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1.1 Negative Transcription Regulation in ProkaryotesHide Citation
By: Kenna Shaw, Ph.D. (Nature Education) 2008 Nature Education
Citation: Shaw, K. (2008) Negative transcription regulation in prokaryotes. Nature Education 1(1):122
A cell generally uses only a fraction of its genome at any given moment in time, so it seems reasonable to predict that
most genes are transcriptionally repressed and that, when required, genes could be switched on, or expressed, but only for
as long as needed. In this way, the cell could avoid wasteful production of unnecessary transcripts and proteins. While
this is essentially the mechanism of gene regulation that has evolved in higher organisms , most bacterial genes are
on by default and must be repressed when not needed. Typical bacterial operons are regulated negatively (that is, using a
repressor protein). Depending upon the small molecule ligand for the repressor, however, they can be inducible (i.e.,
turned on when the signal ligand is present) or repressible (i.e., turned off when the signal ligand is present).
How Is Gene Expression Repressed in Bacteria?
In bacteria, genes are available for expression by default, but they are actively switched off by repressor proteins.
Repressor proteins regulate expression by binding to a DNA sequence, called the operator, which is near the promoter of
an operon, or a cluster of co-regulated genes. Repressor binding blocks RNA polymerase from binding with the
promoter, thereby leading to repression of operon gene expression. Repressor activity is sensitive to a ligand that binds
to the repressor and signals the environmental conditions, such as nutrient levels, which provides a mechanism by which
bacteria can adjust their metabolism accordingly. A classic example of negative repressible regulation of gene expression
involves the trp operon, which is regulated by a negative feedback loop.
Trp Operon Regulation
To better understand how the trp operon works, consider the example of E. coli cells, which can synthesize their own
tryptophan (trp), an amino acid essential for survival. However, if trp is already present in their growth environment,
these bacteria very sensibly cease manufacturing tryptophan. Specifically, within each bacterium, the trp operon contains
genes needed to synthesize trp and, remarkably, expression of these genes is sensitive to levels of trp. When high levels of
trp are present, the repressor protein trpR binds the operator of the trp operon, preventing continued expression of trp-
synthesizing enzymes. However, trpR requires the ligand tryptophan, the product of the enzymes encoded by the operon,
in order to bind the operator. It cannot bind the operator in the absence of trp, thereby allowing continued expression of
the trp operon when the amino acid is needed.
As trp levels increase, trp binds to trpR, causing a conformational change that allows binding to the operator and
repression of gene expression. Trp therefore acts as a self-governor by regulating its own production through a negative
feedback loop. Mutations that disrupt the trpR gene lead to elevated production of trp, even in the presence of trp, thus
reinforcing the notion that negative feedback on the trp operon is trpR-dependent (Oxender et al., 1979).
Attenuation of the Trp Operon
Figure 1: Secondary structure alternatives in the trp
leader transcript.
On the left are the two base-paired structures that are
detected in vitro. The arrows indicate the sites of
RNase TI attack. The G-bonds in the hydrogen-
bonded regions are not cleaved, presumably because
the Gs are base paired. On the right is an alternative
Intro to Microbial Genetics
Unit 1: Operons and Transcriptional Regulation in Bacteria

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secondary structure. Formation of this structure is
thought to prevent transcription termination at the
attenuator. For simplicity we designate the transcript
segments that participate in these hydrogen-bonded
structures strands 1-4 (see center insert). The trp
codons are in strand 1. The trpL75 mutation is
indicated.
1981 Nature Publishing Group Yanofsky, C. At t enuat ion in
t he cont rol of expression of bact erial operons. Nature 289, 753
(1981). All right s reserved.
Figure Detail
Studies have also revealed an additional layer of negative regulation, called attenuation. Attenuation, or dampening, of the
trp operon was discovered by examining E. coli that carried mutations in the trpR gene. As previously described, in the
absence of a functional trpR protein, the trp-sensitive negative feedback loop fails. TrpR mutants continue to produce trp
in the presence of trp. Strangely, however, trpR mutants grown in the absence of trp make even more trp than wild-type
cells starved for trp, suggesting the existence of a secondary mechanism for sensing trp levels (Oxender et al., 1979). This
trpR-independent mechanism for sensing trp levels is an example of attenuation.
Continued molecular analysis revealed that a region within the trp operon mRNA was responsible for attenuation. This
transcribed regulatory region, called the leader of the mRNA and located upstream of all the codons for the trp enzyme
genes, interfered with expression of the trp operon by causing premature termination at an attenuation site located between
the operator and the coding regions of the genes of the trp operon. The mRNA leader can assume different shapes, or
conformations, each one stabilized by base pairing (Figure 1). One of these two conformations allows the rest of the operon
to be transcribed and translated, but the other one does not. But how do these states depend upon tryptophan supply?
The secret to this response lies in a tiny protein, or peptide, encoded by the leader. The leader peptide contains
tryptophan codons, and when tryptophan is plentiful, it is translated easily. This leads to the mRNA pairing that prevents
transcription and translation of the rest of the operon. However, if tryptophan is in short supply, the peptide's translation
stalls. This allows the second shape of the base-paired leader to form, which permits transcription and translation to
continue. Note the base pairing that occurs in the different structures. The pairing is not perfectthere are certain
nucleotides that do not pair. However, enough nucleotide interactions are present to stabilize these secondary structures.
The leader's structure plays a central role in mediating attenuation. That is, in the presence of trp, the newly synthesized trp
operon mRNA adopts a conformation that interferes with continued transcription. Conversely, in the absence of trp, this
conformation changes, allowing read-through.
Overcoming Repression: The Lac Operon
Figure 2: Kinetics of induced enzyme synthesis.
Differential plot expressing culmination of -
galactosidase as a function of increase in mass of cells
in a growing culture of E. coli. Since both axes are
expressed in micrograms of protein, the slope of the
line gives galactosidase production as the fraction (P)
of total protein produced in the presence of the
inducer.
1961 Elsevier Jacob, F. et al., Genetic regulatory
mechanisms in the synthesis of proteins, Journal of
Molecular Biology 3, 318-356 (1961). All rights
reserved.
While repression of genes that are not needed provides clear survival benefits, a mechanism must exist for
overcoming repression. Ideally, this mechanism should be responsive to cues to instigate situation-appropriate
changes in gene expression. In the case of the trp operon, the ligand tryptophan is required for the repressor to work
(repressible negative regulation). But other operons respond to the presence of their small molecule signal ligand; that
is, they are negatively regulated by a repressor protein, but they are inducible (i.e., they can be turned on by a signal).
For example, repression of the lac operon by its repressor, called lacI, is inhibited by the ligand allolactose, to which
the repressor protein directly binds. Thus, lactose, from which allolactose is formed, induces the expression of the lac
operon and of genes required for lactose metabolism.
In the absence of lactose in the environment, the lac operon is transcribed at very low levels (Figure 2). However, when
lactose appears in the environment, a molecule produced from it (allolactose) can bind to the repressor (lacI protein),
thereby causing a conformational change. Now unable to bind to the operator/promoter region, the lacI protein can no
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longer block RNA polymerase from transcribing the operon. Note that there is a short period before the operon is fully
expressed and the cell is fully able to metabolize available lactose. This brief delay from basal expression to induced
expression is called induction. After lactose is removed from the environment, the repressor can once again bind to the
operator/promoter, quickly turning off expression of the operon, returning expression to basal levels.
Experiments by F. Jacob and J. Monod provided much of our foundational knowledge of the mechanisms of lactose
metabolism in bacteria. Working with a panel of mutants that had defects in different components of the lac operon
(Table 1), these researchers were able to determine how the system functioned (Jacob & Monod, 1961).
Table 1: Some Mutants Affecting Lactose Metabolism
Mutant Effect on Repressor Resulting Phenotype
Oc (operator constitutive) Repressor cannot bind
operator.
The lac operon is always
expressed, even in the
absence of lactose.
I- (inhibitor minus) Repressor cannot bind
operator.
The lac operon is always
expressed, even in the
absence of lactose.
Is (super repressor
)
Repressor cannot bind
lactose; thus, it cannot be
released from the operator
site.
The lac operon is never
expressed, even in presence of
lactose.
O
c
Regulates Expression of Genes in Cis
In their research, Jacob and Monod noted that the lacI repressor, formed by a tetramer of the protein encoded by the
lacI gene, binds to specific nucleotides in the operator lacO. When that O sequence is mutated, the repressor can no
longer bind, leaving the entire operon induced or "unrepressed." O
c
mutants are therefore constitutively able to
metabolize lactose, because they are always expressing the enzymes from the operon. Thus, there is no induction time, as
described in Figure 1.
When investigators tried to rescue this phenotype by adding a wild-type copy of the operon to the bacteria, they were
unable to change the behavior of the endogenous mutated operon. Here, the researchers placed the wild-type O
c
operon
on a plasmid that was separate from the bacterial chromosome, and both were present in the same cells. Even when a
wild-type copy was present in the cells and there was no lactose present, the cells expressed the lac operon, so the
mutant O
c
was dominant. This suggested that the operator region controls only the genes adjacent to it, on the same
piece of DNA. In other words, the operator functions in a cis-dominant fashion.
LacI
-
: The Repressor Mutant
The case of the lacI repressor mutant, denoted lacI
-
, was quite different. Constitutive expression of the operon is also
seen in lacI
-
cells. But, contrary to O
c
mutants, the lacI
-
phenotype can be overcome by the addition of a wild-type lacI
gene on a plasmid. This is because the wild-type lacI repressor protein is made correctly from the gene encoded by the
plasmid. The wild-type lacI protein can then bind to any lac operon operator sequence, including the endogenous
version; thus, the repressor can act in trans. Because the wild-type lacI can rescue lacI
-
, the mutant version is
recessive.
LacI
s
: Inhibiting Interactions Between the Repressor and Lactose
In the case of a third mutant, lacI
s
, the result is a repressor that is constitutively bound to the operator. Normally, the
repressor protein has two conformations, or shapes. In one conformation, it is bound to the operator. When lactose is
present, however, the lactose binds to the repressor, causing a change in conformation, and releasing the repressor from
the operator. In lacI
s
mutants, the binding site for lactose is lost in the repressor protein. As a result, no matter how much
lactose is in the system, the operon stays in the "off" state. Moreover, if wild-type lacI is added on a plasmid, it cannot
rescue this mutant. Thus, the mutation is dominant.
Cis-Acting Sequences and Trans-Acting Proteins
Interestingly, the relatively simple mechanisms of gene expression in prokaryotic cells, as exemplified by the trp and lac
operons, provide insight into several general principles involved in regulation in eukaryotes. For example, specific
sequences in DNA serve as binding sites for specific proteins that modulate the binding of RNA polymerase, the enzyme
required for mRNA transcription. These operator sequences in DNA act in cis; in other words, they control the
expression of genes on the same contiguous piece of DNA, generally in fairly close proximity. In contrast, the proteins
that bind those sites act in trans; this means they can be produced by a gene elsewhere in the genome and act wherever
the consensus sequence is located. Furthermore, the ability of E. coli to switch gene expression on and off under
different environmental conditions is an important fundamental example of how cells of all types sense their environment
in order to regulate gene expression.
References and Recommended Reading
Jacob, F., & Monod, J. The operon: A group of genes with expression coordinated by an operator. Comptes Rendus
Biologies 328, 514520 (1960)
---. Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology 3, 318356 (1961)
Oxender, D. L., et al. Attenuation in the Escherichia coli tryptophan operon: Role of RNA secondary structure
involving the tryptophan codon region. Proceedings of the National Academy of Sciences 76, 55245528
(1979)
How Is Gene Expression
Unit 1
Negative Transcription
Regulation in Prokaryotes
Explore Further
Positive Transcription
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Repressed in Bacteria?
Overcoming Repression: The
Lac Operon
Cis-Acting Sequences and
Trans-Acting Proteins
References and Recommended
Reading
Operons and Prokaryotic
Gene Regulation
Simultaneous Gene
Transcription and Translation
in Bacteria
Attenuation in the control of
expression of bacterial
operons
Control: The Glucose Effect
Simultaneous Gene
Transcription and Translation
in Bacteria
Gene Expression and
Regulation Topic Room
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