chapter
28
1. Effect of mRNA and Protein Stability on Regulation E. coli cells are growing in a medium with
glucose as the sole carbon source. Tryptophan is suddenly added. The cells continue to grow, and
divide every 30 min. Describe (qualitatively) how the amount of tryptophan synthase activity in the
cells changes under the following conditions:
(a) The trp mRNA is stable (degraded slowly over many hours).
(b) The trp mRNA is degraded rapidly, but tryptophan synthase is stable.
(c) The trp mRNA and tryptophan synthase are both degraded rapidly.
2. The Lactose Operon A researcher engineers a lac operon on a plasmid but inactivates all parts of
the lac operator (lacO) and the lac promoter, replacing them with the binding site for the LexA
repressor (which acts in the SOS response) and a promoter regulated by LexA. The plasmid is intro-
duced into E. coli cells that have a lac operon with an inactive lacI gene. Under what conditions will
these transformed cells produce b-galactosidase?
Regulation of
Gene Expression
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S-312 Chapter 28 Regulation of Gene Expression
3. Negative Regulation Describe the probable effects on gene expression in the lac operon of a
mutation in (a) the lac operator that deletes most of O
1
; (b) the lacI gene that inactivates the
repressor; and (c) the promoter that alters the region around position 10.
4. Specific DNA Binding by Regulatory Proteins A typical bacterial repressor protein discriminates
between its specific DNA binding site (operator) and nonspecific DNA by a factor of 10
4
to 10
6
. About
10 molecules of repressor per cell are sufficient to ensure a high level of repression. Assume that a
very similar repressor existed in a human cell, with a similar specificity for its binding site. How many
copies of the repressor would be required to elicit a level of repression similar to that in the bacterial
cell? (Hint: The E. coli genome contains about 4.6 million bp; the human haploid genome has about
3.2 billion bp.)
5. Repressor Concentration in E. coli The dissociation constant for a particular repressor-operator
complex is very low, about 10
13
M
. An E. coli cell (volume 2 10
12
mL) contains 10 copies of the
repressor. Calculate the cellular concentration of the repressor protein. How does this value compare
with the dissociation constant of the repressor-operator complex? What is the significance of this
answer?
Chapter 28 Regulation of Gene Expression S-313
6. Catabolite Repression E. coli cells are growing in a medium containing lactose but no glucose.
Indicate whether each of the following changes or conditions would increase, decrease, or not change
the expression of the lac operon. It may be helpful to draw a model depicting what is happening in
each situation.
(a) Addition of a high concentration of glucose
(b) A mutation that prevents dissociation of the Lac repressor from the operator
(c) A mutation that completely inactivates b-galactosidase
(d) A mutation that completely inactivates galactoside permease
(e) A mutation that prevents binding of CRP to its binding site near the lac promoter
7. Transcription Attenuation How would transcription of the E. coli trp operon be affected by the
following manipulations of the leader region of the trp mRNA?
(a) Increasing the distance (number of bases) between the leader peptide gene and sequence 2
(b) Increasing the distance between sequences 2 and 3
(c) Removing sequence 4
(d) Changing the two Trp codons in the leader peptide gene to His codons
(e) Eliminating the ribosome-binding site for the gene that encodes the leader peptide
(f) Changing several nucleotides in sequence 3 so that it can base-pair with sequence 4 but not with
sequence 2
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8. Repressors and Repression How would the SOS response in E. coli be affected by a mutation in
the lexA gene that prevented autocatalytic cleavage of the LexA protein?
9. Regulation by Recombination In the phase variation system of Salmonella, what would happen to
the cell if the Hin recombinase became more active and promoted recombination (DNA inversion) sev-
eral times in each cell generation?
10. Initiation of Transcription in Eukaryotes A new RNA polymerase activity is discovered in crude
extracts of cells derived from an exotic fungus. The RNA polymerase initiates transcription only from a
single, highly specialized promoter. As the polymerase is purified, its activity declines, and the purified
enzyme is completely inactive unless crude extract is added to the reaction mixture. Suggest an
explanation for these observations.
Chapter 28 Regulation of Gene Expression S-315
12. Nucleosome Modification during Transcriptional Activation To prepare genomic regions for
transcription, certain histones in the resident nucleosomes are acetylated and methylated at specific
locations. Once transcription is no longer needed, these modifications need to be reversed. In mam-
mals, the methylation of Arg residues in histones is reversed by peptidylarginine deiminases (PADIs).
The reaction promoted by these enzymes does not yield unmethylated arginine. Instead, it produces
citrulline residues in the histone. What is the other product of the reaction? Suggest a mechanism for
this reaction.
13. Inheritance Mechanisms in Development A Drosophila egg that is bcd
/bcd
may develop nor-
mally, but the adult fruit fly will not be able to produce viable offspring. Explain.
11. Functional Domains in Regulatory Proteins A biochemist replaces the DNA-binding domain of
the yeast Gal4 protein with the DNA-binding domain from the Lac repressor, and finds that the engi-
neered protein no longer regulates transcription of the GAL genes in yeast. Draw a diagram of the
different functional domains you would expect to find in the Gal4 protein and in the engineered protein.
Why does the engineered protein no longer regulate transcription of the GAL genes? What might be
done to the DNA-binding site recognized by this chimeric protein to make it functional in activating
transcription of GAL genes?
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Biochemistry on the Internet
14. TATA-Binding Protein and the TATA Box To examine the roles of hydrogen bonds and hydropho-
bic interactions between transcription factors and DNA, go to FirstGlance in Jmol at
http://firstglance.jmol.org and enter the PDB ID 1TGH. This file models the interactions between a hu-
man TATA-binding protein and a segment of double-stranded DNA. Once the structure loads, click the
“Spin” button to stop the molecule from rotating. When the molecule has reloaded, click the “Con-
tacts” link. With the radio button for “Chains” selected, click on any part of the protein (Chain A, dis-
plays in blue) to select it as the target. Click “Show Atoms Contacting Target” and, in the list of con-
tacts to display, check only “Show putatively hydrogen-bonded non-water” to display hydrogen bonds
between the protein and the TATA box DNA. Then click on the rightmost option for viewing images
(Maximum detail: Target & Contacts Balls and Sticks, Colored by Element). With this view you should
be able to use the zoom and rotate controls and mouse clicks to answer the following questions.
(a) Which of the base pairs in the DNA form hydrogen bonds with the protein? Which of these con-
tribute to the specific recognition of the TATA box by this protein? (Hydrogen-bond length be-
tween hydrogen donor and hydrogen acceptor ranges from 2.5 to 3.3 Å.)
(b) Which amino acid residues in the protein interact with these base pairs?
(c) What is the sequence of the DNA in this model and which portions of the sequence are recog-
nized by the TATA-binding protein?
(d) Examine the hydrophobic interactions in this complex. Are they rare or numerous? To answer
this question, click on “Return to contacts” and check the option to “Show hydrophobic (apolar
van der Waals) interactions.”
Answer
Chapter 28 Regulation of Gene Expression S-317
Data Analysis Problem
15. Engineering a Genetic Toggle Switch in Escherichia coli Gene regulation is often described as
an “on or off” phenomenon—a gene is either fully expressed or not expressed at all. In fact, repression
and activation of a gene involve ligand-binding reactions, so genes can show intermediate levels of
expression when intermediate levels of regulatory molecules are present. For example, for the E. coli
lac operon, consider the binding equilibrium of the Lac repressor, operator DNA, and inducer (see
Fig. 28–8). Although this is a complex, cooperative process, it can be approximately modeled by the
following reaction (R is repressor; IPTG is the inducer isopropyl-–
D
-thiogalactoside):
R IPTG u99999999v R IPTG
Free repressor, R, binds to the operator and prevents transcription of the lac operon; the R IPTG
complex does not bind to the operator and thus transcription of the lac operon can proceed.
(a) Using Equation 5–8, we can calculate the relative expression level of the proteins of the lac
operon as a function of [IPTG]. Use this calculation to determine over what range of [IPTG] the
expression level would vary from 10% to 90%.
(b) Describe qualitatively the level of lac operon proteins present in an E. coli cell before, during,
and after induction with IPTG. You need not give the amounts at exact times—just indicate the
general trends.
Gardner, Cantor, and Collins (2000) set out to make a “genetic toggle switch”—a gene-regulatory
system with two key characteristics of a light switch. (A) It has only two states: it is either fully on
or fully off; it is not a dimmer switch. In biochemical terms, the target gene or gene system (operon)
is either fully expressed or not expressed at all; it cannot be expressed at an intermediate level. (B)
Both states are stable: although you must use a finger to flip the light switch from one state to the
other, once you have flipped it and removed your finger, the switch stays in that state. In biochemical
terms, exposure to an inducer or some other signal changes the expression state of the gene or
operon, and it remains in that state once the signal is removed.
(c) Explain how the lac operon lacks both characteristics A and B.
To make their “toggle switch,” Gardner and coworkers constructed a plasmid from the
following components:
OP
lac
The operator-promoter region of the E. coli lac operon
OP
The operator-promoter region of phage
lacI The gene encoding the lac repressor protein, LacI. In the absence of IPTG, this protein
strongly represses OP
lac
; in the presence of IPTG, it allows full expression from OP
lac
.
rep
ts
The gene encoding a temperature-sensitive mutant repressor protein, rep
ts
. At 37 C this
protein strongly represses OP
; at 42 C it allows full expression from OP
.
GFP The gene for green fluorescent protein (GFP), a highly fluorescent reporter protein (see
Fig. 9–16)
T Transcription terminator
K
d
10
4
M
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The investigators arranged these components (see figure below) so that the two promoters were
reciprocally repressed: OP
lac
controlled expression of rep
ts
, and OP
controlled expression of lacI. The
state of this system was reported by the expression level of GFP, which was also under the control
of OP
lac
.
They noticed that the average GFP expression level was intermediate at concentration X of
IPTG. However, when they measured the GFP expression level in individual cells at [IPTG]
X, they found either a high level or a low level of GFP—no cells showed an intermediate level.
(h) Explain how this finding demonstrates that the system has characteristic A. What is happening
to cause the bimodal distribution of expression levels at [IPTG] X?
mRNA
mRNA
GFP
lac represssor l represssor
lacI repts
GFP
ori T
T
ampR
OPlOPlac
Normalized GFP expression
1.0
[IPTG] (M)
0.8
0.6
0.4
0.2
10-6 10-5 10-4
X10
-3 10-2
0
(d) The constructed system has two states: GFP-on (high level of expression) and GFP-off (low level
of expression). For each state, describe which proteins are present and which promoters are being
expressed.
(e) Treatment with IPTG would be expected to toggle the system from one state to the other. From
which state to which? Explain your reasoning.
(f) Treatment with heat (42 C) would be expected to toggle the system from one state to the other.
From which state to which? Explain your reasoning.
(g) Why would this plasmid be expected to have characteristics A and B as described above?
To confirm that their construct did indeed exhibit these characteristics, Gardner and colleagues first
showed that, once switched, the GFP expression level (high or low) was stable for long periods of time
(characteristic B). Next, they measured GFP level at different concentrations of the inducer IPTG, with
the following results.
Chapter 28 Regulation of Gene Expression S-319
Answer
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