Download Molecular Cell Biology course 1BL320 Spring

Molecular Cell Biology course 1BL320
Spring 2012
Exam, part I, on 2012-02-10
(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
(20 p)
Receptors & signal pathways
1. One central growth regulatory pathway activated by many different receptor types is
the Erk MAP kinase pathway.
a) Describe how the signal is propagated from the cell surface to the nucleus through the
Erk MAP kinase pathway. Include in your answer the following proteins with a
description of their function; Sos, Erk, Grb2, Raf, Mek & Ras.
(3p)
b) The cellular response to Erk activation depends on whether the activation is transient
or sustained. Describe why a sustained Erk activation is necessary to promote cell cycle
progression and why a transient burst of Erk activation fails to do so.
(2p)
c) Signaling pathways often lead to changes in gene expression. Describe two ways to
regulate the activity/function of a transcription factor.
(1p)
d) Two types of post-translational modifications found in proteins involved in signal
transduction are phosphorylation and ubiquitination. Describe these two modifications
including which amino acids that are modified and give examples of cellular functions
that use these modifications.
(4p)
2. Activation of receptor tyrosine kinases (and also other types of receptors) results in
their internalization through clathrin-coated pits. Once internalized they rapidly reach the
endosome where they are sorted towards lysosomal degradation or recycling back to the
plasma membrane. Most receptor tyrosine kinases are sorted toward lysosomal
degradation, explain how this occurs. Include in your answer the role of post-translational
modifications in this process.
(4p)
3. a) Describe the process by which receptor tyrosine kinases (RTK) are activated by
their ligand and how downstream signaling is initiated (discuss in general terms or use a
specific RTK).
(2p)
b) Phospholipase C can be activated by both RTKs and G-protein coupled receptors.
Which reaction does PLC catalyze (include in your answer both substrate(s) and
product(s))? Why is membrane localization important for PLC function?
(1p)
c) Adenylate cyclase (AC) is an important signaling protein downstream G-protein
couples receptors (GPCR). Describe the mechanism by which AC is activated by GPCR?
Which reaction does AC catalyze? And how can activation of AC provoke a change in
gene expression? (Describe the signaling pathway.)
(3p)
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Questions 4-6
Michael Pavlov
Transcription, translation, protein targeting
(15 p)
4.
Capped mRNAs are delivered to the 43S complex by eIF4F initiation factor containing
eIF4E, eIF4A and MNK1 kinase, all bound to the scaffold protein eIF4G.
(a) FMDV (Foot-and Mouth Disease Virus) invades proliferating cell. Why does eIF4G
cleavage by a protease coded by FMDV shut down the translation of cellular mRNAs?
(3p)
Answer: The cleavage separates the N- and C-terminal parts of eIF4G. The N-part has a binding site for
eIF4E that binds 5’ end of capped mRNAs. The C-part has a binding site for eIF3 and uses it to bind the
43S complex. The cleavage disconnects the mRNA binding from the 43S binding and effectively blocks the
translation of capped mRNAs. Viral mRNA uses IRES to bind to the C-part of eIF4G to enter the 43S
complex.
(b) How does the virus maintain the translation of its own mRNAs? Would a
simultaneous stimulation of translation by growth factors through the Ras/Raf signaling
pathway (ending at the MNK1 kinase) make any difference for translation of cellular
mRNAs? Explain why.
(2 p)
Answer: The virus uses IRES (Internal Ribosome Entry Site) to translate its own mRNA. FMDV
uses Lpro to cleave eIF4G. The C-terminus of eIF4G in complex with eIF4A binds the IRES with
high affinity. This results in the delivery of the viral mRNA to the 43S complex through the
interaction between the C-terminus of eIF4G and eIF3 and in the subsequent translation of the
viral mRNA. eIF4E is bound to the N-terminus of eIF4G and its phosphorylation by MNK1
would increase its binding to the mRNA cap-structure. But, since the N-terminus of eIF4G can
not bind to the 43S complex, this would not increase the initiation on capped mRNAs.
5. Initiation of transcription of highly regulated genes often requires some rearrangement
of chromatin structure.
(a) What is the role of histone acetylation by HATs? How can the acetylation of histones
in nucleosomes promote the binding of transcription factors to promoter proximal
regions?
(3p)
Answer: Acetylation of Lys residues in histones of nucleosomes removes their positive charge,
reducing the interaction of negatively charged DNA with nucleosomes. This increases the
mobility of nucleosomes that can now roll more freely alone chromatin DNA. The rolling itself is
carried out by remodeling machines of SWI type. These machines have protein domains
(Bromodomain) that bind to acetylated histones of nucleosomes. Thus, another function of
histone acetylation is to direct chromosome remodeling machines to the acetylated nucleosomes
on the chromatin. Rolling of nucleosomes along DNA makes the TF (transcription factor)
binding sites previously covered by nucleosomes available for the TF binding.
(b) What do the ISWI, SWI and SWR1 chromosome re-modeling machines do and what
is the difference between them? Why do SWI machines have bromo-domains?
(2 p)
Answer: ISWI machines organize nucleosomes by imposing a regular and proper spacing
between nucleosomes facilitating chromatin organization and condensation. SWI/SNF machines
roll nucleosomes on DNA to open the binding sites for TFs. SWR1 machines modulate histone
composition in nucleosomes. They replace H2A histone in nucleosomes with its promoter-
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boundary-specific H2A.Z variant. Bromo-domains direct SWI machines to activated hyper
acylated promoter region.
6. The translocon is a protein-conducting tunnel between the ER lumen and the
cytoplasm of the cell.
(a) Why must the ribosome attached to the translocon maintain a tight seal between the
ER and cytoplasmic compartments? How is the tight seal maintained in the ribosome
absence?
(2 p)
Answer: The ionic composition of the cytoplasm and the ER lumen is quite different. For
example, Ca2+ concentration in much higher in the lumen than in the cytoplasm. Thus,
ions should not travel freely between these two compartments. The resting translocon
maintains the permeability barrier because of the presence of a short helical plug (a
part of the translocon) which is inserted in the central pore of the resting translocon.
After the binding of the ribosome and the resumption of translation the translocon pore
opens when the nascent peptide reaches 60-70 AAs in length. This opening is
accompanied by a major conformational change in the translocon with the plug helix
moving away to the lumen of the ER, opening the protein conducting tunnel. The
ribosome forms a tight seal with the opened translocon so that the permeability barrier is
maintained.
(b) Some proteins are delivered into the ER post-translationally, through the Sec63
modified translocon. This mechanism uses BiP chaperon in the ER. Describe the
Brownian ratchet model of the protein translocation into the ER for this case.
(3 p)
Answer: Post-translational ER translocation occurs on specialized translocons that are
modified by the presence of the tetrameric Sec62/63 complex (on the luminal membrane
of the ER. BiP-ATP binds to Sec63. This binding promotes a conformational change
resulting in ATP hydrolysis and BiP-ADP binding to the luminal portion of the peptide to
be translocated. The peptide with bound BiP-ADP is caught in the ER lumen and can not
slide back into the cytoplasm. Eventually it will slide forward so that the next BiP-ADP
can be loaded by Sec63. Sequential BiP-ADP loading onto the peptide would effectively
pool it into the lumen of the ER and would simultaneously result in its folding by BiP
inside the lumen.
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Questions 7-8
Structural aspects
Lars Liljas
(15 p)
7.
G-proteins are important in many signaling pathways as well as in translation.
a) Describe the three types of G-proteins (two involved in signaling pathways and one in
translation) that were discussed during the course – their fold and their subunit and
domain composition.
(3p)
b) Describe how GDP is released in the case of these three types of G-proteins (other
components involved, mechanism as far as you know)
(3p)
c) All of these proteins have GTPase activity. How is this activity stimulated in the case
of these G-proteins (other components involved, mechanisms as far as you know)? (4p)
8.
Aminoacyl-tRNA synthetases have to recognize all tRNA molecules for a specific amino
acid.
a) Describe in general the design of the synthetases (shape, position of catalytic and
binding regions).
(1p)
b) Which part of the tRNA is in most cases used for this recognition?
(1p)
c) What type of interaction (H-bonding, hydrophobic, ionic etc.) the synthetases use
preferentially to recognize the tRNA?
(1p)
d) In the case of Ser-tRNA this type of recognition is not useful. Why, and how is such a
tRNA recognized?
(2p)
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