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PROGRESS

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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. Attenuation in

the control of expression of bacterial operons. Nature 289, 753

(1981). All rights 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 perfect—there 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 β-

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.

Oc 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." Oc 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 Oc 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 Oc 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 Oc 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.

LacIs: Inhibiting Interactions Between the Repressor and Lactose

In the case of a third mutant, lacIs, 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 lacIs 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 ProteinsInterestingly, 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 ReadingJacob, F., & Monod, J. The operon: A group of genes with expression coordinated by an operator. Comptes Rendus

Biologies 328, 514–520 (1960)

---. Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology 3, 318–356 (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, 5524–5528

(1979)

How Is Gene Expression

Unit 1

Negative Transcription

Regulation in ProkaryotesExplore 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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