BI475: Molecular Biology
Ch. 10: Review Questions & Answer Key
Spring, 2003
1
Circle the letters corresponding to the appropriate terms/phrases that complete items 1–20.
1. Operator constitutive mutants of the lac operon would
a. express the lac repressor constitutively
b.
block the binding of RNA polymerase to the
promoter
c. expr
-galactosidase constitutively d.
prevent the inducer from binding to the repressor
- c + + + 2. An E coli cell that has the genotype I O Z /I O Z would
a.
-galactosidase synthesis upon the addition of lactose b.
galactosidase
c.
-galactosidase
d.
-galactosidase synthesis upon the addition of lactose
3. Which of the following is not used in the electrophoretic mobility shift assay (EMSA)?
a. a radiolabeled DNA fragment
b.
a polyacrylamide gel
c. a DNA binding protein
d.
DNase I
4. Which of the following proteins does not "footprint" the lac operon control region?
a. lac repressor
b.
-galactosidase
c.
RNA polymerase
cAMP-CAP
d.
5. How does binding of the lac repressor to the lac operator block transcription initiation?
a. lac repressor binding blocks RNA polymerase from interacting with DNA at the start site
b. lac repressor binding induces a DNase which cleaves the DNA at the transcription start site
c. lac repressor binding causes a conformational change in RNA polymerase
d. lac repressor binding induces a protease which degrades the sigma subunit of RNA polymerase
6. All of the following statements about the essential carboxy terminal domain (CTD) of RNA polymerase
are true except
a. The CTD is present in RNA polymerase I, II , and III
b.
The
CTD
can
become
phosphorylated
c. The CTD is critical for viability
d.
The CTD of mammals contains more than 50 repeats
of a heptapeptide
7. Most eukaryotic and prokaryotic genes are controlled at the level of
a. transcription initiation b.transcription elongation
c.transcription
d.translation initiation
8. Which of the following is not a step in the nascent-chain (run on) assay?
a. isolation of nuclei
b.
incubation
ribonucleoside triphosphate
c. exposure of cells to a labeled RNA precursor
d.
hybridization
cloned cDNAs
termination
with
of
labeled
9. An enhancer
a. is a DNA element that stimulates transcription of eukaryotic promoters
b. binds to RNA polymerase and stimulates transcription
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BI475: Molecular Biology
Ch. 10: Review Questions & Answer Key
Spring, 2003
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c. acts as a binding site for RNA polymerase
d. interacts with repressor proteins to enhance transcriptional repression
10. The TATA box
a. serves as a promoter sequence for genes transcribed by RNA polymerase III
b. is located approximately 100 base pairs upstream of the start site for mRNAs
c. is present in all eukaryotic genes
d.
acts to position RNA polymerase II for
transcription initiation
11. All of the following elements can function as eukaryotic promoters except
a. a TATA box
b. an initiator element c. CpG islands d. an enhancer
12. All of the following statements about the Wilms’ tumor (WT1) gene or WT1 protein are true except:
a. Loss of functional WT1 protein leads to the development of kidney tumors
b. The WT1 protein has a zinc finger binding domain
c. WT1 protein is a transcription
activator
d. WT1 binds to the control region of the EGR1 gene
13. A leucine zipper motif contains
a. a stretch of five leucine residues in a row
c. a leucine residue complexed with a zinc ion
structure
b. a leucine residue at every seventh position
d. an alternating leucine-alanine-proline
14. Which of the following is not a structural motif found in a DNA binding domain?
a. homeodomain
b.zinc finger
c.helix-loop-helix
d.random
domain
coil
acidic
15. Which of the following is the correct order of binding of general transcription factors to initiate
transcription at RNA polymerase II promoters?
a. TFIID, TFIIB, Pol II, TFIIH
b. PolII, TFIID, TFIIB, TFIIH
c.
TFIIB,
PolII, TFIIH, TFIID
d. TFIID, TFIIH, TFIIB, PolII
16. What is the function of TFIIH in the transcription initiation complex?
a. TFIIH binds to the TATA box
b.
TFIIH unwinds the DNA duplex
c. TFIIH catalyzes the synthesis of RNA d.
all of the above
17. All of the following statements about heterochromatin are true except
a. Heterochromatin stains more darkly with DNA dyes than euchromatin
b. Heterochromatin contains more highly condensed DNA than euchromatin
c. Heterochromatin is associated with inactive genes
d. Heterochromatin is more susceptible to DNaseI than euchromatin
18. All of the following events play a role in yeast mating type switching except
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Ch. 10: Review Questions & Answer Key
a. methylation of the silent mating type locus
the MAT locus
c. chromatin condensation at the silent mating type locus d.
conversion
19. Lipid soluble hormones activate transcription by
a. binding to specific cell-surface receptors
c. binding to a nuclear receptor
d.
b.
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transcription of the gene at
a recombination event known as gene
b.
phosphorylating a protein kinase
inhibiting a histone deacetylase
20. All of the following statements about transcription by archaebacteria are true except
a. Archaens use a single RNA polymerase
b. Archael RNA polymerase has a subunit structure similar in complexity to bacteria
c. Archaens transcribe operons into polycistronic mRNAs
d. Archael promoters have an A/T rich sequence upstream of the transcription start site
21. Define the terms cis-acting DNA sequences and trans-acting proteins.
+
22. Describe the experimental evidence that the product of the lac I gene is trans-acting.
23. Describe how the electrophoretic mobility shift assay (EMSA) and the
techniques are used to identify DNA-protein interactions.
DNase I footprinting
70
24. The major form of bacterial RNA polymerase is com
the role of each of these subunits in the initiation of transcription.
). Describe
25. Most bacterial repressors bind DNA as dimers. Describe how these repressors bind to the operator
sequence.
26. Contrast the transcriptional regulatory elements in prokaryotes and eukaryotes.
27. Describe the similarities and differences between prokaryotic and eukaryotic RNA polymerases.
28. Describe the structure and function of the carboxy terminal domain (CTD) of RNA polymerase II.
29. What is the functional difference between enhancers and promoter proximal elements?
30. Compare the general pattern of cis-acting control elements in yeast and higher eukaryotes.
31. What is an enhancesome?
32. How can transcription factors be purified using sequence-specific DNA-affinity chromatography?
33. Describe the structure and function of a zinc finger motif.
34. Describe the structure of the RNA polymerase II transcription initiation complex.
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35. Describe the functional properties of TFIID and TFIIH.
36. Describe the role of histone deacetylation and hyperacetylation in yeast transcriptional control.
37. Signal transduction is one mechanism for regulating transcription. Describe the model for interferon
gamma-mediated gene activation.
38. Describe how lipid soluble hormones, for example glucocorticoid, regulate gene transcription acting
through nuclear hormone receptors.
39. Describe the structure and function of the yeast mitochondrial RNA polymerase.
40.
Discuss three features that distinguish transcription initiation at Pol I and Pol III promoters from
that at Pol II promoters.
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PART C: Answers
1. c; 2. b; 3. d; 4. b; 5. a; 6. a; 7. d; 8. c; 9. a; 10. d; 11. d; 12. c; 13. b; 14. d; 15. a; 16. b; 17. d; 18. a; 19. c; 20. b
21. Cis-acting DNA elements affect only the expression of genes on the same DNA molecule that are linked to the DNA
element. In contrast, trans-acting proteins are free to diffuse through the cell and can bind to any target DNA sequence.
-
-
+ +
22. An E. coli cell containing a single lac gene (I O Z ) produces an inactive repressor and as a result constitutively
+
synthesizes -galactosidase. The introduction of a lac gene on a plasmid into these E. coli cells leads to loss of galactosidase expression. In this case, the lac repressor can bind to the operator on the chromosome and repress
synthesis of -galactosidase.
23. In the electrophoretic mobility shift assay (EMSA), DNA-protein interactions are detected by changes in the mobility of
a DNA fragment bound to a protein. A DNA fragment containing a putative protein binding site is first radiolabeled and
then incubated in the presence of sequence-specific DNA binding proteins. The DNA fragment containing a bound
protein migrates slower in a gel, causing a shift in the location of the radiolabeled DNA detected by autoradiography. In
32
the DNase I footprinting technique, a DNA fragment is first labeled at only one end with P. The radiolabeled DNA
fragment is incubated with a DNA binding protein and then digested with a limiting concentration of DNaseI. The
DNaseI concentration is set such that on average each DNA molecule is cut only once. The resulting cut DNA
fragments are separated using denaturing gel electrophoresis and visualized using autoradiography. In the absence of a
DNA binding protein, a ladder of DNA bands are detected on the autoradiogram. Binding of a protein to the DNA
prevents DNase I from digesting the radiolabeled DNA at the site of the DNA-protein interaction, resulting in a blank
area (or footprint) in the DNA ladder.
24. The  subunit interacts with the promoter and signals the polymerase to initiate transcription at a specific DNA
sequence. The  and ' subunits polymerize the ribonucleoside triphosphates as directed by the template strand. The 
subunit interacts with regulatory proteins and sometimes with DNA to regulate the rate of transcription initiation.
25. Many bacterial repressors bind to DNA as homodimers, i.e., two copies of the same molecule. In dimeric repressors an
alpha helix in each repressor monomer inserts into the major groove of the operator DNA. Most operator sequences are
short inverted repeats. Each half of the inverted repeat sequence in the operator is called a half-site. Each half site binds
to one monomer of a dimeric repressor. Binding of the repressor to DNA is stabilized by hydrogen bonds, ionic bonds,
and van der Waals interactions.
26. Transcriptional control in eukaryotes is a much more complex process in eukaryotes than in prokaryotes. In prokaryotes
the control elements generally lie close (within 60 base pairs) to the promoters they regulate. In contrast, cis-acting
control elements in eukaryotes often are located many kilobases away from the promoters they regulate. In some cases
these regulatory sites can be tens of thousands of base pairs either “upstream” or “downstream” from the promoter.
27. In prokaryotes there is only one RNA polymerase, which consists of five subunits. In eukaryotes there are three RNA
polymerases. RNA polymerase I synthesizes ribosomal RNA; RNA polymerase II synthesizes messenger RNA; and
RNA polymerase III synthesizes tRNA and other small RNAs. The eukaryotic RNA polymerases are more complex
than the bacterial RNA polymerase. All three contain two large subunits and 12–15 smaller subunits. All three
eukaryotic RNA polymerases contain subunits with some sequence homology to the E. coli RNA polymerase subunits
(, , and ').
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28. The carboxy terminal domain (CTD) of RNA polymerase II consists of a heptapeptide repeat, with a consensus sequence
of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Yeast RNA polymerase II contains 26 or more repeats, while the mammalian RNA
polymerase II contains 52 repeats. The CTD is critical for viability and at least 10 copies of the repeat must be present
for survival. During formation of the transcription initiation complex, the CTD is unphosphorylated. When the RNA
polymerase transcribes away from the promoter, the CTD is phosphorylated at serine and threonine residues. One
hypothesis is that phosphorylation of the CTD causes the release of RNA polymerase from the transcription initiation
complex.
29. Enhancers can stimulate transcription from a promoter tens of thousands of base pairs away. In contrast, promoterproximal elements are located 100- to 200-base pairs upstream of the start site and usually lose the ability to stimulate
transcription from a promoter when moved only several tens of base pairs away.
30. In higher eukaryotes, genes contain both enhancers and promoter proximal elements as well as a TATA box or other
promoter elements such as an initiator element or CpG islands. The promoter proximal elements are usually located
within 200 base pairs of the start site, whereas the enhancers can be located tens of thousands of base pairs either
upstream or downstream of the start site. In contrast, yeast genes contain an upstream activating sequence (UAS) which
functions similarly to enhancers and promoter proximal elements in higher eukaryotes. Most yeast genes contain only
one UAS located a few hundred base pairs upstream of the start site and a TATA box present approximately 90 base
pairs upstream of the start site.
31. An enhancesome is a large nucleoprotein complex bound to an enhancer element. This complex is formed by the
cooperative assembly of transcription factors to their multiple binding sites in an enhancer.
32. Sequence specific DNA affinity chromatography is a technique that takes advantage of the binding specificity of a
protein to a specific DNA sequence. Once the DNA sequence to which a transcription factor binds is identified, this
DNA sequence can be coupled to a bead in a column. A protein mix containing the transcription factor is applied to this
column. Proteins that do not bind to the DNA fragment are washed out of the column. The bound transcription factor
can then be eluted from the column in the presence of high salt.
33. A zinc finger is a structural motif found in DNA binding domains, which consists of a short length of the polypeptide
2+
chain folded around a Zn ion. The two basic classes of zinc finger domains are the C 2H2 and C4 structures. The C2H2
2+
zinc finger domain consists of two cysteine (C) and two histidine (H) residues bound to one Zn ion. The C4 zinc finger
2+
contains four cysteines bound to one Zn ion. The three-dimensional structure of the zinc finger forms a compact
domain, which can insert its  helix into the major groove of DNA.
34. The RNA polymerase II transcription initiation complex is a multiprotein complex. This complex consists of a DNA
promoter element to which general transcription factors (i.e., TFIIA, TFIIB, TFIID, TFIIE, TFIIH) bind along with RNA
polymerase II. This multisubunit nucleoprotein complex consists of 60–70 polypeptides with a mass of approximately 3
Mda and is nearly as large as an eukaryotic ribosome.
35. TFIID is a large multisubunit complex of approximately 750 kDa. TFIID consists of a 38 kDa TATA box-binding
protein (TBP) and 11 TBP-associated factors (TAFs). TBP is the first protein to bind to a TATA box-containing
promoter. TFIIH is the last protein to bind to the initiation complex. TFIIH contains helicase activity which unwinds
the DNA duplex at the start site. As the polymerase transcribes away from the promoter, a subunit of TFIIH
phosphorylates the carboxy terminal domain (CTD) of RNA polymerase II.
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36. Histone deacetylation/hyperacetylation is one mechanism for regulating transcriptional control in yeast. Repressor
proteins can cause deacetylation of histone N-termini in nucleosomes. Unacetylated histones contain positive charges
due to N-terminal lysines and interact strongly with DNA phosphates. These strong interactions may restrict access of
general transcription factors thus leading to transcriptional repression. In contrast, histones that have their N-termini
hyperacetylated are neutralized, eliminating the strong electrostatic interactions with the DNA phosphates. This more
open chromatin configuration facilitates access of general transcription factors and induces transcriptional activation.
37. Signal transduction “translates” a signal from the cell surface to the nucleus. Binding of various polypeptide hormones to
their cell-surface receptors leads to stimulation of transcription of particular genes. For example, several changes in a
transcription factor called Stat1, which is present as an inactive monomer in the cytoplasm, is induced by binding of
interferon gamma. Upon binding interferon gamma, the interferon gamma receptor dimerizes and activates JAK kinase.
The activated JAK kinase phosphorylates a specific tyrosine residue in inactive Stat1 monomers. The phosphorylated
monomers then form a homodimer, which is translocated from the cytoplasm to the nucleus, where it is capable of
activating transcription of interferon gamma regulated genes.
38. Glucocorticoid is a lipid soluble hormone that binds to a member of the nuclear hormone receptor family, the
glucocorticoid receptor (GR), and regulates gene transcription. In the absence of glucocorticoid, the GR in the
cytoplasm is bound to the protein HSP90. When glucocorticoid diffuses through the cell membrane, it binds to the GR
ligand binding domain and causes a conformational change in the GR releasing HSP90. The GR bound to glucocorticoid
is then translocated into the nucleus, where it interacts with glucocorticoid response elements (GRE) and regulates
transcription of responsive genes.
39. A nuclear-encoded RNA polymerase transcribes the circular mitochondrial genome. In yeast the mitochondrial RNA
polymerase consists of a 145 kDa subunit with ribonucleotide-polymerizing activity and a 43 kDa factor that is essential
for initiation at the start site. The large subunit is related to the monomeric RNA polymerases of bacteriophage. The
small subunit is related to  factors in bacterial RNA polymerases. Thus the mitochondrial polymerase appears to be a
hybrid of the simple bacteriophage and bacterial RNA polymerases.
40. Transcription initiation at Pol II promoters requires hydrolysis of ATP to initiate transcription; this is not required at Pol
I or Pol III promoters. The TATA box at –30 is the only control element involved in Pol II complexes; in contrast,
assembly of Pol I complexes involves one upstream element and one promoter control element, and assembly of Pol III
complexes involves two or three internal control elements. Finally, Pol I and Pol III are the final components assembled
into their initiation complexes, whereas several additional transcription factors must be bound after addition of the
polymerase to complete assembly of Pol II complexes.
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