Download Molecular Machines (1MB429) Exam 2011-12-21

Molecular Machines (1MB429) Exam 2011-12-21
Please, write in eligible handwriting!
Answers to the questions may be in Swedish or English.
1.
RNA polymerase is the major machine in the cell for transcription of genes.
(a) What are the roles of the bridge helix and the trigger loop in the mechanism of RNA
polymerase?
(3 p)
(b) How is the accuracy of transcription achieved in RNAP?
(3 p)
2.
Transcription factors are very important molecules involved in the regulation of
transcription.
(a) What is the basis of most sequence specific interactions between transcription factors
and base pairs in the major groove?
(3 p)
(b) Why do most DNA binding domains bind as dimers or as multiple units of similar
domains (as in some Zn-fingers)?
(3p)
3.
Poly-A tail of mRNA is important for its efficient translation into protein.
(a) How does poly-A tail participate in translation? What is the role of the PABP (PolyA Binding Protein) and initiation factor eIF4F in translation initiation? (eIF4F initiation
factor contains eIF4E, eIF4A and MNK1 kinase, all bound to the scaffold protein
eIF4G). How is mRNA:eIF4F complex delivered to the 40S ribosomal subunit (or, more
precisely to the 43S initiation complex)?
(6 p)
Answer: Initiation factor eIF4F binds to the Cap structure on the 5’ end of mRNA. The
4G subunit of 4F complex binds also to PABP proteins that interact with the 3’ poly-A
sequence of mRNA. This leads to the formation of a closed mRNA loop and to a more
efficient translation of mRNA. In addition, PABP binding to 4G protein strengthens the
4E:4G complex, which increase the 4F binding to the Cap. 4G has a binding site for eIF3
that is bound to the 40S subunit. This interaction between 4G and eIF3 brings the mRNA
to the 40S subunit.
(b) Oocytes contain large amounts of un-translated mRNA in complex with Maskin.
Translation of these mRNAs can be activated by hormones and other signals. Explain the
mechanism of translation inhibition by Maskin and how this inhibition is relieved in
response to a hormonal signal. What are the functions of the two important U- and Arich sequences (UUUUAU and AAUAAA) that are normally present close to the 3’ end
of such mRNAs?
(6 p)
Answer: CPE is a U-rich Cytoplasmic Poly-adenylation Element with an UUUUAU
consensus sequence to which CPEB (CPE- binding protein) binds. Maskin binds to
CPEB protein and to eIF4E initiation factor preventing mRNA recruitment to the 43S
1
initiation complex by blocking the eIF4E -eIF4G interaction. Hormone (progesterone)
stimulation of oocytes results in the activation of Eg2 Ser/Thr kinase and
phosphorylation of CPEB which increases CPEB affinity to CPSF. Cleavage-polyadenylation specificity factor (CPSF) (the same as in the nucleus) binds then to a polyadenylation signal AAUAAA and recruits cytoplasmic poly(A) polymerase which adds
poly A-tail to the 3’ of the mRNA to which PABPI binds. PABPI interacts with eIF4G
increasing its affinity to eIF4E. In addition eIF4G is placed in the vicinity of Maskin and
eIF4E which allows eIF4G to replace Maskin to become a eIF4E partner. When
initiation factor eIF4G binds to eIF4E, mRNA can be delivered into the 43S complex.
4.
Actin polymerization in the cell involved in many cell functions, like movement and the
building of the cell cytoskeleton.
(a) Why is the breaking off of F-actin stretches by Cofilin from the (-) end important for
treadmilling in vivo? What are the roles of Profiline and CapZ in actin polymerization in
vivo?
(6p)
Answer: Cofilin causes filaments containing actin-ADP to break into short filaments
increasing the rate of actin disassembly from the (-) end.
Profilin is an AEF (ADP to ATP exchange factor) that accelerates the conversion of G
actin from ADP to ATP form. It also blocks Actin –ATP binding to the (-) end. CapZ
blocks microfilament growth at the (+) end by binding to it with high affinity and
prevents filament disassembly from the (+) end too.
(b) Arp2/3 complex (Arp=Actin related protein) nucleates the branching of actin
filaments. What is the mechanism of this nucleation? What role does the activator domain
of WASP protein play in this process? How does Cdc42 GTPase activate WASP? (6p)
Answer: The Arp2/3 complex contains, apart from other subunits, the two actin-related
proteins: Arp2 and Arp3. In the presence of a WASP activation domain the
conformational change in Arp2/3 places the Arp-proteins so that they mimic the (+) end
of the filament. This initiates the growth of the filament. In addition, the Arp2/3 complex
must be bound to the surface of another filament to initiate the assembly of the next
filament. WASP is activated by Cdc42-GTP which is a small Ras –related GTPase on
the cell surface. WASP exists in a folded inactive conformation in which the activation
domain is hidden. Upon binding to Cdc42, WASP changes its conformation exposing the
activation domain to interact with Arp2/3.
5.
(a) What is a molten globule intermediate? Using energy diagram explain why it is a
stable intermediate?
(4p)
Answer: A molten globule represents an intermediate state in protein folding, which is
relatively stable, has a native like secondary structure but lacks native tertiary
structure and has some hydrophobic residues still exposed to solution.
The molten globule intermediates form local minima in the energy funnel. Its overall
energy being intermediate between the folded (global minima) and the unfolded (top of
the funnel) state. Thus, unlike transient intermediates of protein folding molten globule
intermediates show higher stability.
2
(b) Describe how trigger factors work. What is the role of the PPi (peptidyl-prolylisomerase) domain found in the trigger factor?
(4p)
Answer: The trigger factor associates with the ribosomal large subunit close to the exit
tunnel. The trigger factors work as a molecular cradle which interacts first with the
nascent protein chain. Upon interaction with the nascent chain the trigger factor changes
its conformation and leads the protein to the folding pathway. With the growth of the
nascent chain, the trigger factor leave the association with the ribosome and detaches.
In the next instance another trigger factor binds to the ribosomal exit tunnel and provides
a platform to the next region/domain of the nascent protein to fold. PPiase
(peptidylprolylisomerase) function is to catalyze (accelerate) the cys-trans isomerization
of peptide bonds involving Pro residues.
(c) What are prions? In which way prion pathogen differs from the microbes? Discuss
the similarity and dissimilarity of prion diseases with other neurodegenerative diseases of
mammals.
(4p)
Answer: Prions are infectious protein particles that can cause neurodegenerative
diseases in higher eukaryotes. Usually prion proteins form amyloid fiber in the brain of
the infected animals, which stop neural cell to cell communication and causes the cell
death. These dead cells are removed by the astrocyte action and as a result, several holes
appear in the brain giving it a sponge like appearance. Which factors change the normal
prion proteins to the infection prion fibers are not known.
The prion pathogen is the prion protein without any nucleic acid component. It is unlike
bacteria or virus in that context and it is also not alive.
The prion disease causes amyloid fiber formation in the brain like in other
neurodegenerative disease such as Alzheimer’s or Parkinson’s disease. Unlike those
diseases prion fibers are infectious and can be transmitted to other hosts by many means
including food intake.
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?
(6 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.
3
(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.
(6 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.
4