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Charles Yanofsky - One of the best experts on this subject based on the ideXlab platform.
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Tryptophan Operon of Escherichia coli
Brenner's Encyclopedia of Genetics, 2013Co-Authors: Charles YanofskyAbstract:The Trp Operon of E. coli contains five major structural genes encoding all seven protein functional domains necessary for tryptophan biosynthesis from the common aromatic 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 structure 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 form, and terminate transcription.
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Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and Trp Operon Expression in Bacillus licheniformis
Journal of bacteriology, 2010Co-Authors: Anastasia Levitin, Charles YanofskyAbstract:Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, Trp Operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to Trp Operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at Operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at Operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis.
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Physiological Effects of Anti-TRAP Protein Activity and tRNATrp Charging on Trp Operon Expression in Bacillus subtilis
Journal of bacteriology, 2008Co-Authors: Luis R. Cruz-vera, Ming Gong, Charles YanofskyAbstract:The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to Trp Operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRNATrp. In this study, we examined the roles of AT synthesis and tRNATrp charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRNATrp level from 80% to 40%; the charged level decreased further, to 25%, in an AT-deficient mutant. Adding tryptophan with phenylalanine increased the charged tRNATrp level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRNATrp charging. Changes in the charged tRNATrp level observed during growth with added phenylalanine were associated with increased transcription of the genes of tryptophan metabolism. Nutritional shift experiments, from a medium containing tryptophan to a medium with phenylalanine and tyrosine, showed that wild-type cultures gradually reduced their charged tRNATrp level. When this shift was performed with an AT-deficient mutant, the charged tRNATrp level decreased even further. Growth rates for wild-type and mutant strains deficient in AT or TRAP or that overproduce AT were compared in various media. A lack of TRAP or overproduction of AT resulted in phenylalanine being required for growth. These findings reveal the importance of AT in maintaining a balance between the synthesis of tryptophan versus the synthesis of phenylalanine, with the level of charged tRNATrp acting as the crucial signal regulating AT production.
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Comparison of tryptophan biosynthetic Operon regulation in different Gram-positive bacterial species
Trends in genetics : TIG, 2007Co-Authors: Ana Gutiérrez-preciado, Charles Yanofsky, Enrique MerinoAbstract:The tryptophan biosynthetic Operon has been widely used as a model system for studying transcription regulation. In Bacillus subtilis , the Trp Operon is primarily regulated by a tryptophan-activated RNA-binding protein, TRAP. Here we show that in many other Gram-positive species the Trp Operon is regulated differently, by tRNA Trp sensing by the RNA-based T-box mechanism, with T-boxes arranged in tandem. Our analyses reveal an apparent relationship between Trp Operon organization and the specific regulatory mechanism(s) used.
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Effects of Tryptophan Starvation on Levels of the Trp RNA-Binding Attenuation Protein (TRAP) and Anti-TRAP Regulatory Protein and Their Influence on Trp Operon Expression in Bacillus subtilis
Journal of bacteriology, 2005Co-Authors: Wen-jen Yang, Charles YanofskyAbstract:The anti-TRAP protein (AT), encoded by the rtpA gene of Bacillus subtilis, can bind to and inhibit the tryptophan-activated Trp RNA-binding attenuation protein (TRAP). AT binding can prevent TRAP from promoting transcription termination in the leader region of the Trp Operon, thereby increasing Trp Operon expression. We show here that AT levels continue to increase as tryptophan starvation becomes more severe, whereas the TRAP level remains relatively constant and independent of tryptophan starvation. Assuming that the functional form of AT is a trimer, we estimate that the ratios of AT trimers per TRAP molecule are 0.39 when the cells are grown under mild tryptophan starvation conditions, 0.83 under more severe starvation conditions, and approximately 2.0 when AT is expressed maximally. As the AT level is increased, a corresponding increase is observed in the anthranilate synthase level. When AT is expressed maximally, the anthranilate synthase level is about 70% of the level observed in a strain lacking TRAP. In a nutritional shift experiment where excess phenylalanine and tyrosine could potentially starve cells of tryptophan, both the AT level and anthranilate synthase activity were observed to increase. Expression of the Trp Operon is clearly influenced by the level of AT.
Paul Gollnick - One of the best experts on this subject based on the ideXlab platform.
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Model of transcription attenuation of the B. subtilis Trp Operon.
2014Co-Authors: Natalie M. Mcadams, Paul GollnickAbstract:Bold black letters designate the complementary strands of the attenuator (C/D) (highlighted in blue) and antiterminator (A/B) RNA structures. TRAP is shown in a ribbon diagram with each subunit in a different color. The 11 (G/U)AG repeats of the TRAP binding site are circled and numbered in green. Small black numbers indicate RNA residues relative to the start of transcription. When tryptophan is limiting, the AB antiterminator RNA structure forms, allowing read through of the Trp Operon. In excess tryptophan, TRAP binds to the nascent RNA and prevents formation of the antiterminator structure, which allows formation of the attenuator, leading to transcription termination.
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The rate of TRAP binding to RNA is crucial for transcription attenuation control of the B. subtilis Trp Operon.
Journal of molecular biology, 2007Co-Authors: Maria V. Barbolina, Roman Kristoforov, Amanda Manfredo, Yanling Chen, Paul GollnickAbstract:The Trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic and transport genes in Bacillus subtilis in response to changes in the levels of intracellular tryptophan. Transcription of the TrpEDCFBA Operon is controlled by an attenuation mechanism involving two overlapping RNA secondary structures in the 5′ leader region of the Trp transcript; TRAP binding promotes formation of a transcription terminator structure that halts transcription prior to the structural genes. TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of L-tryptophan. The TRAP binding site in the leader region of the Trp Operon mRNA consists of 11 (G/U)AG repeats. We examined the importance of the rate of TRAP binding to RNA for the transcription attenuation mechanism. We compared the properties of two types of TRAP 11-mers: homo-11-mers composed of 11 wild-type subunits, and hetero-11-mers with only one wild-type subunit and ten mutant subunits defective in binding either RNA or tryptophan. The hetero-11-mers bound RNA with only slightly diminished equilibrium binding affinity but with slower on-rates as compared to WT TRAP. The hetero-11-mers showed significantly decreased ability to induce transcription termination in the Trp leader region when examined using an in vitro attenuation system. Together these results indicate that the rate of TRAP binding to RNA is a crucial factor in TRAP's ability to control attenuation.
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TRAP-RNA interactions involved in regulating transcription attenuation of the Bacillus subtilis Trp Operon.
Methods in enzymology, 2003Co-Authors: Paul GollnickAbstract:Publisher Summary 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 32P-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 [14C] 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.
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regulatory features of the Trp Operon and the crystal structure of the Trp rna binding attenuation protein from bacillus stearothermophilus
Journal of Molecular Biology, 1999Co-Authors: Xiaoping Chen, Alfred A Antson, Min Yang, Chris Baumann, E J Dodson, G G Dodson, Paul GollnickAbstract:Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilusTrp Operon is regulated by an attenuation mechanism similar to that which controls the Trp Operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the Trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.
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Regulation of the Bacillus subtilis Trp Operon by an RNA-binding protein.
Molecular microbiology, 1994Co-Authors: Paul GollnickAbstract:The Bacillus subtilis tryptophan (TrpEDCFBA) Operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L-tryptophan, a transcription terminator forms and the Operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the Operon. The mechanism of selection between these alternative structures involves a trans-acting RNA-binding regulatory protein. This protein is the product of the mtrB gene and is called TRAP for Trp attenuation protein. TRAP has been shown to bind specifically to Trp leader RNA, and to cause transcription of the Trp Operon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is formed.
Paul Babitzke - One of the best experts on this subject based on the ideXlab platform.
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modular organization of the nusa and nusg stimulated rna polymerase pause signal that participates in the bacillus subtilis Trp Operon attenuation mechanism
Journal of Bacteriology, 2017Co-Authors: Smarajit Mondal, Alexander V Yakhnin, Paul BabitzkeAbstract:The Bacillus subtilis TrpEDCFBA Operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the Trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a TrpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the Trp Operon attenuation mechanism by providing additional time for TRAP binding.IMPORTANCE The expression of several bacterial Operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5' UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis Trp Operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.
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Molecular basis of TRAP–5′SL RNA interaction in the Bacillus subtilis Trp Operon transcription attenuation mechanism
RNA (New York N.Y.), 2008Co-Authors: Adam P. Mcgraw, Ali Mokdad, François Major, Philip C. Bevilacqua, Paul BabitzkeAbstract:Expression of the Bacillus subtilis TrpEDCFBA Operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent Trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the Trp Operon structural genes. A 5′-stem–loop (5′SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP–Trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 5′SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 5′SL is in close proximity to the flexible loop region of TRAP during TRAP–5′SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 5′SL generated using the MC-Sym and MC-Fold pipeline imply that the 5′SL binds the protein in an orientation where the helical axis of the 5′SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP–Trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.
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molecular basis of trap 5 sl rna interaction in the bacillus subtilis Trp Operon transcription attenuation mechanism
RNA, 2008Co-Authors: Adam P. Mcgraw, Ali Mokdad, François Major, Philip C. Bevilacqua, Paul BabitzkeAbstract:Expression of the Bacillus subtilis TrpEDCFBA Operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent Trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the Trp Operon structural genes. A 5′-stem–loop (5′SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP–Trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 5′SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 5′SL is in close proximity to the flexible loop region of TRAP during TRAP–5′SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 5′SL generated using the MC-Sym and MC-Fold pipeline imply that the 5′SL binds the protein in an orientation where the helical axis of the 5′SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP–Trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.
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Recycling of a regulatory protein by degradation of the RNA to which it binds.
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Gintaras Deikus, Paul Babitzke, David H. BechhoferAbstract:Abstract 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.
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Trp RNA-binding attenuation protein (TRAP)-Trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis Trp Operon.
Journal of bacteriology, 1995Co-Authors: Enrique Merino, Paul Babitzke, Charles YanofskyAbstract:Expression of the Bacillus subtilis TrpEDCFBA Operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated Trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within Trp leader RNA determines whether transcription will terminate in the leader region of the Operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the TrpE ribosome-binding site. To assess the relative importance of these postulated events in Trp Operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a TrpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the Trp Operon over about an 88-fold range and regulates translation of TrpE over about a 13-fold range. The roles played by different Trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.
Alain Chopin - One of the best experts on this subject based on the ideXlab platform.
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Laboratoire de GenCtique
2015Co-Authors: Microbienne Lnstitut, Hélène Frenkiel, Jacek Bardowski, T Dusko S. Ehrlich, Alain ChopinAbstract:Transcription of the Trp Operon in Lactococcus lactis is controlled by antitermination in the leader reg io
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trnaTrp as a key element of antitermination in the lactococcus lactis Trp Operon
Molecular Microbiology, 1998Co-Authors: Maarten Van De Guchte, Dusko S Ehrlich, Alain ChopinAbstract:The expression of the Trp Operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the Trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the Trp leader transcript, thus allowing transcription and expression of the structural genes of the Operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon–anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated antitermination control, that is based on the processing of the Trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis Trp Operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an Operon of which the structural genes are highly conserved.
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Transcription of the Trp Operon in Lactococcus lactis is controlled by antitermination in the leader region
Microbiology, 1998Co-Authors: Hélène Frenkiel, Jacek Bardowski, S D Ehrlich, Alain ChopinAbstract:The regulatory functions of the leader region preceding the Lactococcus lactis Trp Operon have been studied by mutagenesis analysis. This leader presents striking similarity to 'T-box' leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis Operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U) AG repeats which, in Bacillus, are involved in the binding of the Trp RNA-binding protein (TRAP) which controls Trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the Trp Operon, demonstrating that Trp is indeed controlled by transcription antitermination.
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Multiple transcriptional control of the Lactococcus lactis Trp Operon
Journal of Bacteriology, 1998Co-Authors: R Raya, Paal S. Andersen, Jacek Bardowski, S D Ehrlich, Alain ChopinAbstract:The Lactococcus lactis TrpEGDCFBA Operon is preceded by a noncoding leader region. Transcriptional studies of the Trp Operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire Operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique PTrp promoter, transcription termination at either T1 (upstream of the Trp Operon) or T2 (downstream of the Trp Operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.
Enrique Merino - One of the best experts on this subject based on the ideXlab platform.
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Comparison of tryptophan biosynthetic Operon regulation in different Gram-positive bacterial species
Trends in genetics : TIG, 2007Co-Authors: Ana Gutiérrez-preciado, Charles Yanofsky, Enrique MerinoAbstract:The tryptophan biosynthetic Operon has been widely used as a model system for studying transcription regulation. In Bacillus subtilis , the Trp Operon is primarily regulated by a tryptophan-activated RNA-binding protein, TRAP. Here we show that in many other Gram-positive species the Trp Operon is regulated differently, by tRNA Trp sensing by the RNA-based T-box mechanism, with T-boxes arranged in tandem. Our analyses reveal an apparent relationship between Trp Operon organization and the specific regulatory mechanism(s) used.
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Trp RNA-binding attenuation protein (TRAP)-Trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis Trp Operon.
Journal of bacteriology, 1995Co-Authors: Enrique Merino, Paul Babitzke, Charles YanofskyAbstract:Expression of the Bacillus subtilis TrpEDCFBA Operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated Trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within Trp leader RNA determines whether transcription will terminate in the leader region of the Operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the TrpE ribosome-binding site. To assess the relative importance of these postulated events in Trp Operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a TrpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the Trp Operon over about an 88-fold range and regulates translation of TrpE over about a 13-fold range. The roles played by different Trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.
