Tna Operon Research Articles

Boolean Models of the Transport, Synthesis, and Metabolism of Tryptophan in Escherichia coli

The tryptophan (trp) operon in Escherichia coli codes for the proteins responsible for the synthesis of the amino acid tryptophan from chorismic acid, and has been one of the most well-studied gene networks since its discovery in the 1960s. The tryptophanase (tna) operon codes for proteins needed to transport and metabolize it. Both of these have been modeled individually with delay differential equations under the assumption of mass-action kinetics. Recent work has provided strong evidence for bistable behavior of the tna operon. The authors of Orozco-Gómezetal.(Sci Rep 9(1):5451, 2019) identified a medium range of tryptophan in which the system has two stable steady-states, and they reproduced these experimentally. In this paper, we will show how a Boolean model can capture this bistability. We will also develop and analyze a Boolean model of the trp operon. Finally, we will combine these two to create a single Boolean model of the transport, synthesis, and metabolism of tryptophan. In this amalgamated model, the bistability disappears, presumably reflecting the ability of the trp operon to produce tryptophan and drive the system toward homeostasis. All of these models have longer attractors that we call "artifacts of synchrony", which disappear in the asynchronous automata. This curiously matches the behavior of a recent Boolean model of the arabinose operon in E. coli, and we discuss some open-ended questions that arise along these lines.

The Tryptophan-Induced tnaC Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase (tna) operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the tna operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and E.coli host factors on induction of the tna leader sequence. To do so, we constructed and biochemically validated an experimental assay using the tna operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (e.g., l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the tna leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the nusB gene from strains intended for tryptophan-sensing utilizing the tna leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.

The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA

The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp-dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo l-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. In vitro TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp-dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.

Bistable behaviour and medium-dependent post-translational regulation of the tryptophanase operon regulatory pathway in Echerichia coli

The present work is aimed at studying the dynamic behaviour of the tryptopnanase (tna) operon, which encodes the proteins necessary to uptake and metabolise tryptophan to use it as a carbon source in the absence of glucose. To this end, we designed a micro-bioreactor capable of driving a bacterial culture to a stationary state. This allowed us to explore (at the single cell level) the tna operon steady-state dynamics under multiple culture conditions. Our experimental results suggest that the tna operon is bistable for a specific range of environmental tryptophan and glucose concentrations, and evidence that both reagents play a role on the activation of the enzyme in charge of metabolising tryptophan: tryptophanase (TnaA). Based on our experimental data and the already known regulatory mechanisms, we developed a mathematical model for the tna operon regulatory pathway. Our modelling results reinforce the claim that the tna operon is bistable, and further suggest that the activity of enzyme TnaA is regulated by the environmental levels of glucose and tryptophan via a common signalling pathway. Possible biological implications of our findings are further discussed.

The role of recombination in evolutionary adaptation of Escherichia coli to a novel nutrient.

The benefits and detriments of recombination for adaptive evolution have been studied both theoretically and experimentally, with conflicting predictions and observations. Most pertinent experiments examine recombination's effects in an unchanging environment and do not study its genomewide effects. Here, we evolved six replicate populations of either highly recombining R+ or lowly recombining R- E.coli strains in a changing environment, by introducing the novel nutrients L-arabinose or indole into the environment. The experiment's ancestral strains are not viable on these nutrients, but 130 generations of adaptive evolution were sufficient to render them viable. Recombination conferred a more pronounced advantage to populations adapting to indole. To study the genomic changes associated with this advantage, we sequenced the genomes of 384 clones isolated from selected replicates at the end of the experiment. These genomes harbour complex changes that range from point mutations to large-scale DNA amplifications. Among several candidate adaptive mutations, those in the tryptophanase regulator tnaC stand out, because the tna operon in which it resides has a known role in indole metabolism. One of the highly recombining populations also shows a significant excess of large-scale segmental DNA amplifications that include the tna operon. This lineage also shows a unique and potentially adaptive combination of point mutations and DNA amplifications that may have originated independently from one another, to be joined later by recombination. Our data illustrate that the advantages of recombination for adaptive evolution strongly depend on the environment and that they can be associated with complex genomic changes.

Evidence for Moonlighting Functions of the Subunit of Escherichia coli DNA Polymerase III

The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no detectable homology to other ORFs in a database). It encodes the θ subunit of the DNA polymerase III core complex. The precise function of the θ subunit within this complex is not well established, and loss of holE does not result in a noticeable phenotype. Paralogs of holE are also present on many conjugative plasmids and on phage P1 (hot gene). In this study, we provide evidence indicating that θ (HolE) exhibits structural and functional similarities to a family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins that are also encoded by enterobacterial ORFan genes. Microarray studies comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT mutants revealed highly similar expression patterns for strains harboring holE and ydgT alleles. Among the genes differentially regulated in both mutants were genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE and YdgT downregulate expression of the tna operon by possibly increasing the level of Rho-dependent transcription termination at the tna operon's leader region. Thus, for the first time, a regulatory function can be attributed to HolE, in addition to its role as structural component of the DNA polymerase III complex.

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome’s ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of l-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in l-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased l-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost l-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular l-tryptophan level induced biofilm formation and increase of l-tryptophan repressed biofilm formation. So the effect of the addition of l-tryptophan to the medium on the E. coli biofilm formation was investigated. l-Tryptophan addition at starting culture decreased biofilm formation and furthermore l-tryptophan addition after 16h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular l-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular l-tryptophan concentration triggers degradation of matured biofilm.

Ribosome Recycling Factor and Release Factor 3 Action Promotes TnaC-Peptidyl-tRNA Dropoff and Relieves Ribosome Stalling during Tryptophan Induction of tna Operon Expression in Escherichia coli

Upon tryptophan induction of tna operon expression in Escherichia coli, the leader peptidyl-tRNA, TnaC-tRNA(2)(Pro), resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged tRNA(2)(Pro) availability. Which factors relieve ribosome stalling, facilitate TnaC-tRNA(2)(Pro) cleavage, and relieve growth inhibition were addressed in the current study. In strains containing the chromosomal tna operon and lacking a tnaC plasmid, the overproduction of ribosome recycling factor (RRF) and release factor 3 (RF3) reduced tna operon expression. Their overproduction in vivo also increased the rate of cleavage of TnaC-tRNA(2)(Pro), relieving the growth inhibition associated with plasmid-mediated tnaC overexpression. The overproduction of elongation factor G or initiation factor 3 did not have comparable effects, and tmRNA was incapable of attacking TnaC-tRNA(2)(Pro) in stalled ribosome complexes. The stability of TnaC-tRNA(2)(Pro) was increased appreciably in strains deficient in RRF and RF3 or deficient in peptidyl-tRNA hydrolase. These findings reveal the existence of a natural mechanism whereby an amino acid, tryptophan, binds to ribosomes that have just completed the synthesis of TnaC-tRNA(2)(Pro). Bound tryptophan inhibits RF2-mediated cleavage of TnaC-tRNA(2)(Pro), resulting in the stalling of the ribosome translating tnaC mRNA. This stalling results in increased transcription of the structural genes of the tna operon. RRF and RF3 then bind to this stalled ribosome complex and slowly release TnaC-tRNA(2)(Pro). This release allows ribosome recycling and permits the cleavage of TnaC-tRNA(2)(Pro) by peptidyl-tRNA hydrolase.

Features of Ribosome-Peptidyl-tRNA Interactions Essential for Tryptophan Induction of tna Operon Expression

Certain nascent peptide sequences, when within the ribosomal exit tunnel, can inhibit translation termination and/or peptide elongation. The 24 residue leader peptidyl-tRNA of the tna operon of E. coli, TnaC-tRNA(Pro), in the presence of excess tryptophan, resists cleavage at the tnaC stop codon. TnaC residue Trp12 is crucial for this inhibition. The approximate location of Trp12 in the exit tunnel was determined by crosslinking Lys11 of TnaC-tRNA(Pro) to nucleotide A750 of 23S rRNA. Methylation of nucleotide A788 of 23S rRNA was reduced by the presence of Trp12 in TnaC-tRNA(Pro), implying A788 displacement. Inserting an adenylate at position 751, or introducing the change U2609C in 23S rRNA or the change K90H or K90W in ribosomal protein L22, virtually eliminated tryptophan induction. These modified and mutated regions are mostly located near the putative site occupied by Trp12 of TnaC-tRNA(Pro). These findings identify features of the ribosomal exit tunnel essential for tna operon induction.

Bismuth–dithiol inhibition of the Escherichia coli rho transcription termination factor

Bismuth–dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth–dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay ( I 50 = 60 μM) and as a competitive inhibitor with respect to ribo(C) 10 in the poly(dC)-ribo(C) 10-dependent ATPase assay. The minimum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 μM. Using the tnaA/ lacZ fusion reporter assay we showed that sublethal amounts (3 μM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo β-glactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy.

Influence ofEscherichia coli DnaK and DnaJ molecular chaperones on tryptophanase (TnaA) amount and GreA, GreB stability

The amount of tryptophanase was estimated in Escherichia coli deltadnaJ and deltadnaKdnaJ mutants. Densitometric analysis of polyacrylamide gels demonstrated that the amount of tryptophanase was diminished in both mutants. DnaK and DnaJ molecular chaperones apparently influence the amount of tryptophanase, the expression of which is regulated at all transcription steps, including transcription elongation. The half-life of GreA and GreB proteins (being activators of transcription elongation of the tna operon) are diminished in both mutants suggesting the involvement of DnaK and DnaJ in the stability of these proteins.

Rho's Role in Transcription Attenuation in the tna Operon of E. Coli

Publisher Summary This chapter discusses the role of Rho's in transcription attenuation in the tna Operon of Escherichia coli (E. Coli). The chapter describes methods allowing experimental analysis of the role of the tna operon leader peptide in influencing whether or not Rho-mediated transcription termination will occur. Various E. coli strains have been described as sources of S-30 preparations; S-30s from some strains offer the advantage that they will permit the use of linear DNA templates. Transcription attenuation is advantageous as a regulatory strategy as it allows cellular components and events that do not participate directly in transcription initiation decisions to influence gene expression. Thus, RNA sequences and structures play a decisive role in the process of transcription attenuation. The most widely studied termination factor is the Rho protein of E. Coli. Rho-dependent transcription termination in the leader region of the tna operon can be readily demonstrated by comparing the products of two reactions, one with bicyclomycin addition (50 μg/ml), and the second without this antibiotic.

Reproducing tna Operon Regulation in Vitro in an S-30 System: TRYPTOPHAN INDUCTION INHIBITS CLEAVAGE OF TnaC PEPTIDYL-tRNA

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced inhibition of Rho-mediated transcription termination. Previous studies indicated that tryptophan induction might involve leader peptide inhibition of ribosome release at the stop codon of tnaC, the coding region for the operon-specified leader peptide. In this study we examined tna operon expression in strains in which the structural gene for protein release factor 3, prfC, is either disrupted or overexpressed. We find that prfC inactivation leads to a two- to threefold increase in basal expression of the tna operon and a slight increase in induced expression. Overexpression of prfC has the opposite effect and reduces both basal and induced expression. These effects occur in the presence of glucose and cyclic AMP, and thus Rho-dependent termination rather than catabolite repression appears to be the event influenced by the prfC alterations. prfC inactivation also leads to an increase in basal tna operon expression in various rho and rpoB mutants but not in a particular rho mutant in which the basal level of expression is very high. The effect of prfC inactivation was examined in a variety of mutants with alterations in the tna leader region. Our results suggest that translation of tnaC is essential for the prfC effect. The tryptophan residue specified by tnaC codon 12, which is essential for induction, when replaced by another amino) acid, allows the prfC effect. Introducing UAG or UAA stop codons rather than the normal tnaC UGA stop codon, in a strain with an inactive prfC gene, also leads to an increase in the basal level of expression. Addition of the drug bicyclomycin increases basal operon expression of all mutant strains except a strain with a tnaC'-'lacZ fusion. Expression in the latter strain is unaffected by prfC alterations. Our findings are consistent with the interpretation that ribosome release at the tnaC stop codon can influence tna operon expression.

Some novel transcription attenuation mechanisms used by bacteria

A variety of transcription attenuation mechanisms are used by bacteria to regulate gene and operon expression. This review summarizes previous and current studies designed to elucidate the features of the specific attenuation mechanisms that regulate expression of the tryptophanase ( tna) operon of Escherichia coli and the tryptophan ( trp) operon of Bacillus subtilis. Initiation of transcription in the tna operon is regulated by catabolite repression. Once initiated, transcription is regulated by tryptophan-induced inhibition of Rho-mediated transcription termination in the leader region of the operon. An operon-encoded leader peptide, TnaC, containing a crucial tryptophan residue, plays an essential role in induction. This peptide appears to act in cis on the ribosome translating tnaC to inhibit its release at the tnaC stop codon. The stalled ribosome would block Rho's access to the tna transcript, thereby preventing termination. Transcription of the trp operon of B subtilis is regulated by an attenuation mechanism that responds to a tryptophan-activated eleven subunit RNA-binding regulatory protein, called TRAP. Activated TRAP binds to repeated GAG sequences in the leader segment of the trp operon transcript, disrupting an RNA antiterminator and promoting formation of a terminator. Activated TRAP also regulates translation of trpG in the folate operon by binding to repeat GAG sequences surrounding the trpG ribosome binding site. A temperature sensitive tryptophanyl-tRNA synthetase ( trpS) mutant was previously observed to overexpress the trp operon and trpG, when grown at elevated temperatures in the presence of tryptophan. We have found that the trpS defect increases trp operon and trpG expression by interfering with TRAP's ability to act. We suggest that either accumulation of uncharged tRNA Trp or overproduction of a TRAP-binding transcript reduces the level of functional TRAP in the trpS mutant.

Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Elevated levels of tryptophan induce transcription antitermination at one or more Rho factor-dependent termination sites in the leader region of the operon. Induction requires translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. In the present paper, we show that two bacterial species that lack tryptophanase activity, Enterobacter aerogenes and Salmonella typhimurium, allow tryptophanase induction and tna operon regulation when they carry a plasmid containing the E. coli tna operon. The role of tnaC in induction was examined by introducing mutations in a 24-nucleotide segment of tnaC of E. coli surrounding and including the crucial Trp codon 12. Some mutations resulted in a noninducible phenotype; these mostly introduced nonconservative amino acid substitutions in TnaC. Other mutations had little or no effect; these generally were in third positions of codons or introduced conservative amino acid replacements. A tryptophan-inserting, UGA-reading glutamine suppressor tRNA was observed to restore partial regulation when Trp codon 12 of tnaC was changed to UGA. Stop codons introduced downstream of Trp codon 12 in all three reading frames established that induction requires translation in the natural tnaC reading frame. Our findings suggest that the TnaC leader peptide acts in cis to prevent Rho-dependent termination.

Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli

The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB.

Sequence and characterization of mutT from Proteus vulgaris

A cloned DNA fragment containing the tryptophanase ( tna) operon of Proteus vulgaris was found to contain a gene analogous to mutT of Escherichia coli immediately distal to the tna operon. The presumptive mutT of P. vulgaris was shown to be a functional gene by complemention of a mutT mutant from E. coli. The deduced amino acid sequence of the MutT polypeptide of P. vulgaris was 47% identical and 70% similar to MutT of E. coli. The mutT and tna operons of P. vulgaris were shown to be adjacent on the genome of this organism. These operons are located about 20 min apart in the E. coli genome. Our findings suggest that either or both tna and mutT have different genomic locations in the two organisms.

Inhibition of expression of the tryptophanase operon in Escherichia coli by extrachromosomal copies of the tna leader region.

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Expression is induced by the presence of elevated levels of tryptophan in a growth medium devoid of a catabolite-repressing carbon source. Induction requires the translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. Multicopy plasmids carrying the tnaC leader region were found to inhibit induction of the chromosomal tna operon. Mutational studies established that this inhibition was not due to inhibited transcription initiation, translation initiation, tryptophan transport, or enzyme activity. Rather, multicopy tnaC plasmids inhibited induction by preventing tryptophan-induced transcription antitermination in the leader region of the tna operon. Translation of the single Trp codon in tnaC of the multicopy plasmids was shown to be essential for this inhibition. We hypothesize that translation of the Trp codon of the leader peptide titrates out a trans-acting factor that is essential for tryptophan-induced antitermination in the chromosomal tna operon. We postulate that this factor is an altered form of tRNATrp.