Tryptophan (Trp) Operon

Tryptophan is one of the 20 amino acids that are essential for protein synthesis in living organisms. However, not all organisms can synthesize tryptophan from simpler molecules. For example, humans and other animals must obtain tryptophan from their diet, while some bacteria and plants can produce it by themselves.

One of the most well-studied examples of bacterial tryptophan biosynthesis is the trp operon in Escherichia coli. An operon is a group of genes that are transcribed together as a single unit of messenger RNA (mRNA). The trp operon consists of five structural genes (trpE, trpD, trpC, trpB and trpA) that encode enzymes for the sequential conversion of chorismate to tryptophan. These genes are arranged in a linear order on the bacterial chromosome and are preceded by a regulatory region that contains a promoter and an operator.

The promoter is a DNA sequence that binds the enzyme RNA polymerase, which initiates transcription of the operon. The operator is another DNA sequence that overlaps with the promoter and acts as a switch to control transcription. The operator can bind a protein called the trp repressor, which prevents RNA polymerase from transcribing the operon when it is active. The trp repressor is encoded by a separate gene (trpR) that is not part of the operon.

The regulation of transcription of the trp operon depends on the availability of tryptophan in the cell. When tryptophan is scarce, the cell needs to synthesize more of it to meet its metabolic demands. In this case, the trp repressor is inactive and cannot bind to the operator, allowing RNA polymerase to access the promoter and transcribe the operon. This results in the production of mRNA that contains the coding sequences of all five structural genes, which are then translated into enzymes for tryptophan biosynthesis.

When tryptophan is abundant, however, the cell does not need to produce more of it and can save energy and resources by turning off transcription of the operon. In this case, tryptophan acts as a co-repressor that binds to and activates the trp repressor, enabling it to bind to the operator and block RNA polymerase from accessing the promoter. This results in the repression of transcription of the operon and prevents the synthesis of enzymes for tryptophan biosynthesis.

This type of regulation is called negative feedback, because it involves a product (tryptophan) inhibiting its own synthesis by repressing gene expression. It is also an example of negative control, because it involves a repressor protein preventing transcription.

However, repression by the trp repressor is not the only mechanism that regulates transcription of the trp operon. There is another level of control that fine-tunes expression of the operon based on the availability of tryptophan inside the cell. This mechanism is called attenuation and involves a special region in the mRNA called the leader sequence. The leader sequence contains four segments that can form different base-paired structures that affect transcription termination. The details of attenuation will be explained in point 4.

In summary, the trp operon is a group of genes that encode enzymes for tryptophan biosynthesis in bacteria. It is regulated by two mechanisms: repression by the trp repressor and attenuation by the leader sequence. Both mechanisms depend on the availability of tryptophan in the cell and ensure that transcription of the operon occurs only when it is needed.

The trp operon contains five structural genes that encode enzymes for the biosynthesis of tryptophan from chorismate, a precursor molecule derived from the shikimate pathway. The five structural genes are:

• trpE: It encodes the enzyme anthranilate synthase I, which catalyzes the first step of tryptophan biosynthesis. This enzyme converts chorismate and glutamine into anthranilate and glutamate. Anthranilate is the starting point for the synthesis of the indole ring of tryptophan.
• trpD: It encodes the enzyme anthranilate synthase II, which forms a complex with anthranilate synthase I and regulates its activity. This enzyme also provides feedback inhibition by binding to tryptophan and reducing the affinity of anthranilate synthase I for chorismate.
• trpC: It encodes two enzymes in a single polypeptide chain: N-5`-phosphoribosyl anthranilate isomerase and indole-3-glycerolphosphate synthase. The former enzyme converts anthranilate and 5-phosphoribosyl-1-pyrophosphate (PRPP) into N-5`-phosphoribosyl anthranilate (PRA). The latter enzyme converts PRA into indole-3-glycerolphosphate (IGP), which is the precursor for the synthesis of the pyrrole ring of tryptophan.
• trpB: It encodes the enzyme tryptophan synthase-B subunit, which forms a tetrameric complex with tryptophan synthase-A subunit. This enzyme catalyzes the cleavage of IGP into indole and glyceraldehyde 3-phosphate (G3P). Indole is then transferred to the active site of tryptophan synthase-A subunit.
• trpA: It encodes the enzyme tryptophan synthase-A subunit, which catalyzes the final step of tryptophan biosynthesis. This enzyme condenses indole and serine to form tryptophan and water.

The five structural genes are arranged in an operon, meaning that they are transcribed as a single polycistronic mRNA under the control of a common promoter and operator. The expression of this operon is regulated by two mechanisms: repression by the trp repressor protein and attenuation by the leader sequence. These mechanisms ensure that tryptophan biosynthesis only occurs when tryptophan is needed by the cell.

The trp operon is regulated by a negative feedback mechanism that involves a repressor protein and a co-repressor molecule. The repressor protein is encoded by the trpR gene, which is located outside the trp operon and is constitutively expressed. The co-repressor molecule is tryptophan itself, which binds to the repressor protein and changes its conformation.

In the absence of tryptophan, the repressor protein is inactive and cannot bind to the operator sequence (Otrp) that overlaps with the promoter sequence (Ptrp) of the trp operon. This allows RNA polymerase to bind to the promoter and initiate transcription of the five structural genes (trpE, trpD, trpC, trpB and trpA) that encode the enzymes for tryptophan biosynthesis. The resulting polycistronic mRNA is then translated into proteins that catalyze the conversion of chorismate to tryptophan.

In the presence of tryptophan, the repressor protein binds to tryptophan and becomes active. The active repressor-tryptophan complex then binds to the operator sequence and blocks RNA polymerase from accessing the promoter. This prevents transcription of the structural genes and reduces the production of tryptophan biosynthetic enzymes. This way, the cell can avoid wasting energy and resources on making more tryptophan than it needs.

The regulation of the trp operon by the repressor-tryptophan complex is an example of negative control, because it inhibits gene expression when a specific molecule is present. It is also an example of feedback inhibition, because the end product of a metabolic pathway (tryptophan) inhibits its own synthesis by affecting gene expression.

Attenuation is a mechanism that regulates the expression of the trp operon by controlling the termination of transcription at an early stage. Attenuation depends on the interaction between the leader sequence of the trp mRNA and the ribosome that translates it.

The leader sequence is a short segment of nucleotides at the 5` end of the trp mRNA, upstream of the structural genes. It encodes a 14 amino acid peptide that contains two tryptophan residues. The leader sequence also has four regions that can form base-paired stem-loop structures with each other. These regions are numbered 1 to 4, and region 3 can pair with either region 2 or region 4.

The formation of different stem-loop structures determines whether transcription will continue or terminate. If region 3 pairs with region 4, a 3:4 stem-loop is formed, which acts as a transcription terminator. This means that RNA polymerase will stop transcribing and release the mRNA. This is called attenuation, because transcription is attenuated before reaching the structural genes.

However, if region 3 pairs with region 2, a 2:3 stem-loop is formed, which does not act as a terminator. This means that transcription will proceed to the structural genes and produce a full-length mRNA. This is called anti-termination, because transcription is not terminated by the leader sequence.

The formation of different stem-loop structures depends on the availability of tryptophan in the cell. When tryptophan is abundant, the ribosome that translates the leader sequence will not pause at the two tryptophan codons in region 1. This will allow region 2 to be exposed and pair with region 3, forming a 3:4 terminator loop. Thus, transcription will be attenuated when tryptophan is present.

When tryptophan is scarce, however, the ribosome will pause at the two tryptophan codons in region 1, due to the lack of charged tRNA molecules. This will prevent region 2 from pairing with region 3, and instead allow region 3 to pair with region 4, forming a 2:3 anti-terminator loop. Thus, transcription will continue when tryptophan is absent.

Therefore, attenuation is a way of fine-tuning the expression of the trp operon based on the feedback from translation. It allows the cell to adjust the synthesis of tryptophan according to its demand and supply.

When tryptophan is abundant in the cell, it acts as a co-repressor that binds to the trp repressor protein and activates it. The activated repressor then binds to the trp operator sequence and blocks the binding of RNA polymerase to the trp promoter. This prevents transcription of the structural genes of the trp operon that encode enzymes for tryptophan biosynthesis. This is a negative feedback mechanism that ensures that the cell does not produce more tryptophan than it needs.

In addition to repression, transcription of the trp operon is also regulated by attenuation. Attenuation is a process that involves premature termination of transcription based on the formation of different stem-loop structures in the leader sequence of the trp mRNA. The leader sequence contains four regions that can pair with each other in different ways depending on the availability of tryptophan.

When tryptophan is abundant, the ribosomes that are translating the leader sequence do not pause at the two tryptophan codons in region 1. They quickly synthesize the leader peptide and reach the stop codon between region 1 and 2. This leaves region 3 free to pair with region 4 and form a 3:4 stem-loop structure that acts as a transcription terminator. This causes RNA polymerase to dissociate from the DNA template and stop transcription before reaching the structural genes. Therefore, when tryptophan is abundant, attenuation reduces the expression of the trp operon even further.

The combined effect of repression and attenuation is that transcription of the trp operon is almost completely shut off when tryptophan is abundant in the cell. This saves energy and resources for the cell and prevents wasteful production of tryptophan.

When tryptophan is scarce in the cell, the trp operon needs to be expressed to synthesize more tryptophan. In this case, both the repressor and the attenuator mechanisms are inactive, allowing transcription to proceed.

• The trp repressor protein is synthesized by the trpR gene, but it remains in an inactive form that cannot bind to the operator sequence. Therefore, the operator is free and RNA polymerase can bind to the promoter and initiate transcription of the structural genes.
• The leader sequence of the trp mRNA is also transcribed and translated by a ribosome. However, when the ribosome encounters the two tryptophan codons in region 1, it pauses due to the lack of tryptophanyl-tRNA. This allows region 2 to pair with region 3 and form a 2:3 anti-terminator loop that prevents region 4 from forming a terminator loop. As a result, the 3:4 attenuator loop does not form and transcription does not terminate prematurely. Instead, the ribosome resumes translation after a delay and releases the mRNA, allowing RNA polymerase to continue transcribing the structural genes.

Thus, when tryptophan is scarce, both levels of regulation are relieved and the trp operon is fully expressed to produce more tryptophan.

The trp operon is a classic example of how bacteria can regulate gene expression in response to environmental signals. The trp operon allows the cell to adjust the synthesis of tryptophan biosynthetic enzymes according to the availability of tryptophan in the medium. The trp operon uses two mechanisms to achieve this regulation: repression and attenuation.

Repression is a negative control mechanism that involves the binding of a repressor protein to the operator sequence of the operon, blocking transcription initiation by RNA polymerase. The repressor protein is encoded by a separate gene, trpR, and is activated by tryptophan as a co-repressor. When tryptophan levels are high, the repressor-co-repressor complex binds to the operator and shuts off transcription of the structural genes. When tryptophan levels are low, the repressor is inactive and cannot bind to the operator, allowing transcription to proceed.

Attenuation is a fine-tuning mechanism that involves the premature termination of transcription before reaching the structural genes of the operon. Attenuation depends on the formation of alternative stem-loop structures in the leader sequence of the mRNA, which is transcribed from a region upstream of the first structural gene, trpE. The leader sequence encodes a short peptide with two tryptophan residues and has four regions that can base pair with each other. The ribosome can start translating the leader peptide while transcription is still ongoing, and its speed depends on the availability of tryptophan-charged tRNAs. When tryptophan levels are high, the ribosome quickly translates the leader peptide and stops between regions 1 and 2, allowing regions 3 and 4 to form a terminator loop that signals transcription termination. When tryptophan levels are low, the ribosome stalls at the two tryptophan codons in region 1, preventing regions 3 and 4 from pairing and allowing regions 2 and 3 to form an anti-terminator loop that enables transcription to continue.

By using these two mechanisms, the trp operon can efficiently regulate its expression according to the cellular needs for tryptophan. This saves energy and resources for the cell and prevents wasteful production of enzymes that are not required.

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