Name:
2 points
Chem 465 Biochemistry II
Hour Exam 2
Multiple choice (4 points each):
1. If electron transfer in tightly coupled mitochondria is blocked (with antimycin A)
between cytochrome b and cytochrome c1, then:
A)
all ATP synthesis will stop.
B)
ATP synthesis will continue, but the P/O ratio will drop to one.
C)
electron transfer from NADH will cease, but O2 uptake will continue.
D)
electron transfer from succinate to O2 will continue unabated.
E)
energy diverted from the cytochromes will be used to make ATP, and the
P/O ratio will rise.
2. Which of the following statements about the chemiosmotic theory is false?
A)
Electron transfer in mitochondria is accompanied by an asymmetric
release of protons on one side of the inner mitochondrial membrane.
B)
Energy is conserved as a transmembrane pH gradient.
C)
Oxidative phosphorylation cannot occur in membrane-free preparations.
D)
The effect of uncoupling reagents is a consequence of their ability to carry
protons through membranes.
E)
The membrane ATPase, which plays an important role in other
hypotheses for energy coupling, has no significant role in the
chemiosmotic theory.
3. The DNA in a eukaryotic chromosome is best described as:
A)
a single circular double-helical molecule.
B)
a single linear double-helical molecule.
C)
a single linear single-stranded molecule.
D)
multiple linear double-helical molecules.
E)
multiple linear single-stranded molecules.
4. Histones are _______ that are usually associated with _________.
A)
acidic proteins; DNA
B)
acidic proteins; RNA
C)
basic proteins; DNA
D)
basic proteins; RNA
E)
coenzymes derived from histidine; enzymes
5. Which one of the following statements about enzymes that interact with DNA is true?
A)
E. coli DNA polymerase I is unusual in that it possesses only a 5' 6 3'
exonucleolytic activity.
B)
Endonucleases degrade circular but not linear DNA molecules.
C)
Exonucleases degrade DNA at a free end.
D)
Many DNA polymerases have a proofreading 5' 6 3' exonuclease.
E)
Primases synthesize a short stretch of DNA to prime further synthesis.
6. The function of the eukaryotic DNA replication factor PCNA (proliferating cell nuclear
antigen) is similar to that of the â-subunit of bacterial DNA polymerase III in that it:
A)
facilitates replication of telomeres.
B)
forms a circular sliding clamp to increase the processivity of replication.
C)
has a 3' 6 5' proofreading activity.
D)
increases the speed but not the processivity of the replication complex.
E)
participates in DNA repair.
7. In homologous genetic recombination, RecA protein is involved in:
A)
formation of Holliday intermediates and branch migration.
B)
introduction of negative supercoils into the recombination products.
C)
nicking the two duplex DNA molecules to initiate the reaction.
D)
pairing a DNA strand from one duplex DNA molecule with sequences in
another duplex, regardless of complementarity.
E)
resolution of the Holliday intermediate.
Essay questions - Answer any 5.
1. One of the new theories that was just introduced in this edition of the text has to do
with respirasomes. What is a respirasome and what is the evidence is used to support
the existence of resipirasome?
A respirasome is the assembly of all 4 the electron transport enzyme complexs into a
single membrane bound ‘super’-complex so the individual reactions can be tightly
coupled to each other. Evidence for this theory is that Complex I and III can be isolated
together, and complex III and IV can be visualized as a single unit in the Electron
Microscope. Further, enzyme kinetics shows that electrons are transferred through a
tightly linked solid state. There is also evidence that cardiolipin, a lipid that is especially
abundant in the inner mitochondrial membrane, is essential for the respirasome
structure.
2. Describe the structure of ATP synthase and tell how this enzyme works.
A figure like 19-25 is a good place to start. With that diagram you can cut out
most of the following description of the structure!
ATP synthase consists of two major units; Fo an integral membrane protein and
F1 a peripheral membrane associated with Fo. Fo consists of 3 subunits, ab2 and 10-12
copies of c. C is small, 8000 AA very hydrophobic, and has 2 membrane spanning
helixes and a loop on the matrix side. A and b are to the side of this complex and form
part of an anchor to F1. F1 consists of 3 á units, 3â units, and ãä and å units. The á and
â units form a hexameric ring structure and perform the actual ATP synthesis. The ã
unit runs through the middle of this hexamer and is attached to the Fo complex using
the å subunit. Finally ä is attached to the 2 b units that extend inward from the side of
Fo.
The á and â subunits of F1 have three different conformations, I which binds
ATP, one that binds ADP and Pi, and a third that does not bind either ATP or ADP. All
three of these conformations of á and â exist in the F1 complex, and the conformation
are change as the ã subunit shaft rotates in the center of the complex.
Fo is the transporter that allows protons to cross the membrane. As three
protons cross the membrane they act to physically rotate the ã subunit shaft 120o. As
this shaft rotates a áâ dimer in F1 changes from a form that first binds ADP and Pi, to a
form that binds ATP, making the ADP and Pi combine to form ATP. As another three
proton move through Fo The shaft rotates another 120o and the áâ dimer goes to a
conformation that does not bind ATP so it is released from the complex. Finally another
three protons cross through Fo, and the áâ dimer returns to the form that binds ADP
and Pi again. Since this is occurring at all three sites with each rotation, the net result is
that 3 protons are required to make 1 ATP.
3. Describe the hierarchic structure of Eukaryotic chromosomal material in as much
detail as possible and compare this to the structure of prokaryotic chromosomes.
A figure like 24-33 agains get you past a lot of verbal explanation. The first level of
structure in the Eukaryotic Chromosome comes with wrapping DNA twice around a
histone that consists of 2 copies each of H2A,H2B, H3 and H4 to form a nucleosome.
H1 then links the histones together and they can from together in a helical structure
called a 30nm fiber. These fibers then supercoil around a nuclear scaffold for form
rosettes. The rosettes sold together to form a coil, and the coil becomes part of the
shromosome that can be visible during metosis.
The structure of the prokaryotic chromosome is much less complex. There are DNA
binding proteins that have structures similar to histones, but they never form a stable
nucleosome. Different experiments can be done to show that the DNA is localized to
one particular membrane bound part of the cell, but no hierarchic structure beyond that
is observed.
4. SMC proteins are found in both eukaryotes and prokaryotes. What is their structure
what functions do they perform in Eukayotic cells?
SMC stands for Structural Maintenance of Chromosome. These are additional proteins
used to keep the chromosome together. SMC proteins all have the same basic
structure (shown in figure 24-34), a N terminal domain, a coil domain, a hinge domain,
another coil and finally a C domain. The hinge region folds the protein together so the
two coil domains form a coiled-coil around each other to bring the N and C domains
together for form an ATPase region. The hinge of one SMC then binds to the hinge of
a second SMA to form a V-shaped dimer.
There are two main types of SMC proteins in Eukaryots. Cohesins that link
together sister chromatids as chromosomes condense in metaphase. And Condensins
that Condense DNA as the cell enters mitosis.
5. What is an AAA+ ATPase, and what does it typically do? Several AAA+ ATPases
were mentioned in the chapter on DNA replication. Name these enzymes and tell what
role they played in DNA replication.
AAA+ stands for “ATPase associated with diverse cellular activities”. Typically AAA+
proteins form oligomers that slowly hydrolyze ATP. This ATP hydrolysis switches the
protein between two states, active and inactive so this is used to time various cellular
processes. The first AAA+ protein we saw was DnaA, the protein that first binds to the
oriC sequence to open DNA up and start the replication process. DnaA will only bind to
DNA in when it is in the ATP form once the ATP is hydrolyzed it will stop binding to
DNA so replication won’t begin. The next AAA+ protein we saw was DnaC where the
ATP hydrolysis controlled the timing of loading DnaB helicase onto DNA. Hda was
another AAA+ protein that was involved in making DnaA fall off the DNA after
polymerase III was fulled assembled. The clamp loading par of polymerase III is also a
AAA+ protein. The Eukaryotic MCM2-7 protein complex (Eukaryotic equivalent of
DnaC) is also an AAA+ protein.
6. Below is a table that lists several different key features and enzyme activities that are needed
to replicate DNA. Fill in the table with the appropriate name for each enzyme activity or feature
from prokaryotes and eukaryotes. To make this process easier, here is a list of the names you
need to fill in the table: ARS (autonomously replicating sequence), clamp loading (ã) complex.
(Note: XXX indicates an activity in Eukaryotes where I could not find a name)
Activity
Name in Prokaryotes
Name in Eukaryotes
oriC
ARS (autonomously
replicating sequence)
Sequence on DNA where
replication begins
Uses ATP energy to unwind
DNA
DnaB helicase
MCM 2-7 (minichromosomal
maintenance proteins 2-7)
Relieves torsional stress
when DNA is unwound
DNA topoisomerase II
Binds to single stranded
DNA
SSB (single strand binding)
protein
RPA(replication protein A)
Loads unwinding enzyme
onto the DNA
DnaA
ORC (origin recognition
complex) protein complex
Makes RNA primer
Primase
DNA polymerase á
Adds long pieces of DNA to
an RNA primer
DNA polymerase III
DNA polymerase ä
Keeps polymerase bound to
DNA
â-clamp
PCNA
Loads the ‘clamp’
clamp loading (ã) complex
RFC(replication factor C)
complex
Removes the RNA primer
and replaces with DNA
DNA polymerase I
DNA polymerase å
Seals nick in DNA after the
RNA primer is removed
ligase
XXX
Marks DNA to identify old
and new strands
DAM methylase
XXX
XXX