Is Used To Control Transcription Of Some Anabolic Pathways Involved In Amino Acid Biosynthesis.

Tryptophan Operon of Escherichia coli

C. Yanofsky , in Brenner'due south Encyclopedia of Genetics (Second Edition), 2013

Abstract

The trp operon of E. coli contains five major structural genes encoding all seven protein functional domains necessary for tryptophan biosynthesis from the mutual effluvious precursor, chorismate. Transcription of the trp operon is highly regulated. Initiation of transcription at the trp promoter is regulated by the tryptophan-activated trp repressor. The repressor can bind at multiple operator sites located in the promoter region. Transcription of the structural genes of the trp operon also is regulated by transcription attenuation, in response to the accumulation of uncharged tRNA^Trp. When this uncharged tRNA accumulates, it leads to ribosome stalling during attempted translation of the leader peptide coding region. This leads to the formation of an RNA antiterminator construction that prevents the RNA terminator structure from forming. Absence of the terminator structure allows polymerase to continue transcription into the structural genes of the operon. When there are adequate levels of charged tRNA^Trp in the cell, the ribosome translating the leader peptide coding region completes leader peptide synthesis, allowing the RNA terminator structure to class, and end transcription.

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Factor Expression: Transcription of the Genetic Code

Chang-Hui Shen , in Diagnostic Molecular Biology, 2019

Transcription Attenuation

The trp operon of E. coli codes for the enzymes that the bacterium needs to make the amino acrid tryptophan. Like the lac operon, the trp operon is a negative control mechanism. The lac operon responds to an inducer that causes the repressor to dissociate from the operator, derepressing the operon. The trp operon responds to a repressor protein that binds to 2 molecules of tryptophan. When the tryptophan is plentiful, this repressor-tryptophan circuitous binds to the trp operator. This binding prevents the binding of RNA polymerase, so the operon is not transcribed (Fig. iii.20). On the other hand, when tryptophan levels are reduced, the repressor will not bind the operator, and so the operon is transcribed. This is an example of a arrangement that is repressible and under negative regulation.

In add-on to the standard negative regulation, the trp operon is regulated by some other mechanism of control called transcription attenuation. This mechanism operates by causing premature termination of transcription of the operon when tryptophan is abundant. As shown in Fig. three.20, there are two loci, the trp leader and the trp attenuator, in between the operator and the gene trpE. Secondary structures formed in the mRNA of the leader sequence are responsible for this premature termination. The formation of such secondary structures comes from the transcription terminate signals—an inverted repeat and a string of eight A-T pairs in the attenuator. When tryptophan is scarce, the operon is translated unremarkably. When information technology is plentiful, transcription is terminated prematurely after the leader sequences accept been transcribed. Thus, attenuation imposes an extra level of control on an operon, over and above the repressor-operator system.

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Regulation of Factor Expression

Due north.Five. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry, 2011

Tryptophan (Trp) Operon

The tryptophan operon is responsible for the production of the amino acid tryptophan, whose synthesis occurs in five steps, each requiring a particular enzyme. In Due east. coli, these enzymes are translated from a single polycistronic mRNA. Side by side to the enzyme coding sequences in the DNA are a promoter, an operator, and two regions called the leader and the attenuator (Figure 24-iii). The leader and attenuator sequences are transcribed. Some other gene (trpR) encoding a repressor is located some distance from this gene cluster.

Regulation of the trp operon is determined by the concentration of tryptophan; when adequate tryptophan is present in the growth medium, there is no need for tryptophan biosynthesis. Transcription is turned off when a high concentration of tryptophan is present, and is turned on when tryptophan is absent. The regulatory signal is the concentration of tryptophan itself. In dissimilarity to lactose, tryptophan is agile in repression rather than induction.

The trp operon has two levels of regulation: an on-off machinery and a modulation system. The protein production of the trpR gene (the trp aporepressor) cannot bind to the operator, in contrast to the lac repressor. Even so, if tryptophan synthesis is present, the aporepressor and the tryptophan molecule join together to course an active repressor circuitous that binds to the operator. When the external supply of tryptophan is depleted (or reduced essentially), the operator becomes exposed, and transcription begins. This blazon of on-off mechanism – activation of an aporepressor by the product of the biosynthetic pathway – has been observed in other biosynthetic pathways.

When the trp operon is de-repressed, which is unremarkably the case unless the concentration of tryptophan in the medium is very high, the optimal concentration of tryptophan is maintained past a modulating system in which the enzyme concentration varies with the concentration of tryptophan. This modulation is affected by:

one.: Premature termination of transcription before the start structural gene is reached; and
2.: Regulation of the frequency of premature termination by the concentration of tryptophan.

Located between the five′ end of the trp mRNA molecule and the first codon of the trpE gene is a 162-base of operations segment called the leader (a full general term for such regions). Within the leader is a sequence of bases (bases 123 through 150) with regulatory activity. After initiation of mRNA synthesis, most mRNA molecules are terminated in this region (except in the complete absenteeism of tryptophan), yielding a short RNA molecule consisting of only xl nucleotides and terminating before the structural genes of the operon. This region in which termination occurs is a regulatory region called the attenuator. The base sequence around which termination occurs (Effigy 24-4) has the usual features of a transcription termination site – namely, a possible stem-and-loop configuration in the mRNA followed by a sequence of eight AU pairs.

The leader sequence has an AUG codon that is in-stage with a UGA terminate codon; together these get-go-stop signals encode a polypeptide of 14 amino acids. The leader sequence has an interesting feature – at positions x and eleven are two side by side tryptophan codons.

Premature termination of mRNA synthesis is mediated through translation of the leader peptide. The two tryptophan codons make translation of the leader polypeptide sequence quite sensitive to the concentration of charged tRNA^Trp. If tryptophan is limiting, there will be insufficient charged tRNA^Trp and translation will intermission at the tryptophan codons. Thus biosynthesis of trp depends on two characteristics of factor regulation in leaner:

1.: Transcription and translation are coupled;
2.: Base-pair formation in mRNA is eliminated in any segment of the mRNA that is in contact with the ribosome.

Figure 24-5 shows that the end of the trp leader peptide is in segment one and a ribosome is in contact with most 10 bases in the mRNA by the codons beingness translated. When the final codons are beingness translated, segments 1 and ii are not paired. In a coupled transcription–translation arrangement, the leading ribosome is not far behind the RNA polymerase molecule. Thus, if the ribosome is in contact with segment 2, when synthesis of segment 4 is beingness completed, so segments 3 and iv are free to form the duplex region iii–4 without segment 2 competing for segment iii. The presence of the 3–4 stalk-and-loop configuration allows termination to occur when the terminating sequence of vii U's is reached.

If exogenous tryptophan is not nowadays or is present in very small amounts, the concentration of charged tRNA^Trp will be limiting, and occasionally a translating ribosome volition be stalled for an instant at the tryptophan codons. These codons are located 16 bases earlier the beginning of segment two. Thus, segment 2 will exist free before segment 4 has been translated and the 2–3 duplex volition form. In the absence of the iii–4 stem-and-loop, termination volition non occur and the complete mRNA molecule will exist made, including the coding sequences for the trp genes. Thus one time over again, if tryptophan is present in excess, termination occurs and little enzyme is synthesized; if tryptophan is absent, there is no termination and the enzymes are fabricated. At intermediate concentrations, the frequency of ribosome pausing will be such every bit to maintain the optimal concentrations of enzymes. This tryptophan regulatory mechanism is called attenuation and has been observed for several amino acid biosynthetic operons, eastward.g., histidine and phenylalanine. Some bacterial operons are regulated solely by attenuation without repressor–operator interactions.

Temporal mRNA Regulation in Phage Systems (see website).

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trp Operon and Attenuation

Paul Gollnick , in Encyclopedia of Biological Chemistry, 2004

The Due east. coli trp Operon: Attenuation Based on Translation of a Leader Peptide

The East. coli trpEDCBA operon encodes the enzymes required to synthesize L-tryptophan from chorismic acid. Transcription of the trp operon is regulated in response to changes in intracellular tryptophan levels. When the cells comprise adequate amounts of tryptophan, for example when it is present in the growth medium, transcription of the operon is down-regulated. In contrast, when tryptophan is limiting, the trp operon is actively transcribed in order to express the enzymes required for its synthesis. Initiation of transcription is regulated by the trp repressor, a DNA-binding poly peptide encoded by the trpR gene. In addition, subsequently transcription has initiated, the elongating transcription circuitous is subject to regulation by attenuation. Together, repression (80-fold) and attenuation (8-fold) serve to allow ∼600-fold overall control of transcription of the trp operon in response to various levels of tryptophan availability.

Attenuation Command of the E. coli trp Operon

The E. coli trp operon contains a 162 bp leader region prior to the showtime of the trpE coding sequence. The trp leader transcript contains several inverted repeats, composed of the segments labeled 1–four in Effigy 1, that can form 3 different overlapping base-paired RNA secondary structures. These structures include an intrinsic transcription terminator (3:four), an overlapping antiterminator (2:3), and a pause structure (1:ii). In addition, the leader transcription contains a small open up reading frame (ORF) that encodes a xiv-amino acrid leader peptide, which contains 2 critical tandem UGG Trp codons. The cell's ability to efficiently translate these two Trp codons determines which RNA construction forms in the nascent leader transcript, which in turn controls whether transcription halts in the leader region or continues into the structure genes of the operon.

Shortly later transcription initiates from the trp promoter, the one:two pause hairpin forms (Figure 1). This structure signals RNA polymerase to pause transcription afterwards nucleotide 92. This pausing of RNA polymerase is a disquisitional feature of the attenuation mechanism because it allows fourth dimension for a ribosome to initiate translation of the leader peptide. When the ribosome begins translating the leader peptide this releases the paused RNA polymerase to resume transcription. Transcription and translation are now coupled, with the ribosome closely following RNA polymerase. This state of affairs is essential to allow events involving the ribosome to affect transcription past the associated RNA polymerase.

At this signal there are two possible pathways for the attenuation mechanism to follow depending on the level of tryptophan in the jail cell. The selection depends on how efficiently the tandem Trp codons in the leader peptide are translated. This efficiency reflects the availability of aminoacylated tRNA^Trp in the cell. Under weather where tryptophan is limiting, the amount of charged tRNA^Trp is low. As a effect of this low concentration of tryptophanyl-tRNA^Trp translation of the tandem Trp codons is inefficient and the ribosome stalls at one of these two codons. The associated RNA polymerase continues to transcribe through the trp leader region and transcription and translation go uncoupled. As RNA polymerase proceeds through segments ii and 3 of the leader region, the antiterminator structure (two:3) forms, which prevents germination of the overlapping intrinsic terminator (three:4) structure. Hence transcription continues through the leader region and into the trp structural genes.

When tryptophan is plentiful, the level of charged tRNA^Trp in the prison cell is high. This allows efficient translation of the tandem Trp codons and hence the ribosome proceeds rapidly to the cease of the leader peptide. When the ribosome reaches the leader peptide cease codon, it covers part of RNA segment 2 and thus prevents formation of the ii:3 antiterminator structure as transcription proceeds. This frees RNA segment 3 to base pair with segment 4 and course the terminator. Under these conditions transcription terminates in the leader region prior to the trp structural genes, which are therefore non expressed.

In this attenuation mechanism the regulatory point is the level of charged tRNA^Trp and the sensory event is the efficiency with which the ribosome tin can interpret the tandem Trp codons in the leader peptide. This system can hands be adjusted to regulate other bacterial amino acid biosynthetic operons. The simply needed modification is to alter the identity of the critical codons in the leader peptide, which controls the amino acid the organization will answer to. In that location are numerous examples of such adaptations of this attenuation machinery, specially in enteric bacteria, including the his, phe, and leu operons, which contain vii His, seven Phe and four Leu codons in their respective leader peptides.

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Gene Expression in Bacterial Systems: The trp Operon and Attenuation

P. Gollnick , in Encyclopedia of Biological Chemistry (Second Edition), 2013

The E. coli trp Operon: Attenuation Based on Translation of a Leader Peptide

The E. coli trpEDCBA operon encodes the enzymes required to synthesize L-tryptophan from chorismic acid. Transcription of the trp operon is regulated in response to changes in the levels of tryptophan inside the prison cell. When cells contain adequate amounts of tryptophan, for example, when information technology is present in the growth medium, transcription of the operon is downregulated. Past dissimilarity, when the cell has low levels of tryptophan, the trp operon is actively transcribed in gild to express the enzymes required for synthesizing more. Initiation of transcription is regulated by the trp repressor, a DNA-bounden protein encoded past the trpR gene. In add-on, after transcription has initiated, the elongating transcription complex is subject to regulate by attenuation. Together, repression (80-fold) and attenuation (eightfold) serve to allow approximately 600-fold overall control of transcription of the trp operon in response to various levels of tryptophan availability.

Attenuation Control of the Eastward. coli trp Operon

The E. coli trp operon contains a 162-nucleotide leader region prior to the start of the trpE coding sequence. In that location are several inverted repeats in the trp leader mRNA transcript, which are equanimous of the segments labeled i–4 in Figure one . These segments tin base-pair to form 3 different overlapping base-paired RNA secondary structures ( Figure i ). These structures include an intrinsic transcription terminator (3:4), an overlapping antiterminator (ii:3), and a intermission structure (1:2). In addition, the leader transcription contains a small open reading frame (ORF), which encodes a 14-amino-acid leader peptide. There are two critical tandem UGG Trp codons inside this ORF. How efficiently these ii Trp codons tin be translated determines which RNA structure forms in the nascent leader transcript, which in turn controls whether transcription halts in the leader region or continues into the construction genes of the operon.

Shortly afterwards transcription initiates from the trp promoter, the 1:2 pause hairpin forms in the nascent RNA ( Figure one ). This construction signals RNAP to append transcription afterward nucleotide 92. Pausing of RNAP is a critical feature of the attenuation mechanism because it allows time for a ribosome to initiate translation of the leader peptide. When a ribosome begins translating the leader peptide, this releases the paused RNAP to resume transcription. Transcription and translation are at present coupled, with the ribosome closely following RNAP. This state of affairs is essential to allow events involving the ribosome to impact transcription by the associated RNAP.

At this signal, at that place are two possible pathways for the attenuation mechanism to follow depending on the level of tryptophan in the cell. The choice depends on how efficiently the tandem Trp codons in the leader peptide are translated. This efficiency reflects the availability of aminoacylated tRNA^Trp in the prison cell. Under atmospheric condition where tryptophan is limiting, the amount of charged tRNA^Trp is depression. As a result of the low concentration of tryptophanyl-tRNA^trp, translation of the tandem Trp codons is inefficient and the ribosome stalls at 1 of these ii codons. The associated RNAP continues to transcribe through the trp leader region and transcription and translation get uncoupled. As RNAP continues transcribing through segments 2 and 3 of the leader region, the antiterminator structure (2:iii) forms, which prevents formation of the overlapping intrinsic terminator (three:4) structure. Hence, transcription continues through the leader region and into the structural genes of the operon which encode the tryptophan biosynthetic enzymes.

When tryptophan is plentiful, the level of charged tRNA^Trp in the jail cell is high. This situation allows efficient translation of the tandem Trp codons and hence the ribosome gain rapidly to the finish of the leader peptide. When the ribosome reaches the leader peptide stop codon, it covers office of RNA segment two and prevents germination of the 2:3 antiterminator structure as transcription proceeds. Thus, RNA segment iii is free to base-pair with segment iv and form the transcription terminator. Under these conditions, transcription terminates in the leader region prior to the trp structural genes, which are therefore not expressed.

In this attenuation mechanism the regulatory bespeak is the level of aminoacylated tRNA^Trp and the sensory event is the efficiency with which the ribosome can translate the tandem UGG Trp codons in the leader peptide. This system can easily be adapted to regulate other amino acid biosynthetic operons. The only modification that is needed is to change the identity of the disquisitional codons in the leader peptide, which controls the response of the organization to a specific amino acid. In that location are numerous examples of such adaptations of this attenuation machinery, specially in enteric bacteria, including the his, phe, and leu operons, which contain seven His, seven Phe, and four Leu codons in their respective leader peptides.

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Transcription | Expression of the Bacterial L-Trp Regulon☆

Luis R. Cruz-Vera , in Encyclopedia of Biological Chemistry (3rd Edition), 2021

Sensing L-Trp Related Metabolites

Pseudomonas aeruginosa has its trp operon genes dispersed in three master genetic subunits. P. aeruginosa do not express the TrpR poly peptide. The majority of the trp operon genes are nether the command of the trpL regulatory factor, which senses the accumulation of uncharged tRNA^Trp (Fig. four(A)). However, P. aeruginosa expresses a regulatory protein named TrpI, which instead of detecting complimentary 50-Trp and blocking the expression of genes, this regulatory protein detects indoleglycerol phosphate (InGP), inducing the expression of the trpA and trpB genes that institute the Tryptophan synthase circuitous (Fig. 4(B)) (Manch and Crawford, 1982). InGP is an intermediate metabolite of the L-Trp synthesis pathway, which Tryptophan synthase A utilizes to catalyse the formation of indole (Fig. ane). When 50-Trp concentrations are low, the aggregating of uncharged tRNA^Trp induces the expression of the first four enzymes of the L-Trp anabolic pathway, which results in the aggregating of the InGP intermediate (Fig. 4(A) , <10 µM). InGP interacts with TrpI monomers inducing dimer germination. The dimer binds a promoter region located between the trpI gene and the trpB-A operon, which are transcribed in contrary directions (Fig. 4(B) , >ten µM InGP) (Chang and Crawford, 1990). The interaction of the TrpI-InGP circuitous with its promoter region induces the transcription of the trpB-A operon and represses the transcription of the trpI gene (Fig. four(B) , >10 µM InGP) (Chang and Crawford, 1990). At sufficient or excessive L-Trp concentrations, the majority of the trp operon genes are repressed considering of the depression concentration of uncharged tRNA^Trp (Fig. four(A), ≈/>ten µM). Nether these conditions the concentration of InGP is reduced, the absence of InGP and then reduces the interaction of the TrpI regulator with the promoter region of the trpI gene and trpB-A operon (Fig. 4(B), ≈/<10 µM InGP). These final events decrease the transcription of the trpB-A operon and increment the transcription of the trpI factor (Fig. 4(A), ≈/<ten µM InGP). Therefore, in Pseudomonas the regulation of the trp operon genes is decoupled, for which aggregating of InGP, perhaps produced from other sources, could be utilized to produce 50-Trp without the need to synthesize proteins required for the initial steps in the synthesis of L-Trp.

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Attenuation, Transcriptional

T.M. Henkin , in Encyclopedia of Genetics, 2001

Terminator Proteins

Expression of the Bacillus subtilis trp operon is controlled past TRAP, an unusual RNA binding poly peptide. In the presence of tryptophan, TRAP binds to the trp leader RNA and prevents germination of an antiterminator structure, thereby permitting germination of the competing intrinsic terminator. TRAP assembles into an 11-subunit symmetrical ring, with eleven molecules of tryptophan spaced between the TRAP monomers. The RNA appears to wrap around the outside of the TRAP ring, with contacts betwixt each monomer and GAG/UAG repeats in the RNA binding site. TRAP oligomerization is tryptophan-independent, but RNA binding requires tryptophan, suggesting that tryptophan controls TRAP activeness past causing a conformational change that is required for bounden to its RNA target site. The B. subtilis pyr system is also regulated by bounden of a regulatory protein to the RNA leader region to mediate transcription termination. In this instance, the regulator, PyrR, binds in the presence of UMP, an finish product of pyr operon expression. The target site for PyrR is a complex construction. Bounden to this chemical element precludes germination of an antiterminator construction, which competes with the attenuator. Thus, PyrR causes termination past stabilization of an anti-antiterminator. The trp and pyr systems are similar in that the default state is readthrough of the attenuator in the absence of the stop-product of expression of the operon, and so that transcription volition be prevented only if the required metabolite is present.

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Tryptophan Operon

C. Yanofsky , in Encyclopedia of Genetics, 2001

Regulation of Expression of the trp Operon of Escherichia coli

The five structural genes of the trp operon are preceded by a transcription regulatory region consisting of a promoter/operator, at which transcription initation is regulated, and a transcribed leader segment, within which transcription termination is regulated. Initation at the trp promoter is regulated past the tryptophan-activated trp repressor poly peptide; the extent of repression varies in response to changes in the intracellular concentration of gratuitous tryptophan. Repression regulates operon expression over about an 80-fold range. Polymerase molecules that take initiated transcription at the trp promoter and escaped repression are subject field to a second regulatory mechanism, transcription attenuation. The latter machinery determines whether or not transcription volition finish at a site located in the distal portion of the leader region. This determination is influenced by the intracellular concentration of tryptophan-charged tRNA^Trp. When the Trp-tRNA^Trp concentration is loftier, transcription terminates in the leader region. When tRNA^Trp is mostly uncharged, which occurs when cells experience a severe tryptophan deficiency, termination is avoided and transcription proceeds to the end of the operon. Transcription attenuation in the trp operon of E. coli regulates transcription of the structural genes of the operon over about an eightfold range. The combined action of repression and attenuation regulates transcription of the structural genes of the operon over about a 600-fold range. There is an internal promoter located in the distal portion of trpD (Effigy ane). Transcription initiation at this promoter is unregulated and gain at a frequency less than 10% that attributable to the chief promoter. Tandem sites of transcription termination are located following the trpA structural factor; the first is protein-cistron-independent, a so-called intrinsic terminator, while the second required the protein Rho. Completion of transcription of the operon yields a polycistronic messenger RNA. Ribosomes tin initiate translation at any of the five major ribosome binding sites on this polycistronic messenger.

The trp promoter region of E. coli contains three operators that can bind trp repressor. Operator-leap repressor inhibits transcription initiation. The trp repressor also regulates transcription initiation in several other operons concerned with tryptophan metabolism. The three-dimensional structures of the trp aporepressor (aporepressor lacks bound tryptophan), the trp repressor, and the trp repressor–operator complex, have been determined. These structures have revealed the features of this protein that are responsible for its activation past tryptophan and its recognition of specific operators.

The transcribed leader region of the trp operon of E. coli is most 160 bp in length. Equally mentioned, this genetic segment encodes an mRNA segment that can cause transcription termination in the leader region. The transcript of the leader region tin can fold to class iii RNA structures, termed terminator, antiterminator, and transcription pause structure. The terminator and antiterminator are alternative RNA structures, i.e., they take a sequence of nucleotides in common, thus either, but not both, can be at one fourth dimension. When cells are scarce in charged tRNA^Trp the antiterminator forms; this precludes formation of the terminator. When cells have adequate levels of charged tRNA^Trp, the terminator forms and transcription terminates in the leader region. A deficiency of charged tRNA^Trp is sensed during attempted translation of tandem Trp codons in a fourteen-remainder leader peptide coding region, trpL, located almost the 5′ cease of the trp operon transcript. Coupling of transcription and translation, essential to this mechanism of attenuation, is accomplished by the germination of the transcription pause structure, located nearly the 5′ cease of the transcript. Polymerase pausing allows a ribosome to bind to the transcript and initiate synthesis of the leader peptide. The movement of this ribosome and then releases the paused transcription complex, and transcription and translation keep in unison.

2 of the trp polypeptides, the products of genes trpE and trpA, lack tryptophan, therefore they are synthesized preferentially during severe tryptophan starvation. An boosted regulatory characteristic, translational coupling, insures equimolar synthesis of the polypeptide products of two pairs of adjacent genes, trpE and trpD, and trpB and trpA. As mentioned, the products of these genes form enzyme complexes. The enzyme complex catalyzing the first two reactions in the pathway is feedback-inhibited by tryptophan. The tryptophan binding site is located in the TrpE polypeptide.

The use of ii transcription regulatory mechanisms and feedback inhibition of anthranilate synthase activity allows E. coli to regulate tryptophan biosynthesis efficiently in response to changes in the availability of tryptophan and the charge per unit of protein synthesis.

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The Folding of Proteins and Nucleic Acids

L. Liu , A.M. Gronenborn , in Comprehensive Biophysics, 2012

3.8.3.11 TrpR

The trp repressor (trpR) binds the operator region of the trp operon and prevents the initiation of transcription. Its smallest functional unit is a dimer, ¹⁵⁰ although tetrameric and higher club species are also observed. ^151,152 Under extreme conditions (30% isopropanol), an infinite crystalline three-D supramolecular array was found, ²⁴ possibly involving domain swapping.

Whether or not the trpR dimer constitutes a true domain-swapped case is unclear because a highly intertwined construction was observed and no monomeric homolog is available. Each single polypeptide concatenation in trpR dimer is equanimous of six α helices (A–F) in a relative closed conformation (Figure 14(a)). ²³ In the crystalwide assembly, helices C–E rearrange and form a very long, single helix, resulting in polypeptide termini that are separated by a large distance (Figure 14(b)). Two such dimers come together and create a substructure (half of the tetramer) very similar to that seen in the dimer (Figure fourteen(c)). Therefore, the dimer-like structure is formed past segments of four dissimilar polypeptide bondage, two providing intertwined N-terminal regions and two providing C-terminal regions, ²⁴ with the polymer constituting a branched aggregate rather than a daisy chain-type linear one. Given that the polymeric form was crystallized in the presence of a significant corporeality of alcohol, its relevance to any physiological state is unclear. Indeed, it is well-known that alcohols increment the helical content of flexible peptides and/or destabilize 3rd structures significantly. ^24,153,154

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Chlamydia trachomatis

Huizhou Fan , Guangming Zhong , in Molecular Medical Microbiology (Second Edition), 2015

Variation in the Tryptophan Synthase

Human cells are auxotrophic to tryptophan. In contrast, many microbes are capable of synthesizing tryptophan de novo. Enzymes required for synthesizing tryptophan are encoded on the trp operon. The trp operon in C. trachomatis contains three open reading frames (ORFs), designated trpR, trpB and trpA, which encode proteins TrpR, TrpB and TrpA, respectively. Bacterial tryptophan synthase is generally comprised of two TrpA polypeptides (α₂) and two TrpB polypeptides (β_ii) in an α₂β_ii configuration. The synthase is a bifunctional enzyme, in which the α subunits convert indole glycerol 3-phsophate (IGP) into indole and glyceraldehyde-iii-phosphate, and the β subunits add a serine residual to indole, thereby yielding tryptophan. The two sequential reactions occur in a highly cooperative manner [70–72]; accordingly, removal of ane pair of subunits is sufficient to crusade a near complete halt in the enzyme action of the other pair of subunits [73].

Genetic and biochemical analyses have shown substantial changes in the C. trachomatis tryptophan synthase, as compared with the enzyme in other bacteria. C. trachomatis TrpB shares a relatively high sequence identity with E. coli TrpB (54%), including all critical residues at the active site of the enzyme. In contrast, the caste of amino acid conservation between C. trachomatis TrpA and E. coli TrpA is very depression (27%) [74]. Furthermore, in all the genital serovars, in-frame deletions accept resulted in the deletion of a number of disquisitional residues in TrpA, leading to loss of the ability to catalyse the formation of indole from IGP; all the same, the highly mutated TrpA is still required for the generation of tryptophan by TrpB. In ocular C. trachomatis serovars, there are boosted mutations causing frame-shift, leading to complete inactivation of tryptophan synthesis activity; a complete loss of trpBA has also been institute in an ocular strain. Thus, at that place is a consummate correlation between tryptophan synthesis and C. trachomatis tissue tropism: while all genital serovars can synthesize tryptophan using indole every bit a substrate, ocular serovars cannot.

In addition to TrpA and TrpB ORFs, bacterial trp operons also carry ORFs for enzymes catalysing a series of four additional reactions starting from chorismate to IGP [75,76]. None of these additional genes is plant in the C. trachomatis genome. Thus, C. trachomatis can satisfy the requirement for tryptophan past either salvage from the host cell or synthesizing this amino acid from indole as the but starting fabric. Since human cells cannot produce indole, the source of tryptophan precursor is presumably other microbes, which are in high affluence in the genital tract, but not in the eye. This may explain why genital C. trachomatis serovars take retained a functional tryptophan synthase and the ocular serovars have not.

The tryptophan acquisition mechanism in C. trachomatis is linked to the pathogen's ability to tolerate the antichlamydial cytokine interferon-γ. Human interferon-γ causes tryptophan starvation past inducing the expression of indoleamine-2,3-dioxygenase (IDO), which catalyses the degradation of tryptophan. While indole as well as tryptophan can reverse the inhibitory issue of human interferon-γ on C. trachomatis genital serovars, only tryptophan simply not indole is able to alleviate the growth inhibition in the ocular strains.

Paradoxically, loss of the tryptophan biosynthesis pathway actually contributes to the pathogenesis of ocular strains. Tryptophan starvation can cause chlamydiae to enter persistence, which is well documented in prison cell culture systems [77–79]. Chlamydial persistence is thought to be responsible for trachoma and other chronic eye manifestations. Without the ability to synthesize tryptophan, ocular strains are likely to suffer more severe tryptophan deprivation in the presence of human interferon-γ, and are more forcefully driven to enter persistence, as compared to genital strains.

TrpR is a transcription regulator of the trp operon. The function of TrpR is regulated by the bioavailability of free tryptophan in the microbial cell [76,fourscore–83]. When bacteria are grown in media rich in tryptophan, the homodimeric TrpR is jump to two molecules of tryptophan, forming an active autorepressor complex, which binds to the trp operator, a DNA sequence near a promoter element recognized past the σ factor of the RNA polymerase. Occupation of the trp operator by the autorepressor prevents the RNA polymerase from binding to the trp promoter and initiating trp RNA synthesis. When the concentration of tryptophan drops, tryptophan dissociates from the autorepressor, which in plow dissociates from the trp operator, allowing RNA polymerase to synthesize the trp mRNA [84]. It has been demonstrated that TrpR from C. trachomatis serovar D and L2 binds to the trp promoter in vitro [85,86], and furthermore, TrpR represses transcription of the trp operon in a tryptophan-dependent manner in vitro [85]. As final proof that TrpR acts as a negative regulator of transcription in C. trachomatis, the transcription is no longer regulated by tryptophan in a C. trachomatis L2 variant containing a frameshift mutation in trpR [86].

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