Chemistry 251-002: Principles of Biological Chemistry

Spring 2016 Exam 2

Professors Barry S. Cooperman and E. James Petersson

April 12, 2016: 1:30 PM to 2:50 PM

Full Name (Please print clearly): Anne Swerkey

Signature:

Your above signature certifies that all work written here is your own and that you have received
no help during the exam from anyone other than the Professor or Teaching Assistant. It also
indicates that you have used aids other than the one page approved by the instructors.

Notes:
Only writing on designated test pages will be considered toward your grade on this exam. Initial
at the top left of each page face that you wish to have graded.

Blank pages are provided for scratchwork, but will be discarded after the test period.

Make sure your writing is legible; illegible answers will not receive full credit.

Final answers that are inconsistent with the work shown, or which are obtained through faulty
reasoning will not receive full credit. Appropriate units must be given with a numerical answer.

Calculators may be used during the exam, mobile phones and PDAs may not.

A collection of potentially useful information is appended to the back page of the exam.

The exam will be graded out of 150 total points, point values are given with each question. Point
totals and final grade will be written on the back of this cover page.
Initials:

Points: 1. (12)

2. (20)

3. (20)

4. (24)

5. (18)

6. (8)

7. (24)

8. (24)

Total (150):

2
Initials:

1. Circle the best choice or choices for each prompt. (12 points total, 2 points each)

2.6 Å 20° Base tilt none

A. B form DNA Deep major
20 Å Diameter Rise per normal to of
properties groove
base pair helix axis these

B. Required
for termination Ribosomal
Release all of
of translation Recycling GTP tRNA
Factor these
and ribosome Factor
disassembly

C. Electron
Quinone or
acceptor in Ferridoxin O2 ATP H2 O
Plastoqionone
photosynthesis

D. Active when Pentose

Gluconeo- Glycogen all of
[glucose] is Glycolysis Phosphate
genesis Synthesis these
high Pathway

E. First amino
none
acid in any
Met Lys of
bacterial
these
protein

F. Can donate
all of
phosphate to
these
form NTP

Pentose phosphate okay.

3
Initials:

2. Answer the following questions regarding DNA structure. (20 points total)

A. What is the metabolic function of the enzyme uracil N-glycosylase? How is it related to the
difference in the base composition of DNA vs. RNA? (10 points)

Uracil N-glycolase eliminates U formed from C by mutation of DNA (e.g. HNO2 treatment) (5
pts). This specific reaction can be detected in DNA, because native DNA contains T, but does
not contain U (5 pts).

B. What are the protein and DNA compositions of the core nucleosome particle? Draw and/or
describe. (10 points)

Nucleosome is composed of two copies each of H2A, H2B, H3, H4 (5 pts) and two turns of
supercoiled DNA duplex (3 pts) which are connected by H1 protein (2 pts).

4
Initials:

3. Answer the following questions regarding DNA replication. (20 points total)

A. What is the role of primase in DNA replication? Why is it employed much more extensively
in lagging strand replication than in leading strand replication? (10 points)

The replication of DNA strands requires 1-60 nt RNA segments which are complementary to the
DNA template and act as the primers. These RNA primers are synthesized by primase. (5 pts)

Because lagging strand segments are only 1000-2000 nts long, multiple primers are required for
lagging strand synthesis. Only one primer is required to initiate synthesis of the leading strand.
Thus the primase is employed much more extensively in lagging strand replication. (5 pts)

B. Describe the role of Ter and Tus in the termination of DNA replication. How do the
consequences of DNA polymerase interacting with the permissive and non-permissive faces of
the Ter-Tus complex differ from one another, both structurally and functionally? (10 points)

The Tus protein is bound to Ter sequence. 2 pts When DNA polymerase (in the form of a
replisome that contains helicase activity) approaches the nonpermissive face, the Tus–Ter
complex DNA is unwound, causing cytosine at position 6 of Ter to bind to the Tus protein. This
additional binding interaction increases the affinity of Tus for Ter, leading to DNA polymerase
dissociation. 4pts

When the replisome approaches the permissive face, DNA is unwound, leading to a stepwise
decrease in the affinity of Tus for Ter and eventual dissociation of Tus from Ter, allowing the
bound polymerase to complete replication. 4pts

5
Initials:

4. Answer the following questions regarding RNA transcription and processing. (24 points total)

A. Why is DNA polymerase more accurate in replicating DNA than RNA polymerase is in
transcribing DNA? (6 points)

DNA polymerase has both polymerization and 3’-5’ exonuclease activity. The exonuclease
activity acts as a proofreading step, ensuring the high fidelity of DNA replication. RNA
polymerase lacks 3’-5’ exonuclease activity.

B. Explain how adding 1,6-allolactose to a bacterial cell culture stimulates glucose production (6
points).

1) Gene I encodes the protein lac repressor,

which binds to the lacO region of the operon
and prevents binding of RNAP to the lacP
promoter, preventing transcription. (2 pts)

2) Allolactose is an inducer, binding to lac

repressor and preventing its association
which lac O, thus permitting transcription to
occur. (2 pts)

3) Expression of the ß-galactosidase and

permease proteins encoded in the operon
leads to influx of lactose into the cell and its
hydrolysis to galactose and glucose. (2 pts)

6
Initials:

C. Describe the roles of σ (sigma) and ρ (rho) in bacterial transcription. (6 points)

σ recognizes different promoter regions and determines where initiation of transcription begins.
(2 pts)

ρ (rho): during the termination step, if the hairpins are not strong enough, ρ (rho) will assist the
termination process. (2 pts)

ρ (rho) binds at recognition sequence upstream of termination, migrates 5’ to 3’ until it encounters

stalled RNAP, unwinds RNA: DNA duplex, releases RNA. (2 pts)

D. Diagram the chemical mechanisms for lariat formation during splicing of eukaryotic RNA. In
your answer, use arrows to indicate which bonds are being formed and broken. (6 points)

Lariat splicing during formation of eukaryotic RNA.

7
Initials:

5. An unknown sequence of mRNA is translated in the presence of puromycin, an inhibitor of

protein synthesis, and the product shown below is obtained. Puromycin mimics the 3’ end of an
aminoacyl tRNA and reacts with the growing protein chain to form a product which cannot
undergo further protein elongation and inhibits the ribosome. (18 points total)

A. Write the sequence(s) of ALL mRNA that can encode the product peptide shown. (6 points)

AUG AUU UGU AUG AUU UGC

AUG AUC UGU AUG AUC UGC

AUG AUA UGU AUG AUA UGC

B. Suppose that the enzyme that converts adenine to inosine was inhibited, but that the same
puromycin product was obtained. Write the sequence(s) of mRNA that can encode the product
under these conditions. Explain your reasoning. (6 points)

AUG AUU UGU AUG AUU UGC

If adenosine is not converted to inosine, the anticodon of Ile tRNA will be AAU, so it will pair with
only the AUU codon. The wobble base pairing for Cys tRNA will not be affected.

8
Initials:

C. How does the amide link between the sugar ring and the O-methyl tyrosine group on
puromycin allow it to work as an inhibitor of protein synthesis? Hint: How is this different than a
normal aminoacid-tRNA linkage? (6 points)

The amide linkage cannot easily undergo hydrolysis or reaction with a 3’OH group on an
incoming tRNA like a normal aminoacyl tRNA ester linkage. Therefore, the protein cannot be
elongated or hydrolyzed by the release factor and the ribosome cannot be disassembled by the
ribosome recycling factor. The ribosome will be stuck with the puromycin-peptide in the active
site (peptidyl transfer center).

6. How many turns (n) of the Calvin cycle are necessary to produce one molecule of
glyceraldehyde 3-phosphate (GAP) for energy usage and what are the stoichiometries (n1 – n7)
of the reagents? (8 points)

9
Initials:

7. Answer the following questions regarding the energy production that could occur under
various conditions for a sample of purified mitochondria (where the reactants can be controlled).
A diagram of the respiratory complex is shown for reference. (24 points total)

A. Write a balanced equation for the production of ATP if the reactions are conducted with NADH,
but without succinate or O2. Write your equation in terms of one equivalent of NADH. Remember
to consider proton inventory and explain your reasoning. (10 points)

NADH + H+n + 2 Cytc(Fe3+) + 2 ADP + 2 Pi à NAD+ + 2 Cytc(Fe2+) + 2 ATP + 2 H+n

Without succinate or O2, the only e- transfer occurs between NADH and Q, then between Q and
Cytc at complex III. 4 H+ are pumped at complex I and 4 H+ (2 H+ from QH2) are pumped at
complex III. Since 4 H+ are required for the synthesis of ATP, 2 ATP are made from the 8 H+p.

10
Initials:

B. Identify the electron donor (oxidized) and acceptor (reduced) in the balanced equation in part
A. Write the balanced equation for this redox reaction. Calculate ΔG°’ for the electron transfer
reaction using the redox potentials listed in the table at the end of the exam. (8 points)

NADH + H+ + 2 Cytc(Fe3+) à NAD+ + 2 Cytc(Fe2+) + 2 H+

Cytc(Fe3+) + e- à Cytc(Fe2+) ε’°(acceptor) = 0.235 V

NAD+ + 2 H+ + 2 e- à NADH ε’°(donor) = - 0.315 V

n+ -
NADH equation flipped to be written AOx + n e à ARed with corresponding ε’° by convention.

Δε’° = ε’°(acceptor) - ε’°(donor) = 0.235 V – (- 0.315 V) = 0.550 V

If you plug numbers into this equation, you should not flip any signs. Also, you do not need to
multiply the NADH value by two. You are calculating the difference in potential per e-. The ΔG’°
calculation takes into account the number of e- transferred.

F = - 9.649x104 C•mol-1 = - 9.649x104 J•V •mol-1

ΔG’° = -nF Δε’° = -2 x 9.649x104 J•V•mol-1 x 0.550 V = - 106.1 kJ/mol (- 106 kJ/mol)

C. ΔG°’ for ATP hydrolysis is -30.5 kJ/mol. Calculate the ΔG°’ for the overall reaction written in
part A. Is ATP formation favorable? (6 points)

If ΔG°’ for ATP hydrolysis is -30.5 kJ/mol, ΔG’° for ADP + Pi à ATP is + 30.5 kJ/mol.

So using the answer from part B and adding in the energy change for synthesis of 2 ATP,
ΔG’° = - 106 kJ/mol + (2 X 30.5 kJ/mol) = - 45.0 kJ/mol.

Yes

11
Initials:

8. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of

glyceraldehyde-3-phosphate (GAP) to 1,3-bisphosphoglycerate (1,3-BPG). Answer the following
questions concerning GAPDH. (24 points total)
A. Fill in the state of Xn= NAD+ or NADH for each step. (5 points)

B. Identify three common enzymatic mechanism characteristics in the catalytic cycle of GAPDH.
(6 points)
Covalent intermediate, amino acid acts acid/base, use of cofactor (NAD+/NADH), proximity
acceleration okay

12
Initials:

C. A mutant GAPDH does not bind phosphate well. Instead, water enters this GAPDH and
causes the direct hydrolysis of the acyl thioester intermediate. Draw a scheme similar to steps
4/5. The product of this reaction shows up elsewhere in glycolysis. What is its name? (6 points)

D. Examine the glycolysis diagram included with your test packet. What is the impact of this
mutant GAPDH on energy production during glycolysis and oxidative phosphorylation? Assume
aerobic (i.e., O2 is present) conditions so that mitochondrial reactions utilizing NADH are not
affected. (7 points)
The reaction of this mutant GAPDH would not allow the formation of 1,3-bisphosphoglycerate
(1,3-BPG). Without the formation of the 1,3-BPG substrate, no ATP would be formed by 3-
phosphoglycerate kinase (PGK). The net reaction for glycolysis normally produces 2 ATP per
glucose. In the mutant cell, net production of ATP during glycolysis would be zero. Growth
could not occur under anaerobic conditions. Under aerobic conditions, however, because the
majority of ATP formation occurs from NADH via oxidative phosphorylation, the mutation would
have a minimal effect, decreasing ATP yields from 30/32 to 28/30 (quantities not necessary).

------------------------------------------------------- End of Exam ----------------------------------------------------

13
Initials:

Constants

Speed of light in vacuum c 2.998x108 m•s-1

Elementary charge e 1.602x10-19 C

Hartree energy Eh 4.359x10-18 J

Permittivity of vacuum ε0 8.854x10-12 F•m-1

Faraday constant F 9.649x104 C•mol-1

Plank’s constant h 6.626x10-34 J•s

Boltzmann constant kB 1.381x10-23 J•K-1

Gravitational constant G 6.672x10-11 N•m2•kg-2

Standard acceleration of free fall g 9.807 m•s-1

Permeability of vacuum µ0 4πx10-7 H•m-1 (Exact)

Avagadro’s number NA 6.022x1023 mol-1

Gas constant R 8.315 J•K-1•mol-1

Rydberg constant R∞ 1.097x107 m-1

Conversion Factors

Force N = kg•m•s-2 = J•m-1

Energy J = kg•m2•s-2 = N•m = 0.239 cal

Power W = kg•m2•s-3 = J•s-1

Electrical charge C = A•s = J•V-1

Electric potential V = kg•m2•s-3A-1 = J•C-1

Electric capacitance F = kg-1•m-2•s4•A2 = C•V-1

Temperature °C = K (T/°C = T/K - 273.15)

14
Initials:

Codon Table

tRNA tRNA anticodon Codon Preference

1 Ala1 G*GC GCU; GCC
2 Ala2 UGC GCA; GCG
3 Arg1 CCU AGG
4 Arg2 ICG CGU; CGC; CGA
5 Arg3 U*CU AGA
6 Arg4 CCG CGG
7 Asn GUU AAU; AAC
8 Asp GUC GAU; GAC
9 Cys GCA UGU; UGC
10 Gln UUG CAA; CAG
11 Glu1 S4UUC GAA
12 Glu2 CUC GAG
13 Gly1 GCC GGU; GGC
14 Gly2 U*CC GGA
15 Gly3 CCC GGG
16 His GUG CAU; CAC
17 Ile IAU AUU; AUC; AUA
18 Leu1 IAG CUA; CUU; CUC
19 Leu2 UAA UUA; UUG
20 Leu3 GAG CUC
21 Lys UUU AAA; AAG
22 Met CAU AUG
23 Phe GAA UUU; UUC
24 Pro1 UGG CCA; CCG
25 Pro2 IGG CCU; CCC; CCA
26 Ser1 IGA UCU; UCC; UCA
27 Ser2 GCU AGU; AGC
28 Ser3 CGA UCG
29 Thr1 IGU ACU; ACC; ACA
30 Thr2 CGU ACG
31 Trp C*CA UGG
32 Tyr GψA UAU; UAC
33 Val1 IAC GUU; GUC; GUA
34 Val2 CAC GUG
* Indicates base modification not discussed in Chem251.

15
Initials:

16
Initials:

Glycolysis

17
Initials:

18