MOL 214

Exam 2
April 7, 2015

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Multiple Choice, 2 points each:

1. Below is shown a double-stranded segment of DNA. The transcription start site is

indicated by the nucleotides shown in the gray box. What are the first 10 nucleotides of the
transcript that is produced from this segment of DNA?

A. 5’-CAGUACAGUC-3’
B. 5’-CUGACAUGAC-3’
C. 5’-GUCAUGUCAG-3’
D. 5’-GACUGUACUG-3’

2. Which of the following occurs ONLY in prokaryotes?

A. An mRNA can be translated by more than one ribosome at one time

B. Transcription and translation are separated in space and time
C. Translation begins before transcription is completed
D. One gene can encode more than one polypeptide

3. You are studying a protein from a eukaryotic organism and are hoping to determine its
crystal structure. To do this, you need a large quantity of protein. You generate cDNA
encoding the protein, clone it into a plasmid, and introduce the plasmid into E. coli. When
you run an SDS-PAGE gel to visualize the protein, you find, to your disappointment, that the
protein was not produced by the bacteria. When you extract RNA from the bacterial cells
and do a Northern blot, you find that there is a large quantity of mRNA transcribed from
the recombinant plasmid. What could explain your findings?

A. Your plasmid does not have a promoter

B. Your plasmid does not have a ribosome binding site
C. Your plasmid contains a repressor site upstream of your gene
D. Your plasmid contains an activator site upstream of your gene

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4. Protein X will fold into its correct conformation only when protein Y is present.
However, protein Y is capable of folding into its correct conformation without protein X.
What is the function of protein Y with respect to protein X?

A. Chaperone
B. Ligand
C. Protease
D. Repressor

5. Why were temperature-sensitive mutants used in Schekman’s protein secretion

experiments?

A. He wanted to determine how temperature affects protein folding

B. Protein secretion is always dependent on the temperature
C. A blockage in protein trafficking and secretion leads to cell death
D. He used a mutagen that only generates temperature-sensitive mutations

6. Below is a graph showing the affinity of a receptor for three ligand molecules. Based on
this graph, which of the following choices is true?

A. Ligand “A” has a lower affinity for its receptor than Ligand “B”

B. More molecules of ligand “B” than ligand “C” would be required to reach the same
level of receptor activation

C. It would be more difficult to design a drug that prevented Ligand “C” from
binding to its receptor than a drug that prevented Ligand “A” from binding to its
receptor

D. The KD of Ligand “A” is lower than the KD of Ligand “C”

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7. Which of the following is TRUE, according to the operon model for gene regulation?

A. Genes coding for proteins that participate in the same process are
coordinately regulated
B. Each gene must have its own promoter in order to be transcribed
C. Genes encoding proteins that participate in different processes are often located
in the same operon
D. Each gene must have its own 5’ cap in order to be translated

8. Which of the following mutations would likely prevent Lac Repressor from binding to
the DNA?

A. A mutation in the promoter consensus sequence

B. A 5-base-pair insertion in the middle of the operator sequence
C. The deletion of one of the auxiliary operator sequences
D. A nonsense mutation in the repressor gene that results in only the DNA-binding
domain being translated

9. It is possible to create a pool of genomic DNA from cells by digesting the DNA into
fragments with a restriction enzyme. How will the the pool of genomic DNA differ from a
pool of cDNA generated from those cells?

A. the cDNAs will contain genes with both introns and exons
B. the genomic DNA will be more stable than the cDNA
C. the genomic DNAs will contain only the genes that are being actively expressed in
the cell it is made from
D. the cDNAs generated from this cell type may differ from the cDNAs
generated from another cell type

10. Which of the following questions can be answered by a DNA microarray experiment?

A. How many tyrosine molecules are in a particular protein

B. Which of two samples has greater abundance of a specific protein
C. Which of two samples contains genomic DNA
D. The number of mRNA molecules synthesized per second.
E. The relative abundance of specific mRNAs between two samples

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Short Answer:

11. For each of the following, provide one example of how the process differs in
prokaryotes and eukaryotes: (9 pts.)

(a) Transcription Initiation (3 pts)

Possible Answers (full credit for any of those below):

- Prokaryotes have sigma factors that bind to the promoter,

eukaryotes use transcription factors.

- The structure of the promoter is different in prokaryotes and

eukaryotes.

(b) Transcription Termination (3 pts.)

In prokaryotes, a hairpin forms in the mRNA, leading to release of the

mRNA from the DNA and the polymerase. In eukaryotes, a cleavage site
at the end of the transcription unit is recognized by a specific
endonuclease and cleaved, leading to release of the mRNA. The
polymerase then falls off of the DNA.

Also accepted that eukaryotic transcripts are terminated by

polyadenylation (as on slide 49 of lecture 9).

(c) mRNA Processing (3 pts.)

Possible Answers (full credit for any of those listed below):

- In prokaryotes, mRNA is (essentially) not processed. In eukaryotes,

mRNA is processed to include a 5’ cap, a poly-A tail, and splicing
occurs.

- Prokaryotic mRNA has no introns, while splicing occurs in

eukaryotes and introns are removed.

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12.

ATG TAA
Exon 1 Exon 2 Exon 3

+1 Polyadenylation
5’ UTR 3’ UTR signal
ORF (From ATG
to TAA)
The DNA of a gene is shown above.

(a) Above, draw the longest mRNA that could be produced from this gene (1 pt. for
excluding introns, 1 pt. for including full exons 1, 2, & 3). Be sure to label the
parts of the mRNA, including the 5' untranslated region (1 pt. for correct 5’ UTR)
and the 3' untranslated region (1 pt. for correct 3’ UTR). Under the mRNA, draw a
line to indicate the protein coding region (open reading frame) (1 pt.). (5 pts.)

(b) Draw the shortest mRNA that could be produced. (1 pt.)

The shortest mRNA that could be produced would be alternatively

spliced so that it would not include Exon 2, but must include Exon 1 and
Exon 3.

No additional points taken off for also failing to exclude introns at this
step.

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13. There are 4 tRNAs for glycine. Would it be a problem for the cell if the aminoacyl tRNA
synthetase for one of the four glycine tRNAs was deleted? Why or why not? (3 pts.)

This question was omitted.

14. Explain how the results of Nirenberg and Leder’s experiment, which utilized single
codons, ribosomes, and radioactive amino acids to determine which codons coded for
which amino acids, would have been different if the genetic code did NOT possess the
characteristics listed below. Would their experiment have worked? Why or why not? (6
pts.)

(a) Degenerate

1 pt.: Yes, it would have worked.

1 pt.: If the code were not degenerate, there would almost certainly be codons
that didn’t code for any amino acids (or other statement showing
understanding of “degenerate”).

1 pt.: This would have meant that some of their codons would have resulted in
no ribosomes being retained on the filter, but they still would have been able
to correctly identify the codons for all of the various amino acids.

(b) Unambiguous

1 pt.: If the code were ambiguous, each codon could code for more than one
amino acid.

1 pt.: EITHER Yes, it would have worked, as they would have been able to
identify every codon that coded for each amino acid OR No, it would not have
worked, as they would have been unable to identify a single codon for each
amino acid.

1 pt.: If the code were ambiguous, the ribosomes would be retained on the
filter for more than one codon for each amino acid.

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15. In 1-2 sentences explain…

(a) How prokaryotic ribosomes find the AUG on the mRNA. (3 pts.)

3 pts.: Base pairing between the ribosomal RNA in the small subunit and the
ribosome binding site sequence in the mRNA positions the small ribosomal
subunit at the AUG.

Partial Credit (1 pt.) given for “recognition of upstream sequence”.

(b) How eukaryotic ribosomes find the AUG on the mRNA. (3 pts.)

2 pts.: The small ribosomal subunit is brought to the mRNA by interacting

with proteins that bind to the cap structure at the 5' end of the mRNA.

1 pt.: It then scans along the mRNA from 5' to 3' until it reaches an AUG.

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16. Clindamycin is an antibiotic that inhibits peptide bond formation. (6 pts.)

(a) Is clindamycin more likely to bind to the large or the small ribosomal subunit?
Explain your answer.

1 pt.: The large subunit

2 pts.: That is where the peptidyl transferase center is located (full credit also
given for stating that the large subunit is where the enzymatic function for
peptide bond formation is located OR that the large subunit is the site of
peptide bond formation).

(b) Is clindamycin more likely to bind to ribosomal RNA or protein? Explain your
answer.

1 pt.: RNA

2 pts.: The enzymatic activity of the ribosome is mediated by RNA, not by

protein (full credit also given for: the ribosome is an RNA enzyme/ribozyme).

17. You are a graduate student studying transport of proteins by vesicles. You bathe a
pancreas tissue slice in a solution that contains radioactive leucine for 20 minutes and then
analyze the tissue by electron microscopy. Your electron micrographs show radioactive
protein in the rough ER and the Golgi. You report to your adviser that your protein was
transported from the ER to the Golgi. Why is she skeptical about your conclusion? What
would you have to do to convince her? (4 pts.)

2 pts.: The way the experiment was done, you can't tell whether the protein
went from the ER to the Golgi, from the Golgi to the ER, or from some place
entirely different to both the ER and Golgi.

2 pts.: You would need to do a pulse chase experiment to demonstrate the

path that the protein took.

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18. Below are three mutations in components of various signaling pathways. Would a
pathway that contained each of these mutations function? Why or why not? Be specific
about which step in the pathway would be affected, if any. (12 pts.)

(a) A mutant G Protein that cannot hydrolyze GTP

1 pt.: This pathway would not function properly (it would be turned on all the
time).

3 pts.: The GTP alpha subunit would never return to its inactive state, and
would continually activate the adenylyl cyclase enzyme (or other secondary
messenger).

(b) A mutant Receptor Tyrosine Kinase that cannot form dimers

1 pt.: This pathway would not function properly (would never turn on).

3 pts.: Dimerization of the RTK molecules is required for

autophosphorylation, which is required to activate the phosphorylation
cascade.

(c) A cell in which a response element for a hormone receptor has been deleted (4
pts.)

Either “would function” or “would not function” answers were accepted, IF the
answer was correctly supported with an explanation.

The response elements are sequences in the DNA, and multiple genes may
possess (and be regulated by) response elements for a specific hormone
receptor in their upstream regions. These response elements are recognized
and bound by receptors that are activated by the binding of a hormone
molecule. Thus, the gene downstream of the deleted response element would
not be transcribed.

19. In 2-3 sentences, describe 2 ways that expression of genes in prokaryotes can be
coordinately regulated. (4 pts.)

Possible Answers:

- Operons (1 pt.)- Genes encoding proteins involved in the same process are
transcribed together on one mRNA from one promoter (1 pt.)

- Alternative Sigma Factors (1 pt.)- Different sigma factors bind to RNA

polymerase under certain conditions and recognize different promoter
sequences upstream of genes needed in those conditions (1 pt.)

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- Activators (1 pt.)- Genes encoding proteins involved in the same process can
be regulated together through operons by activator proteins (1 pt.)

- Repressors (1 pt.)- Genes encoding proteins involved in the same process

can be regulated together through operons by repressor proteins (1 pt.)

*NOTE: For full credit, answers 3 & 4 (“activators” and “repressors”) MUST
have included a description of operons

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20. You are investigating the metabolism of a novel sugar in your favorite bacterial species.
You have called this sugar Hershose after your favorite sugary treat, and you are interested
in understanding how the genes needed for Hershose utilization are regulated. Luckily,
you have a number of tools available to investigate this regulation. Below is shown the
upstream region of the DNA for the Hershose utilization operon. You have generated a
number of mutants in the upstream region in which various parts of the upstream region
have been deleted, shown below. The areas enclosed by the brackets ([ ]) represent the
region of the DNA that has been deleted.

You also have identified a compound (similar to X-Gal and called “X-Hersh”) that produces
a bright orange color when cleaved by the same enzyme that is responsible for
metabolizing Hershose. The colony colors that result from growth on plates containing the
X-Hersh compound and Hershose are shown below:

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In order to determine if any regulatory proteins bind to the upstream region of the
Hershose operon, you PCR this area from your mutant cells and perform a Gel-Shift Assay.
The results of this assay are shown below:

(a) Based on the colony color and gel shift data, where does the regulatory protein
most likely bind? Why? (3 pts.) (*NOTE: -0.5 for including incorrect information)

1 pt.: Somewhere in deletion 1 OR base pairs -10 to +1

2 pts.: Because in the deletion 1 mutant, the DNA is not shifted by

protein binding

(b) Based on the colony color and gel shift data, what type of regulatory protein is
the Hershose regulator? Why? (3 pts.)

1 pt.: Activator/Positive Regulatory Protein

2 pts.: Because when the protein does not bind, the operon is not
transcribed, and X-Hersh is not metabolized (OR the inverse
explanation)

(c) What color would the colonies be if the regulatory protein for the Hershose
utilization genes was mutated so that it was always bound to the DNA? Why? (3
pts.)

1 pt.: Orange

2 pts.: Because the Hershose regulatory protein is an activator, it

causes the operon to be transcribed when bound to the DNA

*NOTE: No additional credit taken off if part (b) answer was “repressor”
and the answer here was explained correctly for a repressor protein.

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21. What is the purpose of incorporating a fluorescent label into the cDNA pool as part of a
microarray experiment? (4 pts.)

2 pts.: To be able to differentiate between the cDNA from the 2 types of cells
you want to examine/compare 2 samples

1 pt.: Incorporating the fluorescent label allows you to determine what cDNA
has bound to the spots on the slide/chip OR the label allows you to see the
DNA

1 pt.: Thus, you can determine the relative gene expression of the 2 samples
OR see a signal that is proportional to the relative abundance of an mRNA in
the sample.

22. Briefly explain how DNA rearrangement and mRNA splicing account for antibody
diversity. (5 pts.)

3 pts.: Explanation of DNA rearrangement. By combining various segments of

the genomic DNA coding the variable region of the antibody molecule, many
different primary transcripts can be coded.

2 pts.: Furthermore, the primary transcripts can be alternatively spliced

23. Secreted proteins can be protected from degradation by proteases when they are
inside of vesicles. Propose an experiment that takes advantage of this property to show
that proteins translated on the rough ER end up in the lumen. Be sure to explain how you
will detect the protein. (6 pts.)

1 pt.: Isolate microsomes

1 pt.: Incubate with radioactive amino acids (full credit for any type of
labeling)

1 pt.: Radioactive protein will be produced (or protein produced by

ribosomes will be labeled with other type of label).

3 pts.: Treat the microsomes with protease, remove the ribosomes and show
that the vesicles are radioactive OR extract protein from the microsomes and
run on a gel (or visualize in some other way).

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