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INTRODUCTORY BIOCHEMISTRY
BIOL0280
Third Midterm Examination
April 29, 2014
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your exam after grading it. Please work independently. Read each question carefully before answering.
Unless otherwise indicated, there is only one correct answer for each multiple-choice question. For
multiple choice or matching questions with more than one correct answer, points will be deducted for
missing or incorrect responses. Points are indicated by the question within brackets []. There are no
calculators or other electronic devices needed or allowed on this exam. All hats must be removed
during the exam. Exams will be scanned electronically before being returned.
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1. [2 points] Indicate which statement is true of tetrapyrrole biosynthesis in both animals and plants:
A) δ-aminolevulinate is a tetrapyrrole precursor.
B) Carbon atoms of tetrapyrroles are derived from glycine and succinyl-CoA
C) Carbon atoms of tetrapyrroles are derived from glutamate.
D) A total of 4 δ-aminolevulinate molecules are used to synthesize 1 heme molecule.
Circle the correct answer.
2. [4 points] Draw the structure of adenosine in the space provided below. Your answer must be
completely correct to receive credit. H
4 points for completely correct
3. [4 points] Describe the regulation of ribonucleotide reductase at the primary regulatory sites. Please
include in your description all the molecules that regulate these sites and for each regulator, the
rationale behind the regulation and whether the regulation is positive or negative.
The primary regulatory sites that effect the overall activity of the enzyme and NOT substrate
specificity(-2 if answer describes primary regulation as effecting specificity), are positively
regulated by ATP (1 point) which is an indicator of energy charge (1 point) and negatively by
dATP (1 point) which is a feedback regulator (1 point).
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4. [2 points] Which of the following compounds directly provide atoms to form the purine ring?
A) Aspartate
B) Carbamoyl phosphate
C) Glutamine
D) Histidine
E) Tryptophan
F) Lysine
G) Glycine
H) Ornithine
Circle all correct answer(s) More than one answer may be correct.2 points for all correct, 0.5 point
off for any incorrect or 0.5 point off for any missing answer. 0 points for circling all answers.
5. [4 points] Draw the structure of guanine and cytosine correctly indicating the hydrogen bonding
between them in the space provided below. Your answer must be completely correct to receive
credit. 4 points for completely correct
6. [4 points] Explain how DNA binding proteins can discriminate between AT and TA base pairs.
Make sure to describe exactly where on the DNA proteins would bind and the types of bonds formed
between DNA and proteins and how these bonds provide the specificity to discriminate AT from TA
base pairs.
Proteins make hydrogen bonds to nucleotide bases (1 point) in the major groove of DNA (1 point).
The arrangement of hydrogen bond acceptors and donors differs in AT and TA base pairs,
allowing proteins to differentiate the two possibilities (2 points).
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7. [2 points] Which statement about the carbamoyl phosphate synthetase of mammals, which is used for
pyrmidine biosynthesis, is true?
A) It is located in the mitochondria.
B) It is located in the nucleus.
C) It uses NH4+ as a nitrogen source
D) It uses glutamine as a nitrogen source
Circle the correct answer
8. [2 points] Which of the following statements about topoisomerases is incorrect?
A) Type II topoisomerases change the linking number in increments of 2.
B) They break and reseal phosphodiester bonds.
C) Type I topoisomerases can introduce positive supercoils.
D) Type I topoisomerases form covalent intermediates with their DNA substrates.
E) Type II topoisomerases can use ATP hydrolysis to form negatively supercoiled DNA from
relaxed DNA in E. coli.
Circle the correct answer.
9. [4 points] The DNA of virtually every cell is _under_____-wound (under or over). In eukaryotic
cells, DNA is wrapped around __histone_________ proteins to form a _____nucleosome__ core
particle that can be compacted into a __30 nm__________ fiber.
Please fill in the blanks above with the correct terms.
10. [5 points] For the following statements about DNA replication in E. coli, indicate whether the
statement is true or false (by circling T or F). If the statement is false, provide a brief correction on
the adjacent line.
T or F
It occurs at a replication fork _______________________________________________
T or F
It starts at a unique locus on the chromosome ___________________________________
T or F
It proceeds with a single replication fork_____two replication forks_______________
T or F
It uses RNA transiently as a template strand___RNA is used as a primer___
T or F
strand_
It involves discontinuous synthesis on the leading strand ____discontinuous on lagging
11. [2 points] The enzyme which removes the RNA primer from the Okazaki fragment is
A) Deprimase
B) RNAse H
C) DNA polymerase I
D) DNA polymerase II
E) DNA polymerase III
Circle the correct answer.
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12. [2 points] Which is not a property of E. coli DNA polymerase III?
A) It is responsible for incorporating most of the nucleotides in the lagging strand.
B) It completes synthesis of the entire leading strand prior to aiding in the synthesis of the
lagging strand.
C) It contains a 3' to 5' exonuclease activity.
D) It is a large protein complex containing more than five subunits.
E) It contains a DNA helicase activity for unwinding DNA.
Circle the correct answer.
13. [4 points] Eukaryotic DNA replication initiates replication at multiple origins of replication
simultaneously. How is eukaryotic replication initiated to ensure simultaneous initiation at multiple
origins?
Pre-replication complex is formed at all origins (2 points), Coordinate activation of these
complexes allows for simultaneous initiation at all origins (2 points). Also get credit for
mentioning CDK enzymes off during formation of Pre-RC followed by CDK enzymes on to allow
for coordinate activation,
14. [4 points] Describe the mechanistic difference that distinguishes the splicing of group I introns from
that of nuclear mRNA introns.
Ans: In the group I introns, the initial transesterification in the RNA chain occurs as the
hydroxyl group of a solvent guanosine nucleotide makes a nucleophilic attack on the
phosphodiester bond with no lariat being formed. In mRNA introns, the attacking species is
the 2'-hydroxyl group of an adenylate residue in the intron itself making a lariat.
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15. [2 points] In addition to DNA polymerase's intrinsic 3'-5' exonuclease activity, mismatch repair, and
base excision repair, two other mechanisms for DNA repair include _(oligo)nucleotide excision
repair_ and ____direct repair___.
16. [6 points] In the initiation of transcription in E. coli, the catalytic subunit of RNA polymerase first
combines with the ___σ______ subunit to form the RNA polymerase holoenzyme complex which
then binds to the DNA promoter to form the ____closed_________ complex. Melting of the DNA
duplex by the polymerase then forms the _____open________ complex. After elongation,
termination of transcription can occur with a ρ-independent mechanism with the formation of a
__RNA hairpin______ and ___AU RNA-DNA hybrid duplex_________. Alternatively in the ρdependent mechanism, a rut site is recognized by a ___ρ helicase______ which separates the RNA
from the DNA template.
17. [2 points] Which of the following statements about the poly(A) tails that are found on most
eukaryotic mRNAs are correct?
A) They are added as preformed polyriboadenylate segments to the 3' ends of mRNA precursors by
an RNA ligase activity.
B) They are encoded by stretches of polydeoxythymidylate in the template strand of the gene.
C) They are added by RNA polymerase II in a template-independent reaction using ATP as the sole
nucleotide substrate.
D) They are added by polyadenylate polymerase using ATP as the sole nucleotide substrate.
E) They are cleaved from eukaryotic mRNAs by a sequence specific endonuclease that recognizes
the RNA sequence AAUAAA.
Circle the correct answer.
18. [2 points] Which process listed below is common to the post-transcriptional modification of
eukaryotic mRNA?
A) Addition to the 5' end of a 7-methylguanosine in a 5',5' triphosphate linkage.
B) Addition to the 3' end of a 7-methylguanosine in a 3',5' triphosphate linkage.
C) Addition to the 5' end of a 7-methyladenosine in a 5',5' triphosphate linkage.
D) Methylation of ribose 3'-hydroxyl groups.
E) Phosphorylation of ribose 2'-hydroxyl groups.
Circle the correct answer.
19. [5 points] According to the wobble hypothesis, what is the minimum number of tRNAs required to
decode the six arginine codons CGU, CGC, CGA, CGG, AGA, AGG?
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Minimum number of tRNA molecules: ___3 (2 points)________
Indicate the tRNA anticodon sequences (correctly labeling 5' and 3' ends) that would recognize each
of the six codons for this minimal number of tRNA molecules:
Anticodon sequence (label polarity)
3'-GCI-5' or 3'-GCG-5'
Codon recognized
5'-CGU-3'
3'-GCI-5' or 3'-GCG-5'
5'-CGC-3'
3'-GCI-5' or 3'-GCU-5'
5'-CGA-3'
3'-GCC-5' or 3'-GCU-5'
5'-CGG-3'
3'-UCU-5' (not 3'-UCI-5')
5'-AGA-3'
3'-UCU-5' (not 3'-UCC-5')
5'-AGG-3'
3 points for all correct, -0.5 for each incorrect or missing answer. okay if anticodons are inverted
as long as ends are labeled correctly
20. [4 points] For the following statements about the aminoacyl-tRNA synthetase reaction, indicate
whether the statement is true or false (by circling T or F). If the statement is false, provide a brief
correction on the adjacent line.
T or F GTP hydrolysis is involved in the reaction _____ATP is involved_____________
T or F
An acyl thioester bond is formed _____An acyl ester is formed_____________
T or F
The reaction occurs through two steps ______________________________
T or F
The amino acid is attached to the 2' hydroxyl of an adenosine at the 3' end of the tRNA
molecule ____3' OH of this adenosine is attached to amino acid__________
21. [5 points] E. coli cells are placed in a growth medium containing lactose. Please indicate how the
following perturbations would effect the expression of the lactose operon (write either increase,
decrease, or no change in the spaces provided).
__Increase___ Reduction in the level of glucose in the media
___Increase__ Introduction of a mutation of the -35 and -10 region of the lac promoter to more
closely resemble the σ70 consensus sequence
___Decrease_ Introduction of a mutation that inactivates lactose permease (product of lacY gene)
___Decrease_ Introduction of a mutation of adenylate cyclase that reduces cellular levels of cAMP
___Decrease__Introduction of a mutation in CRP protein to cause it to bind to the operator sequence
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22. [5 points] For the following statements about prokaryotic translation, indicate whether the statement
is true or false (by circling T or F). If the statement is false, provide a brief correction on the adjacent
line.
T or F
Amino acids are added to the amino terminus of the growing polypeptide chain
_____added to c terminus_____________________
T or F
Specific sequences within the mRNA ensure that translation begins at the correct codon
____________________________
T or F
Peptide bonds form between an aminoacyl-tRNA and a peptidyl-tRNA positioned at the
A and P sites, respectively, of the ribosome ______________________________
T or F
Termination involves the binding of a terminator tRNA to a stop codon on the mRNA
_____release factor binds to ribosome for termination___________
T or F
Puromycin binds to the A site leading to hydrolysis of peptide from peptidyl-tRNA in the
P site _an amide bond is formed between puromycin and peptide_
23. [4 points] Give two examples of coactivators and explain how they are involved in transcriptional
activation in eukaryotes. Your answer should include the identities of the binding partners of the
coactivators.
Two examples are TFIID and Mediator (2 points). They are involved in activation when bound to
a transactivator where they help to recruit the basal transcription machinery and RNAP II to the
promoter. (2 points)
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24. [5 points] For the following statements about the trp operon leader RNA region shown below,
indicate whether each statement is true or false (by circling T or F).
1
5ʹ′
2
3
4
trp
structural
genes
T or F A short open reading frame, containing Trp codons among others, exists within sequence 1 of the
leader RNA
T or F The structure of the leader RNA in vivo depends on the position of the ribosomes translating it.
T or F Base pairing of sequence 1 and 2 blocks association of sequence 2 and 3, leading to attenuation
T or F Base pairing between sequence 2 and 3 forms a stem loop that acts as a ρ-independent terminator
T or F Covering of sequence 2 by ribosome when tryptophan levels are high prevents ρ-independent
termination
25. [8 points]
Shown to the right are
the recognition
sequences for several
type II restriction
endonucleases:
ClaI:
NotI:
EcoRV
A) Draw the structure of both ends of a linear DNA fragment that are produced by a ClaI restriction
digest. (Label all 5' and 3' ends).
AT-3'
and
5'-CGAT
TAGC-5'
3'-TA
B) Draw the structure resulting from the reaction of the end sequences in A) with DNA polymerase
and the four deoxynucleoside triphosphates.
ATCG-3'
and
5'-CGAT
TAGC-5'
3'-GCTA
C) Draw the structure of both ends of a linear DNA fragment that are produced by a EcoRV
restriction digest. (Label all 5' and 3' ends).
GAT-3'
and
5'-ATC
CTA-5'
3'-TAG
D) Draw the structure of the two possible junctions produced if the ends with the structures in B) are
ligated to the ends with the structures in C). (Label all 5' and 3' ends).
5'GATCGAT
-3'
and 5'ATCGATC
-3'
3'CTAGCTA
-5'
3'TAGCTAG
-5'
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26. [3 points] Which of the following partial amino acid sequences from a protein whose gene you wish
to clone would be most useful in designing an oligonucleotide probe to screen a cDNA library?
A) Met-LeuArg-Leu
B) Met-Trp-Cys-Trp
Explain why you selected your answer:
B) is minimally degenerate so less oligonucleotides would need to be synthesized to find the
gene. (1 point)
27. [4 points] A scientist wishes to produce a mammalian protein in E. coli. The protein is a
glycoprotein with a molecular weight of 40,000. Approximately 20% of its mass is polysaccharide.
The isolated protein is usually phosphorylated and contains three disulfide bonds. The cloned gene
contains no introns.
(a) What sequences or sites will be required in the vector to get this gene regulated, transcribed, and
translated in E. coli?
The cloned gene must be preceded by a good E. coli promoter and its associated operator and by a
ribosome-binding (Shine-Dalgarno) sequence. The other end of the gene should have a
transcription terminator sequence.
(b) List two problems in E. coli that might arise in producing a protein identical to that isolated from
mammalian cells and describe each problem in no more than two sentences.
Potential problems are that (1) E. coli enzymes may not glycosylate the protein, which may
affect its folding and activity, and (2) the protein kinases that phosphorylate the protein in
mammalian cells are probably absent in E. coli; therefore, the engineered protein will not be
phosphorylated.
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