The repressible operons in the bacteria such as E coli involve amino acid biosynthesis. These operons work the opposite way of lac operons. The lac operons get activated in the presence of lactose in the medium. The repressible operons get activated when the respective amino acid is not present in the medium. The bacterial cells require certain amino acids for their cellular processes. Hence, they synthesize these amino acids using amino acid biosynthetic pathway. Tryptophan operon is one of the most important amino acid operons in the E. coli. It consists of genes encoding tryptophan biosynthesis enzymes. In all, there are five structural genes. They are known as trpA, trpB, trpC, trpD, and trpE respectively. There is a leader region known as trpL. Upstream regions of trpE include promoter and operator regions. Within the trpL lies the attenuator (att) site. It plays a crucial role in tryptophan operon regulation. There is a separate region known as trpR for repression.
The trp-repressor gene gets activated in the absence of tryptophan in the medium. The gene synthesizes a repressor protein which remains inactive. It does not bind with the trp- operator. As soon as the trp-repressor protein lacks binding with the operator, the trp- structural genes get expressed to give rise to enzymes involved in the synthesis of tryptophan. In the presence of tryptophan, the enzymes responsible for tryptophan production do not get synthesized.
Organization of genes in a tryptophan operon:
There are two types of genes such as regulatory genes and structural genes. As discussed earlier, the regulatory genes include a promoter, the operator, the trpL leader region with a transcription pause site, the termination site, and the attenuator site. Upon translation, the trpA-E genes help in synthesizing enzymes. The transcription of trpE followed by translation gives rise to Anthranilate synthetase component-I. The transcription of trpD followed by translation gives rise to Anthranilate synthetase component-II. The transcription of trpC followed by translation gives rise to PRA isomerase INGP synthetase. The word PRA indicates phosphoribosyl anthranilate. The InGP indicates Indole-3-glycerol phosphate. The transcription of trpB gene followed by translation gives rise to tryptophan synthetase-β. The transcription of trpA gene followed by translation gives rise to tryptophan synthetase-α. The Anthranilate synthetase component-I and Anthranilate synthetase component-II (PRA synthetase) form an enzyme complex known as I2II2. The tryptophan synthetase-β and tryptophan synthetase-α form an enzyme complex known as α2β2 enzyme complex.
There is a chain of reactions leading to the synthesis of tryptophan. Chorismate gives rise to Anthranilate in the presence of enzyme complex I2II2 and glutamine. The Anthranilate gives rise to phosphoribosyl anthranilate in the presence of I2II2 enzyme complex and phosphoribosyl pyrophosphate. The phosphoribosyl anthranilate gives rise to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP). This step requires the activity of an enzyme known as PRA isomerase InGP synthetase. The CdRP gets converted to InGP (Indole-3-glycerol phosphate) in the presence of the enzyme PRA isomerase InGP synthetase. The InGP produces L-tryptophan. It requires serine and α2β2 enzyme complex.
The trpR gene produces an aporepressor protein. It is an inactive protein. It requires the tryptophan for activation. Upon activation, it binds to the operator. Hence, it prevents the initiation of transcription. The enzymes for tryptophan biosynthesis do not get produced. This type of mechanism is known as repression. It reduces the expression of structural genes to seventy-fold. Abundant active aporepressor indicates an abundant tryptophan. The amino acid tryptophan mimics the activity of the allolactose. Both belong to the examples of effector molecules. Sometimes, tryptophan occurs in very low concentrations. This condition is known as tryptophan starvation. The trp gene expression shoots in tryptophan starvation conditions. All the structural genes and the trpL attenuator get expressed. The transcript proportion directly relates to the amount of tryptophan. The termination of short transcripts also gets achieved. This process is known as attenuation. High amounts of tryptophan lead to an increased proportion of short transcripts.
Image: Tryptophan operon
A molecular model for attenuation:
The leader region of the mRNA consists of a sequence. It synthesizes short polypeptide chains. There is a stop codon in the transcript. There are two adjacent codons before it. They play a crucial role in attenuation. A control region at the promoter end is known as an attenuator. It exerts a transcriptional control based on the translation of a small peptide gene. The presence of tryptophan in this region helps in reducing the rate of transcription of structural genes. The leader peptide mRNA consists of four regions. These regions fold and form secondary structures by complementary base pairing.
The transcription pause signal arises due to the pairing of the first and the second regions. The termination signal arises due to the pairing of the third and the fourth regions. Antitermination signal arises due to the pairing between the second and the third regions. Antitermination prevents the termination and allows the process of transcription to proceed further. The signaling of transcription done by the specific configuration of the leader transcript is known as an attenuator stem. There is a great sign of the pause signal in the bacteria. The bacterial cells show a coupled transcription and translation processes. The coupling of transcription and translation occurs due to the pause of RNA polymerase due to the pairing of the first and the second regions. The pause signal lasts longer. Since the process undergoes a lengthy pause, the ribosomes get adequate time in assembling and initiating the process of translation.
The process of translation requires the tRNAs, the ribosomes, and the RNA transcript. Due to the starvation of tryptophan, the charged tryptophanyl tRNAs reduce dramatically. It happens due to the unavailability of the tryptophan in the cells for aminoacylation. Since the first region gets covered due to the ribosome, the pairing of the first and the second region does not occur. As a result, the regions two and three pairs with each other leading to antitermination signal. Hence, the RNA polymerase transcribes the structural genes.
Consider a condition in which the bacterial cells have abundant tryptophan. These conditions are known as non-starved conditions. Now the ribosome covers the region two. Hence, the second region cannot pair with the third region. However, the third region pairs with the fourth one. Hence, the signal arising due to the pairing between the third and fourth region leads to termination. The 3:4 pairing gives rise to a structure known as an attenuator.
References:
[1] High-yield Cell and Molecular Biology, Volume 845, Ronald W. Dudek
[1] High-yield Cell and Molecular Biology, Volume 845, Ronald W. Dudek
[2] Instant Notes in Biochemistry, David Hames, Nigel Hooper
[3] Lewin's Essential GENES, Benjamin Lewin
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