What happens if there is a mutation in the operator gene in the trp operon?

How do bacteria avoid wasteful production of unnecessary proteins when their genes are always on? The answer lies in regulating the operon.

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.

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


© 1981 Yanofsky, C. Attenuation in the control of expression of bacterial operons. Nature 289, 753 (1981). All rights reserved.

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


© 1961 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 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

MutantEffect on RepressorResulting PhenotypeOc (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 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.


Jacob, 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)

What happens if the operator region is mutated?

a) Most mutations in the operator, the binding site for repressor, lead to lower affinity for the repressor and hence less binding. Thus these mutations allow continued transcription (and thus expression) of the lac operon even in the absence of inducer; this is referred to constitutive expression.

What is the role of operator gene in operon?

Operator genes contain the code necessary to begin the process of transcribing the DNA message of one or more structural genes into mRNA. Thus, structural genes are linked to an operator gene in a functional unit called an operon.

What is the function of the operator region of the trp operon?

What does the operator do? This stretch of DNA is recognized by a regulatory protein known as the trp repressor. When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by physically getting in the way of RNA polymerase, the transcription enzyme.

What happens if the operator is removed from the operon?

According to the lac operon model proposed by Jacob and Monod, what is predicted to occur if the operator is removed from the operon? The lac operon would be transcribed continuously.