Tryptophan operon

   

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

[2] Instant Notes in Biochemistry, David Hames, Nigel Hooper

[3] Lewin's Essential GENES, Benjamin Lewin

© Copyright, 2018 All Rights Reserved.

Transcription in Prokaryotes

DNA is a special part of the cell. Without DNA, there would not have been life, evolution, survival, and existence. The base of synthesizing RNA starts from a DNA strand. Hence, DNA has a strict impact on the processes occurring in the body. DNA gets transcribed into RNA. Studying transcription helps to investigate how mutations affecting transcription cause inherited diseases. The first step involved in gene expression is transcription. Interested in cloning or treating a genetic disorder with DNA? Then keeping updates regarding transcription processes is helpful. RNA is a key nucleic acid involved in synthesizing proteins. Watson and Crick proposed the central dogma. It is a two-step process denoted as DNA leading to transcription of RNA and RNA leading to translation of the protein. Not all genes encode for proteins. Hence, not all DNA gets transcribed to RNA.

A gist of DNA replication:

Initiation of replication starts at a point where the unwinding of DNA takes place. Enzymes synthesize an RNA primer and the fragments so that new nucleotides get added. The segments get elongated through a process of elongation followed by removal of the primers. The unjoined fragments get ligated to end the process of replication. Using DNA as a template, the transcription starts.

RNA synthesis:

Genes are known as ordered sequences of nucleotide bases encoding polypeptide chains via RNA molecules. They are nothing but the pieces of DNA that consist of specific information for making a particular protein. Each gene associates with regulatory sequences known as gene regulatory elements. They are involved in regulation of transcription. The process of transcription starts with denaturation of the DNA double helix. The enzyme known as RNA polymerase catalyzes the process of transcription. In prokaryotes, RNA polymerase is responsible for unwinding. The process of unwinding in eukaryotes occurs with the help of other proteins. The DNA starts unwinding next to the gene involved the process. Hence, RNA polymerase starts catalyzing the RNA synthesis. The RNA gets synthesized from 5’-3’ direction along the 3’-5’ template strand. Out of the two DNA strands, only one strand participates in the process of transcription. Total four nucleotide phosphates act as precursors for transcription. They include ATP, GTP, CTP, and UTP. Recall that DNA synthesis requires RNA primers. However, RNA synthesis does not require primers. RNA polymerases are efficient in initiating the synthesis of new polynucleotide chains without any primers. Participant molecules in RNA synthesis are DNA strand, RNA polymerases, nucleoside triphosphates, elongation and termination factors. Also, there is a requirement of specific gene sequences for the initiation of transcription. In both prokaryotes and eukaryotes, the process of transcription occurs in three consecutive steps such as initiation, elongation, and termination. The eukaryotic transcription needs an understanding of prokaryotic transcription first. 
A prokaryotic gene responsible for transcription needs the three following sequences:

·    The promoter sequence is an upstream sequence present at the start of the RNA coding gene sequences.

·   RNA coding gene sequences are known as DNA sequences capable of getting transcribed to RNA.

·        The terminator specifies the destination point of transcription.

The genes specifying the initiation process have two promoter sequences known as -35 and -10. Each promoter has a specific consensus sequence.

Promoter region	No. of base pairs upstream	Consensus sequence
-35	35	5’-TTGACA-3’
-10	10	5’-TATAAT-3’

Table: Promoters -35 and -10 respectively.

Initiation:

RNA synthesis gets initiated by the recruitment of RNA polymerase holoenzyme. It binds to the promoter region. The holoenzyme consists of the core enzyme form of RNA polymerase. It has four polypeptides such as two α, one β, and one β’ polypeptides bound to a sigma factor. It recognizes both the promoter regions. It first recognizes -35 region where the DNA is a close promoter complex. Then the holoenzyme unwinds the DNA. The untwisted form of the promoter is known as an open promoter complex. The RNA polymerase orients itself to the Pribnow box for further process. Thus, the process of initiation includes two main steps. First, RNA polymerase gets attached to its core promoter. Second, the closed promoter gets converted into an open promoter complex. Then the RNA gets synthesized. For a successful initiation, RNA polymerase now moves away from the promoter region.

Image: Transcription in prokaryotes

Elongation and termination

 The RNA polymerase approximately includes a 30 base pair of DNA. It includes a transcription bubble of 12-14 base pairs. The RNA gets attached to the template strand of the DNA. RNA-DNA base pairs assist the RNA in the attachment. The main feature of RNA polymerase observed during the elongation process does not constantly synthesize the transcript. Instead, it follows a discontinuous synthesis. The process of elongation although rapid gets interspersed by brief pauses. During brief pauses, the active site of the polymerase undergoes a slight structural conformation or gets rearranged. A pause lasts for a few milliseconds and accompanies backtracking thereby occurring randomly. A pause helps in termination of the transcript synthesis. The termination of transcription follows a signal given by terminator sequences. An important protein involved in the process of termination is known as rho (ρ). Hence, there are two types of terminator sequences known as Rho-dependent and Rho-independent terminators. The Rho-dependent terminators are known as type II terminators. They lack A-T string and avoid forming hairpin loops. Rho has RNA binding ATPase domain. Rho-independent terminators, on the other hand, form hairpin loops and consist of inverted repeat sequences. A string of AT base pairs transcribes a string of Us. When the inverted palindrome (with a hairpin loop) gets transcribed, there arises RNA-RNA base pairing. Studies revealed the presence of a flap structure on the surface of RNA polymerase which mediates in termination.

Anti-termination:

It is a process beyond termination. There is an anti-termination process too common in prokaryotes. Anti-termination occurs when RNA polymerase ignores a terminator signal and continues to elongate the transcript until the next signal. Anti-termination provides a mechanism through which few genes at the end of the operon get switched off. Anti-termination protein attaches to the DNA and transfers to the RNA polymerase. However, the reason behind switching off those genes remains unclear.

Premature termination and attenuation:

The primary transcript produced by RNA polymerase is known as a mature mRNA. The bacteria involve coupled transcription and translation. It allows a special type of control known as attenuation. It is a process in which the expression of amino acid biosynthesis gets regulated. Some of the bacteria get bacteriophage infection. Bacteriophages transcribe their genomes using bacterial RNA polymerases.

References:

[1] Transcription, William M. Brown, Philip M. Brown
[2] Molecular Biology of the Cell, Bruce Alberts

© Copyright, 2018  All Rights Reserved