MOL 214

Exam 2
December 3, 2015

Your exam code number is:

Write this number on each
page of your exam.

DO NOT write everything you know about a topic, this will waste your time. If you provide
more than one answer for a question only your first answer will be graded.

This exam is 13 pages, please check that you have all pages in your exam. There is a codon
table on the last page of the exam.

If you need extra space, continue only on the back of the page that the question is written
on. Clearly label that you are using the back for your answer.

Remember to write legibly, if we can’t read it, we can’t grade it!

I pledge my honor that I have not violated the honor code

during this examination.

Signature: __________________________________

Printed Name: _______________________________

 Exam Code Number: ____________________

Multiple Choice Questions (2 points each)

1. tRNAs are linked to their corresponding amino acid via which of the following bonds?
a. Hydrogen bond
b. Ionic bond
c. Covalent bond
d. Phosphodiester bond

2. CpG islands are often associated with:

a. Regulatory regions such as promoters
b. Termination sequences
c. The coding region of genes
d. CpG islands are evenly distributed throughout the genome

3. The addition of ubiquitin, a small polypeptide, is a type of covalent modification that can
affect protein function. Ubiquitination often results in ______________.
a. Membrane association
b. Protein degradation
c. Protein secretion
d. Nuclear translocation

4. How is initiator tRNA unique among tRNAs?

a. It is the only tRNA that never sits in the A site of the ribosome.
b. It is the only tRNA that never sits the P site of the ribosome.
c. It is the only tRNA with arginine.
d. It is the only tRNA without an amino acid attached.
e. It is the only tRNA with serine attached.

5. During this (or these) process(es) chromosomes separate to form two nuclei that are
genetically identical.
a. Mitosis
b. Meiosis I
c. Meiosis II
d. Mitosis and meiosis I
e. Mitosis and meiosis II

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6. How are most eukaryotic transcription regulators able to affect transcription when their
binding sites are far from the promoter?
a. by binding to their binding site and sliding to the site of RNA polymerase assembly
b. by looping out the intervening DNA between their binding site and the promoter
c. by unwinding the DNA between their binding site and the promoter
d. by attracting RNA polymerase and modifying it before it can bind to the promoter

7. A poison added to an in vitro translation mixture containing mRNA molecules with the
sequence 5′-AUGAAAAAAAAAAAAUAA-3′ has the following effect: the only product
made is a Met-Lys dipeptide that remains attached to the ribosome. What is the most likely
way in which the poison acts to inhibit protein synthesis?
a. It inhibits peptidyl transferase activity.
b. It inhibits translocation of the ribosome to the next codon.
c. It inhibits release factor.
d. It mimics release factor.

8. A protein is synthesized from its _______ as the ribosome moves toward the ______ end of
the mRNA.
a. N to C terminus; 5’
b. C to N terminus; 5’
c. N to C terminus; 3’
d. C to N terminus; 3’

9. How do cells of mammalian immune systems generate antibody diversity?

a. Post-translational modifications
b. Incorporation of viral DNA into the mammalian genome
c. Homologous recombination during meiosis
d. Random excision of and ligation of V, J, and D regions

10. Based on your knowledge of the chemical properties of RNA and amino acids, which amino
acids would be most likely found in the RNA binding site on a protein?
a. Lysine, phenylalanine, and glycine
b. Cysteine, alanine, and methionine,
c. Lysine, arginine, and serine
d. Arginine, valine, and isoleucine

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11. The parent cell shown below-left undergoes meiosis. In the box are three different gametes,
numbered1 through 3.

Which of these gametes could have come from this parent cell?
a. gamete 1
b. gamete 2
c. gamete 3
d. All (1, 2, 3)

Short Answer Questions

12. Leucine has 6 codons: UUA, UUG, CUU, CUC, CUA, CUG. What is the minimal number of
tRNAs needed to decode Leu? Briefly explain your answer. (3 points)
3 (1 point) Because of the wobble position in the anticodon one tRNA can be used for
multiple codons (2 points)
For reference:
Codons UUA and UUG base pair with anticodon 3’-AAU-5’
CUU, CUC, and CUA bp with anticodon 3’-GAI-5’
CUG pairs with 3’-GAC-5’

13. Why is meiosis I known as a reduction division and meiosis II is known as a separation
division?(4 points)

During meiosis I chromosomes separate, resulting in a reduction of the chromosome

number from diploid (cell has 2 versions of each chromosome, one maternal and one
paternal chromosome) to haploid (cell has only one version of each chromosome, either
maternally-derived or paternally-derived chromosome).

During meiosis II sister chromatids of a replicated chromosome separate, thus it is a

separation division (closely resembling mitosis).

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14. You have performed a microarray on the tumors of a number of patients who have been
diagnosed with leukemia. Your graduate advisor asks you to “cluster” the results of the
microarray, which are shown below.

What does your advisor want you to do, and how would the patients be located relative to
one another after this was performed? (3 points)
Group patients that have similar gene expression patterns together (1 point)
This could also include grouping genes that have similar expression patterns together.
Patients 1 and 3 should be next to each other (1 point).
Patients 2 and 5 should be next because they are the next closest (1 point)

Your advisor wants to know how much of each gene is being transcribed in Patient 5 in
mcg/L. Can you use the data you have already collected to determine this? Why or why
not? (2 points)

No, because microarray results only help you to determine relative amounts.

15. What is the difference between the way bacteria and eukaryotes recognize the start codon for
translation? (4 points)

Eukaryotes: the small unit will scan starting from the 5’ cap until it finds AUG and start
translation there.
Bacteria: mRNA has no cap to help recruit the small subunit; instead the small subunit of
the ribosome binds to ribosomal binding sites (or Shine-Delgarno sequences).

What is the functional significance of this difference? (2 points)

Bacterial can be polycistronic, so multiple proteins can be translated from the same
mRNA as long as there are multiple RBS.

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16. The diagram below shows a gene and three different splice variants from mice

DNA E1 E2 E3 E4 E5

Splice variant 1 E1 E3 E5

Splice variant 2 E1 E4 E5

E1 E2 E4 E5
Splice variant 3

A microarray is designed with separate spots for exon 2, exon 3, and exon 4. Then,
mRNA from newborn mice is used to make green probes, and mRNA from post-
pubescent is used to make red probes. When red and green probes are present at equal
concentrations, yellow fluorescence is produced. The probes are mixed together and
hybridized to the microarray, giving the results below.

Spot Color of Fluoresce

Exon 2 Red
Exon 3 Green
Exon 4 Yellow

Based on the data presented, indicate how expression of each splice variant changes as
mice develop. (3 points)

Splice variant 1: Decreases or is expressed more in newborn mice

Splice variant 2: Decreases or is expressed more in newborn mice

Splice variant 3: Increases or is expressed more in post-pubescent mice

Name two other techniques could you perform to determine if splice variants are present?
(4 points)
Note: only ONE technique is required!
Northern Blot or
RT-PCR with primers to splice variants or
Western blot with antibodies to specific splice variants or
Sequencing of cDNA made from mRNA or
Hybridizing mRNA with original DNA and examine intron loops
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17. Depicted below is a gene and associated regulatory region. Examine the image below and
predict the level of RNA transcription you would expect from each construct: (4 points)

highly transcribed (+ +), basal transcription (+) or no transcription (-)

18. You are working in your senior thesis lab studying the function of your gene of interest, yfg,
with the sequence shown below. You believe the methylation state of the cytosines in yfg is
important for function. To determine the methylation state of all the cytosines of the
sequence you treat DNA harvested from the cell with sodium bisulfite.

What will the sequence of yfg be after treatment with sodium bisulfite? (2 points)

5’-ATGUGCGTATGCGUATCGATTCGATTUGAGUUCGTAA-3’

-1 for Us in the wrong position.

What will the sequence of the PCR product of yfg be after treatment with sodium
bisulfite? (2 points)

5’-ATGTGCGTATGCGTATCGATTCGATTTGAGTTCGTAA-3’

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19. Below is the sequence from the 3′ end of an mRNA.

5′-CCGUUACCAGGCCUCAUUAUUGGUAACGGAAAAAAAAAAAAAA-3′

This sequence contains the stop codon for the protein encoded by this mRNA. What is
the anticodon on the tRNA in the P-site of the ribosome when the release factor binds to
the A-site? Be sure to indicate 5’ and 3’. (2 points)

The stop codon is UAA. The codon preceding the stop codon is UGG and will be bound
to a tRNA in the P-site of the ribosome when release factor binds to the A-site. The
anticodon of the tRNA will bind to the codon UGG and will be 5’-CCA-3’.

20. Give two reasons why chemicals that inhibit GTP hydrolysis prevent translation. Indicate the
step in the translation process that is affected. (4 points)

GTP hydrolysis is required: (any two of the following)

1) Binding of GTP to initiation factors induces conformation that can bind to small subunit of
ribosome so it in turn can bind mRNA.
2) initiation factors that bring the charged initiator tRNA to the small ribosomal subunit.
3) initiation factors that bring the large ribosomal subunit to join the small subunit
4) to release the tRNA into the A site to allow peptide bond during elongation.
5) during elongation for translocation of the mRNA to move the next codon into the A site.
6) in termination and breaking of the amide bond to free the nascent polypeptide.

21. Pictured below is the domain organization of a fragment of an unfolded transmembrane

protein. The boxed H-domains are composed of hydrophobic amino acids. If you know that
the N- terminus is found on the outside of the cell, draw the expected structure of the
fragment of this transmembrane protein as it would appear in a membrane. (3 points)

outside

H H H

cytoplasm

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22. What type of chemical interaction is required for the formation of α helices and β sheets?
(2 points)

Hydrogen bonds

Given this common interaction underlies the formation of α helices and β sheets, what
differentiates between α helices and β sheets? (3 points)

α helices form when Hydrogen bonds form locally (or within the same strand),
β sheets form when hydrogen bonding occurs between different regions of the protein,
resulting in strands of protein that lie adjacent to one another, interacting laterally via
Hydrogen bonds.

23. You are interested in studying the chaperone protein SurA in E. coli. You introduce two
different mutations to the coding sequence; one mutates aspartic acid critical for function to
arginine and the other mutates the same aspartic acid to serine. Which mutation would you
predict would have a more deleterious effect on the function of SurA? Briefly explain your
answer. (3 points)

You would expect the Aspartic acid to arginine mutation to have the stronger impact on
function since you are changing a negatively charge amino acid to a positive charge. In
contrast the change from aspartic acid to serine is a change from a negative to an uncharged
polar amino acid should have less severe phenotypes.

24. Describe and briefly explain the role of “strand invasion”. (4 points)

Strand invasion is important for homologous recombination via crossing over in meiosis. Strand
invasion is when a broken strand of DNA invades the complementary region of a homologous
region of DNA (the sister chromatid).

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25. You are a graduate student studying the expression of Gene X that has been implicated in
human cancers. As part of your experiment you treat normal and cancer cells with Drug Y.
After the treatment is complete, you analyze Gene X mature mRNA levels by northern blot
and its protein expression by western blot. The results are shown below:

Considering that you probed the blot for the mature mRNA of Gene X, what is the most
likely explanation of the bands marked ‘A’ and ‘B’? (2 points)

A and B are different splice forms of Gene X

What could explain the increase in Gene X’s protein levels, marked ‘C’? (2 points)
The increase in Gene X’s protein levels could be caused by increased translation or
increased protein half-life/stability or decreased proteolysis/degredation.

Transcription is incorrect because the mRNA levels are the same by northern blot

26. Predict the location within the cell the following proteins would be targeted to based upon
the characteristics described below. Assume that the only targeting sequences possessed by
each protein are those listed. Briefly explain your answer (6 points)

a. A protein with an internal sequence highly enriched for positively charged residues.

This is similar to the NLS, likely destination the nucleus

b. A protein with a non-polar cleavable N-terminal sequence.

This is like the signal sequence, likely final destination is the ER, Golgi, or it will be
secreted

c. A protein with a non-polar cleavable N-terminal sequence and a stop transfer sequence.

This is like the signal sequence, but with the addition of the stop transfer sequence.
Likely final destination is the membrane of the ER (or Golgi or plasma membrane).

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27. You are studying a 20 kDA protein and suspect that it self-associates. To investigate further
you make cell extracts and conduct size exclusion chromatography (also known as gel
filtration) in two different conditions. Afterwards you perform western blot analysis on the
chromatography fractions. The results and positions of size standards are shown below:

Explain the behavior of your protein on the size exclusion column. Why does its
behavior change in condition 1 vs. condition 2? (2 points)

Protein is 80 kDa /in a complex/self-association in condition 1. In condition 2 these interactions are

disrupted/protein is denatured and so the protein behaves as a smaller unit or 20kDa protein.

If it is clear that the student understands that the protein is somehow associating with something
such that condition 1 has the protein in a complex (or polymer), then full credit was given.

Partial credit given for an answer indicating that the conditions affect the protein size or for
acknowledging difference in size but giving no indication as to which condition results in a larger
sized protein.

Assume that your hypothesis of self-association is correct and that your protein does not
interact with proteins other than itself. How many copies of your protein are self-
associating? (1 point)

4 (the monomer is 20 kDa and the complex migrates at 80 kDa. 80/20 = 4)

Assume that your hypothesis is incorrect. What technique could you use to identify other
proteins with which your protein of interest interacts? (2 points)

Acceptable answers are Co-IP or affinity chromatography.

Partial credit: protein-protein interaction assay (not specific enough for full credit). Other
chromatographies.

Western blot is not acceptable because 1) it requires ahead of time that you know what the
interacting proteins are and that you have antibodies against them. Moreover, western blotting on
the size exclusion column will only inform you of which cellular proteins behave similarly on the size
exclusion column, not which protein are specifically interacting with your 20 kDa protein.

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28. What would be the likely consequence of mutating Ran so that it can no longer interact with
importin? (2 points)

Importin would not release the cargo protein (1 point) and would not be recycled back
out to the cytoplasm (1 point).

29. The eukaryotic cell cycle is tightly regulated by many proteins to maintain the proper control
of cell growth and division. Experiments using a cell free system in Xenopus oocytes,
partially purified an activity that is crucial in determining whether cells undergo mitosis.
What were the two proteins in this activity? (2 points)

Cyclin and Cdk

The graph below depicts the changes in protein level over time of the two proteins
responsible for the activity that is crucial in determining if cells undergo mitosis. On the
graph below label the name of the protein that corresponds with the correct curve. (2 points)

Protein 1= CDK
Protein 2= cyclin

Name a protein that can regulate the activity of these two proteins and explain how this
regulation affects progression through the cell cycle. (3 points)
(1.5pt for protein name; 1.5pts for affect on cell cycle)
Wee 1 kinase, adds an inhibitory phosphate onto CDK, which leads to blocks the cell
cycle progression
Or Cak kinase adds an activating phosphate onto CDK that can contribute to cell cycle
progression once Cdc25 removes the inhibitory phosphate.
Or Cdc25 phosphatase, removes the inhibitory phosphate on CDK which allows the CDK
to be active and the cell cycle to progress.
Or Ubiquitin ligase adds ubiquitin groups to cyclin to target for degredation which halts
the cell cycle.

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