Download Chapter 16: Gene Regulation in Bacteria

Chapter 16: Gene Regulation in Bacteria
Student Learning Objectives
Upon completion of this chapter you should be able to:
1. Distinguish between the various mechanisms of transcriptional regulation.
2. Understand the regulation of the lac operon, including the roles of the lac repressor
and activator proteins.
3. Understand the regulation of the trp operon, including the molecular details of
attenuation.
4. Recognize how repressors and antisense RNAs are used in translational regulation.
5. Recognize how feedback inhibition is used in posttranslational regulation.
6. Know how riboswitches can regulate gene expression.
16.1 Overview of Transcriptional Regulation
Overview
Gene regulation can occur at a number of levels (Figure 16.1), but the most common is at
the transcriptional level. The first section of this chapter examines the variety of means by which
genes may be transcriptionally regulated in bacteria. In most cases, transcriptional regulation
involves the actions of regulatory proteins that can bind to the DNA and affect the rate of
transcription of one or more nearby genes. These proteins include activators and repressors which
exert positive and negative control, by small effector proteins which include inducers, inhibitors,
and corepressors. It is important that you understand the primary mechanisms of regulation and
the terminology associated with each. An overview of these is provided in Figure 16.2.
Outline of Key Terms
Effector molecules
Inducer
Corepressor
Inhibitor
Gene regulation
Constitutive genes
Inducible genes
Repressible genes
Transcriptional regulation
Negative control
Repressors
Positive control
Activators
Focal Points


Common points where regulation of gene expression in bacteria occurs (Figure 16.1)
Binding sites on a genetic regulatory protein (Figure 16.2)
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Exercises and Problems
For questions 1 to 7, match each regulatory term with its correct definition.
_____ 1. Activator
_____ 2. Inhibitor
_____ 3. Repressor
_____ 4. Inducer
_____ 5. Negative control
_____ 6. Positive control
_____ 7. Corepressor
a.
b.
c.
d.
e.
f.
g.
Gene regulation by repressor proteins.
Binds to an activator protein and inhibits it from binding to the DNA.
Gene regulation by activator proteins.
A protein that binds to the DNA and inhibits transcription.
A protein that binds to the repressor and causes it to bind to the DNA.
An effector molecule that increases transcription.
A protein that increases transcription.
16.2 Regulation of the Lac Operon
Overview
This section takes a closer look at a specific example of gene regulation in E. coli.
Indeed, the lac operon was the first gene regulation system to have its molecular mechanism
worked out. Our initial understanding of gene regulation can be traced back to the 1950s and the
creative minds of two French scientists: François Jacob and Jacques Monod. They were interested
in the phenomenon of enzyme adaptation, which refers to the observation that a particular
enzyme appears within a living cell only after the cell has been exposed to the substrate of that
enzyme. To investigate this phenomenon, Jacob and Monod focused their attention on lactose
metabolism in E. coli, and the rest is history!
The lac operon encodes a polycistronic mRNA for proteins that are involved in the
uptake and metabolism of lactose (See Figure 16.3). The operon can be transcriptionally
regulated in two main ways. The first mechanism is one that is inducible and under negative
control. This form of regulation involves the lac repressor protein which binds to the operator
site. Once bound, the repressor can prevent RNA polymerase from transcribing the genes and so
the operon is off. However, this binding is reversible. When allolactose is bound to the repressor,
it causes a conformational change that prevents the repressor from binding to the operator. This
event induces transcription (See Figure 16.4). The regulation of the lac operon enables E. coli to
respond to changes in the level of lactose in it environment (See Figure 16.5).
The second mechanism by which the lac operon can be transcriptionally regulated
involves an activator protein called CAP. This protein can bind cAMP and the complex will bind
to the CAP site. This stimulates the ability of RNA polymerase to transcribe the lac operon.
Glucose inhibits the enzyme adenylyl cyclase which catalyzes the synthesis of cAMP. Thus, in
the presence of glucose, the lac operon is turned off (Refer to Figure 16.8).
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Outline of Key Terms
Lac operon
Lac repressor
Allosteric regulation
Allosteric site
CAP site
Catabolite activator protein
Enzyme adaptation
Merozygote
Trans-acting factor
Trans-effect
cis-acting element
cis-effect
Catabolite repression
Diauxic growth
Cyclic AMP (cAMP)
Operon
Polycistronic mRNA
Promoter
Terminator
Operator
Focal Points





Organization of the lac operon (Figure 16.3)
Mechanism of induction of the lac operon (Figure 16.4)
The cycle of lac operon induction and repression (Figure 16.5)
Evidence that the lacI gene encodes a diffusible repressor protein (Figure 16.7)
The role of the CAP protein in the regulation of the lac operon (Figure 16.8)
Exercises and Problems
For questions 1 to 11, complete the sentence with the most appropriate term(s):
1. _______ refers to the phenomenon in which the enzymes that metabolize a molecule are made
only when that molecule is present in the environment.
2. The two French scientists that elucidated the molecular mechanism of the lac operon are
_______ and _______.
3. The lacY gene encodes the protein _______, which allows the uptake of lactose into the cell.
4. The lacZ gene encodes the enzyme _______, which cleaves lactose into ______ and ______.
5. The lacI gene encodes the _______.
6. The operator site is also called _______, and it is the site where the ________ binds.
7. The promoter of the lac operon is the site where the ________ binds.
8. The lac operon is ________, meaning that it encodes more than one structural gene.
9. The actual inducer of the lac operon is _______.
10. Effector molecules bind to a protein’s _______ site, which is distinct from the active site.
11. The CAP protein can bind to the CAP site of the lac operon only if it binds ________ first.
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For questions 12 to 18, indicate whether the lac operon will be ON or OFF under the described
conditions.
_____ 12. Wild-type operon; lactose present; glucose absent
_____ 13. Wild-type operon; lactose present; glucose present
_____ 14. Mutation in the lac repressor that prevents it from binding to its normal DNA site;
lactose absent; glucose absent
_____ 15. Mutation in the lac repressor that prevents it from binding to allolactose
_____ 16. Mutation in the CAP protein that prevents it from binding to its normal DNA site
_____ 17. Mutation in the CAP protein that prevents it from binding to cAMP
_____ 18. Mutation in the lacP region of the operon
16.3 Regulation of the Trp Operon
Overview
This section examines a second operon in E. coli called the trp operon, which encodes
enzymes involved in the synthesis of the amino acid tryptophan. While the lac operon is an
inducible operon, the trp operon is a repressible one. The trp operon can be transcriptionally
regulated in two main ways. The first mechanism involves the trp repressor. In the absence of
tryptophan, this protein is inactive and cannot bind to the operator site. Therefore, RNA
polymerase can bind to the promoter and transcribe the genes of the trp operon. When tryptophan
levels are high, tryptophan acts as a corepressor. It binds to the trp repressor and activates it. The
complex can now bind to the trp operator site to inhibit transcription (See Figures 16.11A and B).
The second mechanism by which the trp operon can be transcriptionally regulated is
termed attenuation. This mechanism operates under conditions with high levels of tryptophan
(See Figure 16.11C). Attenuation involves the formation of a stem-loop structure which causes
early termination of transcription (Refer to Figures 16.12 and 16.13).
By studying the genetic regulation of many operons, geneticists have discovered a
general trend concerning inducible versus repressible regulation. Inducible operons typically
encode catabolic enzymes. The substance to be broken down or a related compound often acts as
the inducer. Repressible operons, on the other hand, typically encode anabolic enzymes. The
corepressor or inhibitor is commonly the small molecule that is the product of the enzymes’
biosynthetic activities.
Outline of Key Terms
Trp repressor
Attenuation
Attenuator sequence
Focal Points



Organization of the trp operon (Figure 16.11)
Sequence of the trpL mRNA produced during attenuation (Figure 16.12)
Mechanism of attenuation of the trp operon (Figure 16.13)
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Exercises and Problems
For questions 1 to 7, complete the sentence with the most appropriate term(s):
1. The trp operon contains _______ structural genes that encode enzymes involved in the
biosynthesis of tryptophan.
2. The trp operon also contains a gene called ______, which plays a regulatory role. This gene
encodes a 14-amino acid protein called the _______.
3. The trpR gene, which is not part of the trp operon, encodes the _______.
4. The operator site is also called _______, and it is the site where the ________ binds.
5. The promoter of the trp operon is the site where the ________ binds.
6. In the trp operon, the corepressor is ________.
7. Attenuation involves the formation of a stem-loop structure between regions 3 and 4of the
attenuator sequence. This stem-loop structure acts as a _______ terminator of transcription.
For questions 8 to 10, indicate whether the trp operon will be ON or OFF under the described
conditions.
_____ 8. Wild-type operon; low levels of tryptophan
_____ 9. Wild-type operon; high levels of tryptophan
_____ 10. Mutation in the trp repressor that prevents it from binding to tryptophan
11. Why are attenuation systems not found in eukaryotes?
16.4 Translational and Posttranslational Regulation
Overview
Gene regulation can also occur at the translational and posttranslational levels, although
much less commonly than at the transcriptional level. Translational regulation typically involves
interactions with the machinery of initiation. The majority of cases involve translational
repressors and antisense RNA (Figure 16.14). In posttranslational regulation, the target of
regulation is the functional protein. This can occur via feedback inhibition (Figure 16.15) or
covalent modifications.
Outline of Key Terms
Posttranslational regulation
Feedback inhibition
Allosteric enzyme
Posttranslational covalent
modification
Translational regulation
Translational regulatory protein
Translational repressor
Antisense RNA
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Focal Points


Gene regulation via antisense RNA (Figure 16.14)
Feedback inhibition (Figure 16.15)
Exercises and Problems
For the following questions, match the definition with the appropriate term.
_____ 1. Any regulatory mechanism that acts on the functional protein.
_____ 2. The final product in a metabolic pathway influences an earlier enzyme in the
pathway.
_____ 3. An RNA strand that is complementary to the mRNA strand.
_____ 4. Binds to the mRNA and prevents it from interacting with the ribosome.
_____ 5. An enzyme with two different binding sites, one catalytic and one regulatory.
_____ 6. A regulatory mechanism that interacts with the initiation, elongation, or termination
of protein synthesis.
a.
b.
c.
d.
e.
f.
antisense RNA
translational repressors
allosteric enzyme
feedback inhibition
translational regulation
posttranslational regulation
16.5 Riboswitches
Overview
The last decade saw the discovery of riboswitches, which mediate a novel mechanism of
gene regulation. A riboswitch involves an RNA molecule that can exist in two different
secondary structures, based on the binding of a small molecule. Riboswitches are widespread in
bacteria; however, they are also found in archaea, algae, fungi, and plants. A riboswitch can
regulate gene expression at several levels, including transcription, translation, and RNA stability
(See Table 16.2).
Outline of Key Terms
Riboswitches
Focal Points


Riboswitch regulation of transcription in bacteria (Figure 16.16)
Riboswitch regulation of translation in bacteria (Figure 16.17)
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Exercises and Problems
Characterize the following four statements as True (T) or False (F). If False, change the statement
to make it correct.
_____
_____
_____
_____
1.
2.
3.
4.
Riboswitches are proteins involved in gene regulation.
Riboswitches are found in both prokaryotes and eukaryotes.
Riboswitches exist in two alternate secondary structures.
Riboswitches regulate gene expression at the levels of transcription, translation,
RNA stability, and RNA splicing.
5. Briefly describe how riboswitches differ from antisense RNAs?
Chapter Quiz
1. Cyclic AMP is involved in the regulation of which of the following bacterial operons?
a. the lac operon
b. the trp operon
c. Both A and B
d. Neither A nor B
2. The ______ operon utilizes attenuation as a regulatory mechanism.
a. the lac operon
b. the trp operon
c. Both A and B
d. Neither A nor B
3. Feedback inhibition is a mechanism to regulate gene expression at the ________ level.
a. transcriptional
b. posttranscriptional
c. translational
d. posttranslational
4. The lac operon is turned off in the presence of what?
a. lactose
b. glucose
c. arabinose
d. any sugar
5. The presence of tryptophan does what to the trp repressor?
a. inactivates it
b. activates it
c. degrades it
d. initiates transcription of
e. none of the above
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6. A mutation in the lac repressor protein that prevents it from binding to allolactose will cause
which of the following? Assume that glucose is not present.
a. expression of the lacZ gene to be constitutively high
b. expression of the lacZ gene to be constitutively low
c. expression of the lacZ gene to be high in the presence of lactose and low in the absence
of lactose
d. expression of the lacZ gene to be low in the presence of lactose and high in the absence
of lactose
7. An enzyme that contains both a catalytic site and a regulatory site is called _______.
a. antisense
b. inducible
c. allosteric
d. cis-acting
8. The lac repressor protein binds to the ________ site.
a. CAP
b. terminator
c. promoter
d. silencer
e. operator
9. The genes required for the breakdown of cellobiose are likely to be
I
and the genes required for the synthesis of isoleucine are expected to be
II
a. I = induced
;
II = repressed
b. I = induced
;
II = induced
c. I = repressed
;
II = induced
d. I = repressed
;
II = repressed
by cellobiose,
by isoleucine.
10. Riboswitches have been found in all of the following organisms EXCEPT
a. archaea.
b. bacteria.
c. fungi.
d. plants.
e. humans.
Answer Key for Study Guide Questions
This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers
in parentheses ( ) represent possible alternate answers to a problem, while answers marked with
an asterisk (*) indicate that the response to the question may vary.
16.1
1. g
2. b
3. d
4. f
5. a
6. c
7. e
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10. allosteric site
11. cAMP
12. ON
13. OFF
14. ON
15. OFF
16. OFF
17. OFF
18. OFF
16.2
1. Enzyme adaptation
2. Jacob and Monod
3. lac permease
4. -galactosidase; glucose and
galactose
5. lac repressor
6. lacO; lac repressor
7. RNA polymerase
8. polycistronic
9. allolactose
16.3
1. five
2. trpL; leader peptide
3. trp repressor
4. trpO; lac repressor
5. RNA polymerase
6. tryptophan
7. rho-independent
8. ON
9. OFF
10. ON
11. Attenuation requires that transcription and translation be coupled. In eukaryotes, this
is not possible because transcription occurs in the nucleus, while translation takes place in
the cytoplasm
16.4
1. f
2. d
3. a
16.5
1. F, are RNA
2. T
3. T
4. T
5. Riboswitches are encoded within the transcript they regulate, and act in cis to control
expression of the gene(s) within that transcript, Antisense RNAs, on the other hand, act in
trans to regulate the activity of other RNA transcripts.
4. b
5. c
6. e
Quiz
6. b
7. c
8. e
9. a
10. e
1. a
2. b
3. d
4. b
5. b
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