Download Derived copy of Bis2A 14.1 Bacterial Gene

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Derived copy of Bis2A 14.1 Bacterial
∗
Gene Regulation
Erin Easlon
Based on Bis2A 14.1 Bacterial Gene Regulation† by
OpenStax College
Mitch Singer
This work is produced by OpenStax-CNX and licensed under the
Creative Commons Attribution License 4.0‡
Abstract
By the end of this section, you will be able to:
• Describe the steps involved in prokaryotic gene regulation
• Explain the roles of activators, inducers, and repressors in gene regulation
The DNA of bacteria and archaea is usually (there are a few known exceptions to the circular chromosome
in bacteria) organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm.
Proteins that are needed for a specic function, or that are involved in the same biochemical pathway, are
often times encoded together in blocks called operons. Therefore, operons are single transcription units,
encoding for multiple genes. Expression of these genes is organized from a single regulatory region and all
genes in the operon are therefore regulated as a single unit. For example, all of the genes needed to use
lactose as an energy source are coded next to each other in the lactose (or lac ) operon.
In bacteria, all transcription is controlled through RNA polymerase, a multiprotein complex that recognizes the promoter region and initiates transcription, elongates the transcript, and terminates transcription.
Therefore, gene expression can be regulated at any of these steps, initiation, elongation, or termination;
however, in bacteria, the majority of the regulation is at the level of transcription initiation.
The Role of the Promoter
The rst level of control of gene expression is at the promoter itself. There are two ways a promoter controls
gene expression. First is which RNA polymerase holoenzyme (sigma + Core RNA polymerase) recognizes
the promoter. Remember, bacteria have a number of sigma factors many of which control gene expression only under certain conditions, such as Sigma-S during stationary phase. The second level of control
is promoter strenght, some promoters are considered "strong", while others are considered "weak". The
basis of promoter strength is the specicity the promoter has to RNA polymerase. Each dierent sigma
factor has a dierent recognition sequence, for example, the sigm-70 protein in E. coli has the recognition sequence 5'-TTGACA-(16-17 nucleotides)-TATAAT-3'. Strong promoters have sequences close to the
consensus recognition sequence, weak promoters have sequences more divergent to the consensus.
∗ Version
1.1: Jul 17, 2015 2:51 pm -0500
† http://cnx.org/content/m56075/1.2/
‡ http://creativecommons.org/licenses/by/4.0/
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Regulator Proteins
The next layer of control is the addition of regulatory proteins. These proteins can either act to increase
transcription, and are often called activators or activator proteins. These proteins bind to the promoter
region and aid RNA polymerase to recognize a promoter and initiate transcription. Alternatively, regulatory proteins that inhibit transcription are often referred to as repressors or repressor proteins. Some
regulatory proteins can act both as a repressor or an activator depending upon how they interact with RNA
polymerase and the promoter. For example the regulatory protein called CAP can act to activate some genes
and repress other genes. Therefore the terms "activator" and "repressor" should be used depending upon
the situation or condition, and may not truely reect the role of the protein in question.
Allosteric modulators of Protein Regulators
Many regulatory proteins do not function independently, instead they rely on an allosteric regulatory molecule
to control their activity. These small molecules are often referred to as inducers or co-repressors or coactivators. These small molecules are often metabolites, such as lactose or tryptophan or small regulatory
molecules, such as cAMP or GTP. Below are some examples of regulatory systems that are controlled by
repression and by activation. In some instances the presence of the small molecule activates or enhances
the DNA-binding activity of the protein, thereby allowing the protein to bind better and regulate (either
activate or repress) transcription initiation. Alternatively, the small molecule interacts with the regulatory
protein and inhibits or decreases its ability to interact with the DNA or RNA polymerase, which can lead
to either activation or repression of the gene it is controlling.
1 Repressors vs Activators: How do you tell
Expression of the gene
In general there are three states that can be used to described the expression of a gene or operon. The rst is
constitutive. That the level of expression observed under most conditions. This level of expression could
be very high, if the promoter is strong, or expression could be very little, if the promoter is weak. Regardless
of the amount of expression, we observe very little change in expression under a variety of conditions. The
second state is activation or induction, that is under a specic set of conditions, the expression of a gene
or operon is increased or activated. Finally, expression of a gene can be decreased under a certain set of
conditions, this is called repression. Mechanistically, in the last two cases, regulatory proteins are required
to change the constitutive expression pattern. Whether a regulatory protein acts in a positive way, that is
as an activator or acts in a negative way, as a repressor is not necessarily obvious.
A simple test
So how does one determine if a regulatory protein functions in a positive or negative way? A simple genetic
test is to ask "what happens to expression if the regulatory protein is absent?" If a regulatory protein is acting
positively, then its presence is required to activate gene expression. In its absence, there is no regulatory
protein, therefore no activation, and the out come is no transcription. The phenotype of a null mutation in
a regulatory protein is no activation. The opposite is true for a regulatory protein acting negatively. In its
absence expression should be increased, because the gene keeping expression low is no longer around.
Repression vs Activation
What should become clear, is that how a regulatory protein functions: negatively or positively, may be
independent as to what its eect is on gene expression. As you will see below, when E. coli is grown in
the presence of lactose (and in the absence of glucose) expression of the lac operon is induced or activated.
Yet, the protein regulator that is responsible for this expression phenotype is a repressor; it binds to the
DNA to prevent transcription and a null mutation that removes the gene (lacI ) increases expression of the
lac operon. In other words, the mechanism by which a regulatory protein work (positively or negatively)
is independent as to how the gene or operon is expressed and behaves. This apparent contradiction can be
rationalized when you incorporate the role of the allosteric regulator; in this case lactose. As you will see in
the examples below, the key to the regulation is two fold: the mechanism by which the regulator works and
the role and nature of the allosteric regulator.
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2 The
trp
3
Operon: A Repressor Operon
Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli
can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded
by ve genes. These ve genes are next to each other in what is called the tryptophan (trp ) operon
(Figure 1). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the
switch controlling the activation of the genes in the trp operon is switched o. However, when tryptophan
availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are
expressed, and tryptophan is synthesized.
Figure 1: The ve genes that are needed to synthesize tryptophan in E. coli are located next to each
other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor
protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the
tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and
the genes are transcribed.
A DNA sequence that codes for proteins is referred to as the coding region. The ve coding regions for the
tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before
the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase
binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site; each
operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and
regulate transcription.
A DNA sequence called the operator sequence is encoded between the promoter region and the rst
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coding gene. This operator contains the DNA code to which the repressor protein can bind. When
tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to
bind to the trp operator. Binding of the tryptophanrepressor complex at the operator physically prevents
the RNA polymerase from binding, and transcribing the downstream genes. It should be noted that the
term "operator" is limited to just a few systems and almost always refers to the binding site for a repressor.
Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory
proteins allowing them to perform there appropriate function, repress transcription or activate transcription.
These regulatory protein binding sites can vary as to location, but all control or regulate how RNA polymerase
initiates transcription.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore,
the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the
operator to keep the genes turned o, the trp operon is negatively regulated and the proteins that bind to
the operator to silence trp expression are negative regulators.
trp
:
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Watch this video1 to learn more about the
5
trp
operon.
3 Catabolite Activator Protein (CAP): An Activator Regulator
Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the
operator sequences that act as a positive regulator to turn genes on and activate them. For example,
when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to
process these alternate genes must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to
accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy
metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive
regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that
control the processing of alternative sugars. When cAMP binds to CAP, the complex binds to the promoter
region of the genes that are needed to use the alternate sugar sources (Figure 2). In these operons, a CAP
binding site is located upstream of the RNA polymerase binding site in the promoter. This increases the
binding ability of RNA polymerase to the promoter region and the transcription of the genes. Please note,
CAP-cAMP complex can also act as a repressor, depending upon where the binding site for CAP-cAMP is
located. In the case of the Lactose operon, its position allows it to act as an activator; but in other operons
it is positioned 3' or downstream from the promoter and can act as a repression.
Figure 2: When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes
to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the CAP
protein, a positive regulator that binds to an operator region upstream of the genes required to use other
sugar sources.
1 http://openstaxcollege.org/l/trp_operon
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4 The
lac
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Operon: An Inducer Operon
The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have
proteins that bind to activate or repress transcription depending on the local environment and the needs of
the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other
sugars as energy sources when glucose concentrations are low. To do so, the cAMPCAP protein complex
serves as a positive regulator to induce transcription. One such sugar source is lactose. The lac operon
encodes the genes necessary to acquire and process the lactose from the local environment. CAP binds to
the operator sequence upstream of the promoter that initiates transcription of the lac operon. However, for
the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low
or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will
the lac operon be transcribed (Figure 3). This makes sense for the cell, because it would be energetically
wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available.
:
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Figure 3: Transcription of the lac operon is carefully regulated so that its expression only occurs when
glucose is limited and lactose is present to serve as an alternative fuel source.
In E. coli, the
case?
trp
operon is on by default, while the
lac
operon is o. Why do you think this is the
If glucose is absent, then CAP can bind to the operator sequence to activate transcription. If lactose is
absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is
met, then transcription remains o. Only when both conditions are satised is the lac operon transcribed
(Table 1).
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Signals that Induce or Repress Transcription of the lac Operon
Glucose CAP binds Lactose Repressor binds Transcription
+
-
-
+
No
+
-
+
-
Some
-
+
-
+
No
-
+
+
-
Yes
Table 1
:
Watch an animated tutorial2 about the workings of
2 http://openstaxcollege.org/l/lac_operon
http://cnx.org/content/m56876/1.1/
lac
operon here.
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5 Section Summary
The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are three
ways to control the transcription of an operon: repressive control, activator control, and inducible control.
Repressive control, typied by the trp operon, uses proteins bound to the operator sequence to physically
prevent the binding of RNA polymerase and the activation of transcription. Therefore, if tryptophan is not
needed, the repressor is bound to the operator and transcription remains o. Activator control, typied by
the action of CAP, increases the binding ability of RNA polymerase to the promoter when CAP is bound.
In this case, low levels of glucose result in the binding of cAMP to CAP. CAP then binds the promoter,
which allows RNA polymerase to bind to the promoter better. In the last examplethe lac operontwo
conditions must be met to initiate transcription. Glucose must not be present, and lactose must be available
for the lac operon to be transcribed. If glucose is absent, CAP binds to the operator. If lactose is present,
the repressor protein does not bind to its operator. Only when both conditions are met will RNA polymerase
bind to the promoter to induce transcription.
6 Art Connections
Exercise 1
Figure 3 In E. coli, the
that this is the case?
trp
operon is on by default, while the
(Solution on p. 10.)
lac
operon is o. Why do you think
7 Review Questions
Exercise 2
If glucose is absent, but so is lactose, the
a.
b.
c.
d.
lac
operon will be ________.
(Solution on p. 10.)
activated
repressed
activated, but only partially
mutated
Exercise 3
(Solution on p. 10.)
Bacteria and archaea lack a nucleus. Therefore, the genes in bacteria and archaea are:
a.
b.
c.
d.
all expressed, all of the time
transcribed and translated almost simultaneously
transcriptionally controlled because translation begins before transcription ends
b and c are both true
8 Free Response
Exercise 4
(Solution on p. 10.)
Exercise 5
(Solution on p. 10.)
Exercise 6: Exercise 6
(Solution on p. 10.)
Describe how transcription in bacteria can be altered by external stimulation such as excess lactose
in the environment.
What is the dierence between a repressible and an inducible operon?
In the lac operon detailed above, LacI acts as a repressor and its allosteric regulator, lactose, acts
as an inducer of the system. Redesign the lacI gene such that it no longer acts as a repressor but
instead acts as an activator. What basic properties of the protein would need to change?
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Solutions to Exercises in this Module
to Exercise (p. 9)
Figure 3 Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some
on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the
trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make
the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on
when lactose is present.
to Exercise (p. 9)
B
to Exercise (p. 9)
D
to Exercise (p. 9)
Environmental stimuli can increase or induce transcription in bacteria. In this example, lactose in the
environment will induce the transcription of the lac operon, but only if glucose is not available in the
environment.
to Exercise (p. 9)
A repressible operon uses a protein bound to the promoter region of a gene to keep the gene repressed or
silent. This repressor must be actively removed in order to transcribe the gene. An inducible operon is either
activated or repressed depending on the needs of the cell and what is available in the local environment.
Solution to Exercise (p. 9)
First the Lac promoter would need to be much weaker promoter. Second, the LacI binding site (operator)
would most likely need to move to the 5' end of the promoter. Third, when LacI binds to lactose, instead
of decreasing the anity of the protein to the DNA, it would need to increase the binding anity. Similar
to the way CAP-cAMP enhances the binding of CAP. In this hypothetical model, in the presence of lactose,
lactose would bind LacI, cause a conrmation change that now stimulates LacI to bind to the promoter
region and activate transcription. Hence, the expression pattern would look identical to as it does in the
example above, except the mechanism of action by the regulator has changed from negative to positive.
Glossary
Denition 1: activator
protein that binds to prokaryotic operators to increase transcription
Denition 2: catabolite activator protein (CAP)
protein that complexes with cAMP to bind to the promoter sequences of operons that control sugar
processing when glucose is not available
Denition 3: inducible operon
operon that can be activated or repressed depending on cellular needs and the surrounding environment
Denition 4: lac operon
operon in prokaryotic cells that encodes genes required for processing and intake of lactose
Denition 5: negative regulator
protein that prevents transcription
Denition 6: operator
region of DNA outside of the promoter region that binds activators or repressors that control gene
expression in prokaryotic cells
Denition 7: operon
collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells
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Denition 8: positive regulator
protein that increases transcription
Denition 9: repressor
protein that binds to the operator of prokaryotic genes to prevent transcription
Denition 10: transcriptional start site
site at which transcription begins
Denition 11: trp operon
series of genes necessary to synthesize tryptophan in prokaryotic cells
Denition 12: tryptophan
amino acid that can be synthesized by prokaryotic cells when necessary
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