase pathway leads to cell
proliferation, whereas a transient activation fails to do so. Please provide a molecular
explanation for this observation.
(4p)
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Questions 4-7
Michael Pavlov
(16 p)
Transcription, translation, protein targeting
4. Transformer protein (Tra) in Drosophila determines the alternative splicing of Dsx
genes. Male type Tra mRNA has a stop (UAG) codon in exon 2 which is followed by
other exons in this mRNA. This mRNA is transported into the cytoplasm but rapidly
degraded there. Describe the mechanism of the Nonsense Mediated Decay (NMD) that
operates on this type of mRNAs paying special attention to the roles of Upf factors.
(3 p)
Answer: When mRNA is delivered into the cytoplasm it contains Exon Junction
Complexes (EJC) positioned about 20 nucleotides before exon-exon junctions in mRNA.
They mark splicing events. The translating ribosome removes all EJCs occurring before
the stop codon. If the stop codon is in the last exon then all EJC-s are removed from the
mRNA and it can not be a substrate for NMD. However, if the stop codon is a premature
one, i.e. if it occurs not in the last exon, then after the termination the ribosome would
have non-displaced EJCs behind it. EJC contains Upf3 to which Upf2 binds in the
cytoplasm. The release factor arrives to the ribosome in complex with Upf1 protein . The
Upf1 on the ribosome forms a bridge with Upf2/3 in the EJC and this induces mRNA decapping by a de-capping factor. The 5’-decaped mRNA is then degraded by XRN1
exonuclease.
5. Splicing of exons in pre-mRNA is done by Spliceosome.
(a) How does spliceosome recognize the 5’ of introns in pre-mRNA? What is the role of
U1 particle in this process?
(1p)
(b) Describe the roles SF1/BBP and U2AF in the recognition of the 3’ end of intron and
their interplay with U2 RNP. How is the branching A selected in intron?
(4 p)
Answer:(a) Spliceosome uses its U1 particle to recognize the 5’ of the intron while its
U2 particle together with its auxiliary factors is used to recognize the 3’end. U1 particle
recognizes the 5’ through the WC complementarity between the 5’ sequence of intron
RNA and the sequence of U1 snRNA.
(b)U2 particle uses its U2 snRNA to guide U2 particle binding to the branch point of the
intron that is situated near the 3’ intron end. The 3’ end itself and the initial recognition
of the branch point sequence is, however, done by proteins. The 3’ is initially recognized
by U2AF factor while the branch point sequence is recognized by BBP protein. U2AF35
interacts with AG 3’ intron boundary directly and also with SR proteins of the following
exon.
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6. EJCs (exon junction complexes) contain several different proteins including Aly and
Upf3. What is the role of EJCs in mRNA transport into the cytoplasm? Describe the
normal transport of mRNA from the nucleus that relies on the use of Tap/p15
transporter. What is the role of Dbp5 helicase in the Tap/p15 dependent mRNA
transport? How does the Brownian ratchet work?
(3 p)
Answer: EJC is placed on mRNA during splicing, about 20 nucleotides upstream of exonexon junction. It serves as a binding site for the Tap/p15 mRNA transporter. Aly protein
is believed to bridge Tap/p15 and EJC. Tap/p15 transporter interacts with FG-(Phe-Gly)
rich hydrophobic sequences of the proteins forming the center on Nucleo-pore-complex
(NPC). This interaction is important for passive transport of charged mRNA through the
NPC. The directionality of the transport is due to the removal of Tap/p15 from mRNA on
the cytoplasmic side of NPC by Dbp5 helicase with the use of ATP energy. The removal
of Tap/p15 opens charged patches on mRNA which prevents mRNA back-sliding to the
nucleus since it can not pass back through the oily FG repeat regions of the NPC. This
mechanism is called Brownian ratchet.
7.
Capped mRNAs are delivered to the 43S complex by eIF4F initiation factor containing
eIF4E, eIF4A and MNK1 kinase, all bound to the scaffold eIF4G protein. A mutation in
MNK1 kinase leads to MNK1 inactivation. The cell acquired, however, a compensatory
mutation in a phosphotase gene resulting in the reduced levels of the phosphotase that
dephosphorylates 4E-BP proteins. Explain in details the mechanism of such a
compensation. What is the normal role of active (wild type) MNK1?
(5 p)
Answer: Translation initiation rate depends on the amount of eIF4E:eIF4G (4E:4G)
complexes and their activity. The activity of 4E:4G complexes is increased when 4E is
phosphorylated at Ser 209 by MNK1 kinase due to higher affinity of phosphorylated 4E
to cap-containing mRNAs. The amount of the 4E:4G complexes depends on the presence
of active 4E-BP (4E- Binding Proteins). 4E-BP proteins bind eIF4E in a competitive
manner effectively removing 4E from complex with 4G. 4E-BPs are inactive when
phosphorylated. The level of inactive 4E-BP would increase if the phosphotase
concentration is decreased, leading to increased amounts of 4E:4G complexes. Thus, the
lower activity of 4E:4G complexes in the absence of 4E phosphorylation is compensated
by the increased amounts of 4E:4G complexes when the phosphotase level is reduced.
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Questions 8-12
Structural aspects
Stefan Knight Maria Selmer
(16 p)
Signaling
(8) You have discovered a mutated cell line where activation of a number of metabolic
pathways in response to signaling through a GPCR remains in effect much longer than in
normal cells. Suggest a point mutation that could cause this effect, and motivate your
suggestion based on your knowledge of GPCR signaling.
(3p)
(9) Growth hormone is a small alpha helical protein. Explain how this asymmetric
molecule binds to and activates growth hormone receptor.
(3p)
(10) Epidermal growth factor, like several other growth factors, transmit signals through
a receptor tyrosine kinase pathway involving the G protein Ras. Signaling through these
pathways result in cell growth and division, and overactive Ras signaling can ultimately
lead to cancer. Everything else being the same, what effect do you think the presence of a
hyperactive Ras GAP would have on the potential for cancer development? (Motivate
your answer).
(2p)
Transcription/Translation
Transcription
(11) Metabolite levels are used to regulate transcription of the bacterial lac operon.
Outline the structural mechanism for how the levels of two metabolites affect the two
involved regulatory proteins and how these proteins regulate transcription.
(4p)
Translation
(12) (a)During the elongation cycle of protein synthesis, the tRNAs move through the
ribosome. Describe the course of events to bring an aminoacyl tRNA matching the A-site
codon to the ribosomal P site.
(3p)
(b) Present one major difference between the mitochondrial and bacterial ribosomes.
(1p)
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