Trp Operon Research Articles

Metabolic design based on a coupled gene expression—metabolic network model of tryptophan production in Escherichia coli

The presumably high potential of a holistic design approach for complex biochemical reaction networks is exemplified here for the network of tryptophan biosynthesis from glucose, a system whose components have been investigated thoroughly before. A dynamic model that combines the behavior of the trp operon gene expression with the metabolic network of central carbon metabolism and tryptophan biosynthesis is investigated. This model is analyzed in terms of metabolic fluxes, metabolic control, and nonlinear optimization. We compare two models for a wild-type strain and another model for a tryptophan producer.An integrated optimization of the whole network leads to a significant increase in tryptophan production rate for all systems under study. This enhancement is well above the increase that can be achieved by an optimization of subsystems. A constant ratio of control coefficients on tryptophan synthesis rate has been identified for the models regarding or disregarding trp operon expression. Although we found some examples where flux control coefficients even contradict the trends of enzyme activity changes in an optimized profile, flux control can be used as an indication for enzymes that have to be taken into account in optimization.

Tryptophan recycling is responsible for the interferon-gamma resistance of Chlamydia psittaci GPIC in indoleamine dioxygenase-expressing host cells.

Comparative genomics indicates that vast differences in Chlamydia sp. host range and disease characteristics can be traced back to subtle variations in gene content within a region of the chromosome termed the plasticity zone. Genes required for tryptophan biosynthesis are located in the plasticity zone; however, the complement of genes encoded varies depending on the chlamydial species examined. Of the sequenced chlamydia genomes, Chlamydia psittaci GPIC contains the most complete tryptophan biosynthesis operon, encoding trpRDCFBA. Immediately downstream of the trp operon are genes encoding kynureninase and ribose phosphate pyrophosphokinase. Here, we show that, in GPIC, these genes are transcribed as a single transcript, the expression of which is regulated by tryptophan. Complementation analyses, using various mutant Escherichia coli isolates, indicate that the tryptophan biosynthesis, kynureninase and ribose phosphate pyrophosphokinase gene products are functional. Furthermore, growth of C. psittaci GPIC in HeLa cells, cultured in tryptophan-free medium, could be rescued by the addition of anthranilate, kynurenine or indole. In total, our results indicate that this complement of genes enables GPIC to recycle tryptophan and thus accounts for the interferon-gamma resistant phenotype displayed in indoleamine-2,3-dioxygenase-expressing host cells.

Modeling operon dynamics: the tryptophan and lactose operons as paradigms

Understanding the regulation of gene control networks and their ensuing dynamics will be a critical component in the understanding of the mountain of genomic data being currently collected. This paper reviews recent mathematical modeling work on the tryptophan and lactose operons which are, respectively, the classical paradigms for repressible and inducible operons. To cite this article: M.C. Mackey et al., C. R. Biologies 327 (2004).

Features of a Leader Peptide Coding Region that Regulate Translation Initiation for the Anti-TRAP Protein of B. subtilis

The rtpA gene of Bacillus subtilis encodes the Anti-TRAP protein, AT. AT can bind and inhibit the TRAP regulatory protein, preventing TRAP from promoting transcription termination in the trpEDCFBA operon leader region. AT synthesis is upregulated transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). Here we analyze AT's translational regulation by rtpLP, a 10 residue leader peptide coding region located immediately preceding the rtpA Shine-Dalgarno sequence. Our findings suggest that, whenever the charged tRNA(Trp) level is sufficient to allow the ribosome translating rtpLP to reach its stop codon, it blocks the adjacent rtpA Shine-Dalgarno sequence, inhibiting AT synthesis. However, when there is a charged tRNA(Trp) deficiency, the translating ribosome presumably stalls at one of three adjacent rtpLP Trp codons. This stalling exposes the rtpA Shine-Dalgarno sequence, permitting AT synthesis. RNA-RNA pairing may also influence AT synthesis. Production of AT would inactivate TRAP, thereby increasing trp operon expression.

Recycling of a regulatory protein by degradation of the RNA to which it binds.

When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.

Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy

BackgroundThe growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis.ResultsIn 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions. A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource. The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway. Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis. Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently).Conclusions(i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing. There are currently seven tryptophan congruency groups in the Bacteria. Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer). (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced. The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis. Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Ancient origin of the tryptophan operon and the dynamics of evolutionary change.

The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons.

514 Influence of initial electron beam characteristics on Monte Carlo calculated absorbed dose distribution for linac photon beam

The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

A Mg2+-dependent RNA Tertiary Structure Forms in the Bacillus subtilis trp Operon Leader Transcript and Appears to Interfere with trpE Translation Control by Inhibiting TRAP Binding

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg2+-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5′-p-azidophenacyl-containing transcripts revealed a Mg2+-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg2+ inhibits TRAP binding, which may be partially due to the effect of Mg2+ on RNA tertiary structure formation. Results from expression studies using trpE′-′lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg2+-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

A Double Dose of Trp Regulation

Amino acid synthesis is tightly controlled in bacteria by a system in which the amino acids themselves, as well as their corresponding transfer RNAs (tRNAs), regulate production. For example, in Bacillus subtilis , the mechanism for regulating tryptophan (Trp) synthesis involves an antiterminator protein called TRAP, which is activated by Trp. However, TRAP activity is antagonized by a protein called anti-TRAP (AT), where the synthesis of AT is induced by uncharged tRNA Trp . Chen and Yanofsky now show another level of regulation for the Trp operon in which uncharged tRNA Trp also regulates the translation of AT through tandem Trp codons in a leader peptide coding sequence. This mechanism is somewhat reminiscent of the classical Trp attenuation system in Escherchia coli ; however, the leader peptide in B. subtilis exerts its effect at the level of translation instead of transcription termination, as seen in E. coli . The authors term the B. subtilis system "regulatory sophistication" because it shows both transcriptional and translational regulation of AT in response to tRNA Trp . G. Chen, C. Yanofsky, Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA. Science 301 , 211-213 (2003). [Abstract] [Full Text]

Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA.

The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon. Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp). Here we show that translation of AT also is regulated by uncharged tRNATrp. A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene. Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production. Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression.

Roles of the RecJ and RecQ proteins in spontaneous formation of deletion mutations in the Escherichia coli K12 endogenous tonB gene.

The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from recJ(-) and recQ(-) cells. Large deletions, due to simultaneous mutations of the trp operon, were also isolated. The rates of tonB mutation were 2.77 x 10(-8), 4.13 x 10(-8) and 5.00 x 10(-8) for rec(+), recJ(-) and recQ(-) cells, respectively. We analyzed 94 and 99 tonB mutants from the recJ(-) and recQ(-) cells, respectively, by sequencing. We found that IS insertion dominated, followed by base substitutions, frameshifts and deletions in both recJ(-) and recQ(-) strains. We then analyzed 55 tonB-trp deletions, ranging in size from 5907 to 20,832 bp, from the recJ(-) strains and 47 tonB-trp deletions, ranging in size from 4,959 to 16,390 bp from the recQ(-) strains. About one-third of tonB-trp deletions from both the recJ(-) and the recQ(-) cells were found to have occurred between short sequence repeats at the deletion termini. About one-third of tonB-trp deletions from both mutants showed 2-4 bp repeats in the immediate vicinity of the endpoints, which appeared to indicate no clear association with deletion. The remaining one-third of tonB-trp deletions had no homology at the endpoint. These results were similar to those for the rec(+) cells. Hanada and colleagues demonstrated that structually similar rearrangements arising during lambda bio phage formation (illegitimate recombination) increased in the recQ(-) strain. To explain this discrepancy, we interpreted as distinctive the mechanism for rearrangement during transducing phage formation which is recQ-dependent and that for deletions formed in chromosomes which is recQ-independent.

Dynamic model of Escherichia coli tryptophan operon shows an optimal structural design.

A mathematical model has been developed to study the effect of external tryptophan on the trp operon. The model accounts for the effect of feedback repression by tryptophan through the Hill equation. We demonstrate that the trp operon maintains an intracellular steady-state concentration in a fivefold range irrespective of extracellular conditions. Dynamic behavior of the trp operon corresponding to varying levels of extracellular tryptophan illustrates the adaptive nature of regulation. Depending on the external tryptophan level in the medium, the transient response ranges from a rapid and underdamped to a sluggish and highly overdamped response. To test model fidelity, simulation results are compared with experimental data available in the literature. We further demonstrate the significance of the biological structure of the operon on the overall performance. Our analysis suggests that the tryptophan operon has evolved to a truly optimal design.

TRAP–RNA Interactions Involved in Regulating Transcription Attenuation of the Bacillus subtilis trp Operon

This chapter describes the methods that have been used to characterize the interaction of trp RNA-binding attenuation protein (TRAP) with RNA, and to study how these interactions are involved in regulating transcription attenuation of the trp operon. TRAP is encoded by mtrB, the second gene of the mtrAB operon. MtrA encodes GTP cyclohydrolase I, an enzyme involved in folic acid biosynthesis. Both Bacillus subtilis and Bacillus stearothermophilus TRAP have been expressed in Escherichia coli (E.Coli) using the T7 expression system. Several assays have been used to examine the interaction of TRAP with RNA. These assays use 32 P-labeled transcripts containing either the trp leader sequence or artificial TRAP binding sites. One of the quickest and most reliable methods to quantitatively analyze TRAP binding to RNA is the nitrocellulose filter binding assay. Equilibrium dialysis was first used to demonstrate that tryptophan binds to TRAP using an assay. This simple method involves placing TRAP on one side of a dialysis membrane and [ 14 C] L-tryptophan on the other side and then allowing dialysis to proceed until equilibrium is reached. Both transcriptional and translational gene fusions containing the trp promoter and leader region fused to lacZ have been used extensively to examine TRAP function in vivo.

Search for Alternative RNA Secondary Structures Regulating Expression of Bacterial Genes

Expression of many bacterial genes is regulated by formation of alternative secondary RNA structure within the leader mRNA sequence. Our algorithm designed to search for these structures (basing on analysis of one nucleotide sequence) was applied to analyze operons of amino acid biosynthesis in alpha- and gamma-proteobacteria. The attenuators of these operons are predicted for genomes of some poorly known gamma-proteobacteria including Shewanella putrefaciens, attenuators of the tryptophan operon in some alpha-proteobacteria are also predicted.

Lateral gene transfer and ancient paralogy of operons containing redundant copies of tryptophan-pathway genes in Xylella species and in heterocystous cyanobacteria

Tryptophan-pathway genes that exist within an apparent operon-like organization were evaluated. A seven-gene cluster in Xylella fastidiosa exhibits a sharply delineated low-GC content. This strongly implicates lateral gene transfer. In contrast, parametric studies and protein tree phylogenies did not support the origination of a gene block in the Anabaena/Nostoc lineage by lateral gene transfer.

Zinc Is Required for Assembly and Function of the Anti-trp RNA-binding Attenuation Protein, AT

The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism. AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp). Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ. Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity. AT is shown to contain Zn(II) at a ratio of one ion per monomer. Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers. All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination. Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers. Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo. We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.

TROSY-NMR studies of the 91kDa TRAP protein reveal allosteric control of a gene regulatory protein by ligand-altered flexibility.

The tryptophan biosynthesis genes of several Bacilli are controlled through terminator/anti-terminator transcriptional attenuation. This process is regulated by tryptophan-dependent binding of the trp RNA-binding attenuation protein (TRAP) to the leader region of the trp operon mRNA, precluding formation of the antiterminator RNA hairpin, and allowing formation of the less stable terminator hairpin. Crystal structures are available of TRAP in complex with tryptophan and in ternary complex with tryptophan and RNA. However, no structure of TRAP in the absence of tryptophan is available; thus, the mechanism of allostery remains unclear. We have used transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments to study the mechanism of ligand-mediated allosteric regulation in the 90.6kDa 11-mer TRAP. By recording a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP from the thermophile Bacillus stearothermophilus over an extended range of temperatures, we have found tryptophan binding to be temperature-dependent, in agreement with the previously observed temperature-dependent RNA binding. Triple-resonance TROSY-based NMR spectra recorded at 55 degrees C have allowed us to obtain backbone resonance assignments for uniformly 2H,13C,15N-labeled TRAP in the inactive form and in the active form (free and bound to tryptophan). On the basis of ligand-dependent differential line-broadening and chemical shift perturbations, coupled with the results of proteolytic sensitivity measurements, we infer that tryptophan-modulated protein flexibility (dynamics) plays a central role in TRAP function by altering its RNA-binding affinity. Furthermore, because the crystal structures show that the ligand is buried completely in the bound state, we speculate that such dynamic behavior may be important to enable rapid response to changes in intracellular tryptophan levels. Thus, we propose that allosteric control of TRAP is accomplished by ligand-altered protein dynamics.

Modified model for the regulation of the tryptophan operon in Escherichia coli.

This article proposes a modification of the model developed by Sinha (1988) and Sen and Liu (1990) for the regulation dynamics of the tryptophan operon in E. coli based on a consistent overall balance of the agent repressing the mRNA transcription. The dynamics of the model are analyzed by means of continuation techniques and the influence of periodic fluctuations in the intracellular demand for tryptophan is addressed. The analysis provides deeper insight into the dynamics of this operon system and the results obtained may be a useful starting point for the optimization of tryptophan yield in bacterial cultures.

Specificity of TRAP-RNA interactions: crystal structures of two complexes with different RNA sequences.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes in bacilli by binding to the leader region of the nascent trp operon mRNA. When activated by binding tryptophan, the 11-subunit circular TRAP molecule binds to a target sequence consisting of 11 (G/U)AG repeats, separated by two or three variable 'spacer' nucleotides. Reported here are two crystal structures of TRAP bound to RNAs containing 11 GAG repeats separated by UU and CC spacer nucleotides, determined at 1.75 and 2.50 A resolution, respectively. These show the spacer regions of the RNA molecules to be highly flexible, making no direct hydrogen-bonding contacts with the protein. Comparison of these structures with the previous structure of TRAP bound to (GAGAU)(10)GAG RNA, in which the spacer nucleotides stack with each other close to the protein surface, shows that the RNA can adopt different conformations depending on the sequence of the spacer regions. This gives insight into the structural basis of the specificity of TRAP and into the mechanism of binding.