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trp Operon Organization and Regulation in Different Bacterial Species
trp Operon Organization and Regulation in Different Bacterial Species E Merino, Universidad Nacional Autonoma de Mexico, Morelos, Mexico RA Jensen, University of Florida, Gainesville, FL, USA C Yanofsky, Stanford University, Stanford, CA, USA © 2013 Elsevier Inc. All rights reserved.
Glossary Operator A DNA segment with a specific nucleotide sequence recognized by a DNA-binding regulatory protein. Operon A transcribed unit of genetic material, generally containing one or more structural genes. Riboswitch An RNA element, commonly located in the 5′ leader segment of a transcript, that is used to regulate expression of the genes within its transcription unit, in response to the presence or absence of a specific metabolite. Recognition of these metabolites by riboswitches can be performed without the participation of any protein.
Tryptophan is one of the key amino acids influencing the structure and function of many proteins. The metabolic path way responsible for tryptophan biosynthesis from chorismate, the common precursor of the three aromatic amino acids, requires the enzymatic activities of seven enzyme functional domains encoded by the trp genes. The organization of these trp genes within operons and the regulatory mechanisms used to control trp operon expression vary appreciably, undoubt edly reflecting organismal divergence in response to different metabolic demands. This article describes trp operon organiza tional and regulatory differences in diverse bacterial species, illustrating the flexible range of choices that organisms must have had during evolution of their tryptophan biosynthetic capability.
Regulation of the Biosynthetic trp Operon of Bacteria Tryptophan biosynthesis is complex and remarkably costly. Accordingly, expression of the genes encoding the proteins responsible for this process is generally highly regulated. The elements and factors responsible for trp gene regulation vary significantly from species to species, and include DNA and RNA elements, DNA- and RNA-binding proteins, protein-binding proteins, and translating ribosomes. It is evident that many of these regulatory features were under evolutionary selection to allow an organism to respond to changes in the intracellular levels of free tryptophan and/or uncharged transfer RNATrp (tRNATrp). Some of these ele ments influence the action of RNA polymerase, while others affect premature termination of transcription. In any event, clear phylogenetic preferences can be seen in the types of regulatory strategies used in controlling trp operon expression. In Gram-negative bacteria, such as Escherichia coli, expres sion of the trp operon is finely adjusted through recognition
208
Transcription attenuation A regulatory mechanism used by bacteria to sense – and respond – to a specific metabolic signal, by regulating premature termination of transcription of the transcribed operon. Attenuation is based on cell selection of one of two mutually exclusive secondary structures, one of which is commonly a Rhoindependent transcription terminator. TrpR An aporepressor, which, upon binding two L-tryptophan molecules, undergoes conformational changes to become an active repressor. The active repressor can bind at specific operator sites in its target promoters, blocking initiation of downstream gene transcription.
of the intracellular levels of both free tryptophan and uncharged tRNATrp as signaling cues. The tryptophan concentration is assessed by the trp repressor protein, TrpR, which regulates transcription initiation (Figure 1; Boxes 1(ia) and 1(ib)). When intracellular levels of trypto phan are low, TrpR exists in an inactive conformation that cannot bind to trp operator DNA. Consequently, RNA poly merase is able to initiate trp operon transcription (Box 1 (ia)). As the intracellular level of tryptophan rises, it binds to the TrpR aporepressor, altering its conformation, and activating its DNA-binding ability. In this active form, TrpR binds to specific DNA operator sequences that overlap the trp promoter, preventing initiation of trp operon transcrip tion (Box 1(ib)). In addition, transcription of the trp genes of this bacterial group is regulated by transcriptional attenuation, in response to the level of uncharged tRNATrp. Using this regulatory mechanism, under growth conditions where the charged tRNATrp level is low, the ribosome trans lating the trp operon’s leader peptide coding sequence, trpL, stalls at either of its two adjacent trp codons. The position of the stalled ribosome favors the formation of an RNA antiterminator structure, and, hence, transcription con tinues into the structural gene region of the operon (Box 1(iia)). When the level of charged tRNATrp is high, transla tion of trpL messenger RNA (mRNA) is completed without ribosome pausing, allowing the leader RNA to fold and form a Rho-independent terminator, promoting premature transcription termination (Box 1(iib)). Interestingly, the trp operons of some members of the Chlamydiales, which are phylogenetically remote from Gammaproteobacteria, are also regulated by TrpR repression and ribosome-mediated transcriptional attenuation (Figure 1). A member of the Gammaproteobacteria, Pseudomonas aeru ginosa, and its close relatives possess a trpBA operon, encoding the two subunits of the tryptophan synthase enzyme complex. This operon is positively regulated by the product of the
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 7
doi:10.1016/B978-0-12-374984-0.01591-6
trp Operon Organization and Regulation in Different Bacterial Species
Escherichia coli K12 Haemophilus influenzae
trpL
TrpR TrpI
Pseudomonas aeruginosa
trpL
TrpR
Psychrobacter cryohalolentis
TrpR
Proteobacteria
Gamma
Beta Alpha Epsilon Epsilon Delta
trpR
Thermotogae Thermotogae Actino bacte teria ria tinobac
Plan Planctomy omycetes Bacteroi detes s Bacteroidete TrpR
AT
Bacillales
trpC
trpG
trpD
trpC
trpE
trpG
trpD
trpC
trpE
aroH
trpG
trpD
trpL
trpE G
Brucella melitensis Gluconobacter oxydans Campylobacter jejuni Helicobacter pylori
trpL
trpE G
ppiD trpE
trpL
truA
trpF truA
ppiD
trpD
trpC
moaC
trpG D
trpF
trpG
trpE
trpG
trpB
trpC F
trpD
trpC
trpF aroA trpB
trpD
trpF
trpE
trpG
trpD
trpC
trpF aroA trpB
trpE
trpG
trpD
trpC
trpF
trpB
trpA aroH aroA
Thermotoga maritima
trpE
trpC
trpF
trpB
trpA
Mycobacterium tuberculosis Streptomyces coelicolor
trpE
trpL
trpE
trpE
trpG
Leptospira interrogans
trpE
trpG
Pirellula sp.
trpE trpB
Chlamydia trachomatis mtrB Bacillus subtilis Bacillus thuringiensis
trpE trpL
trpR aroF aroB aroH
Staphylococcus aureus Streptococcus pneumoniae Leuconostoc mesenteroides
trpC
trpD
priA
trpD trpA
trpG
trpD
trpB
trpC
trpA
trpA
trpC
trpF
trpB
trpA
hisC
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
ltbR
trpC
phzE
) trpB
(17 trpB
trpA trpA
trpC
trpD
tyrA
aroE
TrpI mtrB
trpA
trpB trpA
trpF
purH
trpF trpF
trpA
trpC
trpC
trpD trpD
trpB trpB
trpD
trpG
trpB
trpA
trpA
trpG
trpE
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
aroA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
aroF
trpE
trpG
trpD
trpC
trpF
trpB
trpA
Clostridium thermocellum Clostridium hydrogenoformans
trpD
trpA
trpC F
trpB trpA
trpC
trpB
trpA accD folC
trpB trpA
trpB
trpF
trpG
hisI
trpA accD
trpC
Dehalococcoides sp. Sphaerobacter thermophilus
trpE G
trpA accD
trpB
trpA
trpE
trpL
trpB trpF
trpF trpB
moeA
trpA trpB
trpC
trpC
trpA trpA
trpF trpB trpA accD folC
Synechococcus sp.
trpG D
trpB trpB
trpF
trpD
trpD
trpI
trpF
trpC moaC moeA
trpE
trpR
trpA
trpF
trpC
trpE
trpG
trpB
trpD
trpC
trpE
trpR
trpA
ppiD
trpG trpD
trpE G
Oceanobacillus iheyensis Lactobaci llales cillales
trpG
trpD
trpL
Bacteroides fragilis
Chla Chlamydiae
Firmicutes Firmicutes
trpE
trpB
trpC F
trpG
trpL
Corynebacterium glutamicum Spirochaetes Spirochaetes
trpL
Acidovorax sp. Agrobacterium tumefaciens
Deinococcus radiodurans
Chlorofle Chloroflexi
trpC F trpD
Bordetella parapertussis
Solibacte rusitatus
Deino coc ccus Deinoco Cyanobacter obacteriia
trpG D trpG
trpE
Pelobacter carbinolicus
Aci Acidobacteria
Clostridia
trpE trpE
209
trpB
TrpR
pabB
trpG ( 7
rtpA
)
TrpI activator and its binding site TrpR and its DNA-binding sites TRAP and its RNA-binding sites trpL
trpA mtrB 14
( )
trpL leader peptide gene Transcription attenuator T-box element AT
Anti-TRAP
Figure 1 Operon organization and transcription regulation of trp genes. The dendogram shown is based on the phylogenetic classification of the different bacterial species. Selected species are color coded according to major phylogenetic groupings. The different trp genes are color coded. trp gene fusions are indicated by single arrows with more than one color. For large operons with more than five consecutive non-trp genes (white arrows), the number of these genes is indicated. mtrB, rtpA, and trpI are, respectively, the structural genes for the TRAP, AT, and TrpI regulatory proteins.
trpI gene in response to the accumulation of indoleglycerol phosphate (InGP), a tryptophan biosynthetic intermediate (Figure 1). In these organisms, the trpI and trpBA operons are divergently transcribed from overlapping promoters. When the intracellular level of tryptophan is low, InGP is overproduced and it binds to TrpI, inducing a conformational change that favors its binding to DNA operator sequences located in the trpI–trpBA intergenic region. TrpI binding simultaneously represses transcription of its own gene, trpI, and activates tran scription of the trpBA operon, either by direct interaction with RNA polymerase or by inducing DNA bending (Box 1(iiia)). Under growth conditions where there is excess tryptophan, the InGP concentration is low and TrpI can only bind to its highaffinity binding site; thus, transcription of trpBA cannot be activated (Box 1(iiib)). Although the biosynthetic trp operons of Gram-positive bacteria are also regulated in response to the intracellular level of free tryptophan and the availability of charged tRNATrp, quite different molecular mechanisms are used by
these organisms (Figure 1). For example, in the vast majority of Gram-positive bacteria, transcription initiation of the trp operon is regulated in response to uncharged tRNATrp by the T-box riboswitch mechanism (Figure 1). This element regulates expression of a large number of amino acid-related genes, such as those specifying aminoacyl-tRNA synthetases, amino acid transporters, and biosynthetic proteins, including those involved in tryptophan biosynthesis. For these genes, the RNA leader region of the trp operon can be folded into a set of highly conserved RNA secondary structures that are able to interact, with great specificity and affinity, with uncharged tRNATrp. This interaction stabilizes an RNA antiterminator structure, prevent ing transcription termination in the respective operon, thereby promoting transcription of the remaining segment of the operon (Box 1(iva)). When the pool of charged tRNATrp becomes elevated, this charged tRNA is incapable of stabilizing the antiterminator structure. Thus, the more energetically favored secondary structure – the transcription terminator – forms, and transcription of the trp operon is terminated
Box 1 Molecular mechanisms used in regulating transcription of the trp biosynthetic operons of bacteria. The variety of factors and elements participating in the regulation of transcription of trp biosynthetic operons are shown. These factors include the DNA-binding proteins TrpR and TrpI; translating ribosomes; the RNA-binding protein TRAP; protein–protein interactions by the anti-TRAP (AT) protein; and the riboswitch T-box. (i) Transcription regulation based on tryptophan activation of the TrpR aporepressor. The TrpR protein is found mainly in Gammaproteobacteria and in some Chlamydiales (Figure 1). (a) When intracellular levels of tryptophan are low, TrpR exists in its aporepressor conformation and thus transcription of the trp operon proceeds. (b) As the intracellular level of tryptophan rises, the binding of free tryptophan to TrpR converts it to an active conformation, which allows it to bind specific operator DNA sequences that overlap the trp promoter. As a result of this binding, transcription of the trp operon is repressed. (ii) Ribosome-mediated transcription attenuation. Regulation of the trp operon by transcriptional attenuation is observed in different Proteobacteria, as well as in some Chlamydiales, Actinobacteridae, and Deinococci (Figure 1). The intracellular level of charged tRNATrp determines whether a ribosome pauses while translating the trpL leader peptide coding region. (a) At low intracellular levels of charged tRNATrp, the ribosome translating trpL mRNA stalls at one of its adjacent Trp codons. This pausing favors formation of the RNA antiterminator structure (B:C). (b) At high intracellular levels of charged tRNATrp, the translating ribosome does not stall, allowing formation of the transcription terminator structure (C:D). (iii) Regulation based on the activation of the TrpI protein. The TrpI protein appears to be restricted to Pseudomonas aeruginosa, P. putida, and P. syringae (Figure 1). The corresponding structural gene, trpI, is adjacent to and transcribed divergently from the trpBA operon (Figure 1). (a) Whenever the intracellular level of tryptophan is low, InGP is overproduced and TrpI assumes its active conformation, which is able to bind at two operator sites in the trpI–trpBA intergenic region. TrpI binding activates transcription of the trpBA operon. (b) When the tryptophan concentration is high, InGP is not overproduced and TrpI is only able to bind at its high-affinity binding site in the trpI–trpBA intergenic region. This binding represses transcription of trpI but is insufficient to activate transcription of the trpBA operon. (iv) Regulation based on the T-box riboswitch. Regulation of biosynthetic trp operons by the T-box riboswitch is observed in most of the Bacillales, Lactobacillales, and some members of the Clostridia group (Figure 1). (a) Uncharged tRNA can pair with the trp leader RNA stabilizing an antiterminator structure (B:C). (b) When tRNATrp is charged, it is no longer able to interact with the trp RNA leader, thereby favoring formation of the terminator structure (C:D), causing Rho-independent transcription termination. (v) Regulation based on the action of the TRAP protein. TRAP is an RNA-binding protein found in some Bacillales, including Bacillus subtilis, and in a few Clostridia (Figure 1). (a) Whenever tryptophan is growth limiting, TRAP is inactive. Since TRAP is inactive, it cannot bind to leader RNA and the antiterminator structure (B:C) is formed. (b) When there is an excess of tryptophan, TRAP is activated, and it binds to the leader RNA disrupting the antiterminator structure, allowing the terminator structure (C:D) to be formed. (vi) Regulation based on the action of the anti-TRAP protein AT. (a) In organisms with an at gene, whenever the uncharged tRNATrp level is high, AT is synthesized and it binds to tryptophan-activated TRAP, inhibiting its activity. (b) As the level of uncharged tRNATrp decreases, AT synthesis is arrested and thus active TRAP is able to regulate the trp operon.
(ia)
(ib)
(iia)
(iib) B C D
A
C D uuuu
A
(iiia)
B
(iiib)
trpI
trpI
trpBA
(iva)
trpBA
(ivb) B
C
C D uuuu B
D
(va)
(vb) B C C D
A (via)
A
B
D trpEDCBA uuuu
(vib) B C A
D
A
B
C D trpEDCBA uuuu
trp Operon Organization and Regulation in Different Bacterial Species (Box 1(ivb)). Interestingly, T-box elements regulating trp bio synthetic genes are often arranged in tandem in the upstreamtranscribed region of the trp operon. However, in some species, only a single T-box element is present (Figure 1). It has been postulated that the existence of tandem T-boxes may expand the regulatory range of these elements for the respective operon because pairing of two uncharged tRNATrp molecules, instead of one, would be required to stabilize the two antiterminator structures of these T-boxes. This regulatory advantage might compensate for the lack of a separate regulatory mechanism sensing free tryptophan. In some Bacillales, including Bacillus subtilis, and in a few Clostridia, regulation of the trp genes is not mediated by the T-box mechanism; instead, the trp operon is regulated by an attenuation mechanism mediated by the RNA-binding pro tein, trp RNA-binding attenuation protein (TRAP) (Figure 1; Boxes 1(va) and 1(vb)). In these organisms, the leader RNA sequence of the trp operon can be folded into two mutually exclusive secondary structures, one corresponding to a Rhoindependent transcription terminator, and the other corre sponding to an antiterminator. Whenever tryptophan is growth limiting, TRAP is inactive and cannot bind to RNA. In this case, the leader RNA of the trp operon folds to form the energetically favored antiterminator secondary structure. This structure prevents the formation of the transcription termina tor, and consequently, transcription proceeds into the structural gene region of the operon (Box 1(va)). When there is an excess of tryptophan, TRAP is activated and it binds to leader RNA, disrupting the antiterminator structure and favor ing formation of the terminator structure. This leads to premature termination of transcription in the leader region of the operon (Box 1(vb)). In addition to this mechanism of regulation, it has been demonstrated that the few transcripts that escape transcription termination are subject to transla tional regulation via formation of a secondary structure that sequesters the ribosome-binding site of the first gene of the operon, trpE. Also, in B. subtilis, expression of trpG, located in the unlinked folate operon, is coordinately regulated along with the other trp genes by TRAP, which, in its tryptophanactivated conformation, binds at the trpG mRNA ribosomebinding site, inhibiting trpG translation. In B. subtilis, the accumulation of uncharged tRNATrp is also sensed in regulating trp operon and trpG expression. This regulation is due to the action of an additional regulatory protein, anti-TRAP, or AT. Expression of the structural gene for AT, rtpA, is regulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. Transcription of the at operon is regulated by a T-box element, and transcription proceeds whenever the level of uncharged tRNATrp is high. The at operon also contains a leader peptide coding region. Whenever the intracellular level of uncharged tRNATrp is high, translation of the leader peptide coding region stalls at one of its Trp codons. AT is then synthesized, and it is capable of binding to tryptophan-activated TRAP, inhibiting its function (Box 1(via)). AT appears to have evolved very recently because it has been identified only in B. subtilis and very closely related organisms (Figure 1). In the other TRAP-containing organisms, it is not known whether the accumulation of uncharged tRNATrp is sensed by some other regulatory mechanism.
211
Operon Organization of the trp Genes All organisms capable of synthesizing tryptophan from choris mate share a similar metabolic pathway that involves the activities of the seven enzyme functional domains encoded by the trp genes. Genome organization of these genes appears to have diverged significantly between different bacterial groupings, although spe cific gene arrangements are observed among phylogenetically related organisms, as described for the following groups. In the lower Gammaproteobacteria, including E. coli, the seven trp biosynthetic genes are organized within a single tran scriptional unit, the trp operon. In this phylogenetic group, the trp operon always contains a trpCF gene fusion, and, less often, a trpGD fusion (Figure 1). Interestingly, differences in gene arrangements are not restricted to the genes encoding the trp enzymes. Genome organizational plasticity has also played an important role in determining the location of trp genes coding for regulatory proteins, such as trpR. For example, in the Gammaproteobacteria group, trpR is commonly transcribed as a monocistronic operon. However, it is also found within larger operons containing other trp genes, allowing coordinate expression of trpR and biosynthetic genes. Examples of this type are found in Psychrobacter cryohalolentis and Chlamydia trachomatis, where trpR is located on the trpREG and trpRLBA operons, respectively (Figure 1). As previously mentioned, in some organisms, the intracellu lar level of charged tRNATrp is sensed during ribosome translation of a small leader peptide coding region, specified by the gene trpL. This small coding region is located almost invariably as the first gene of the trp operon, although, on occa sion, trpL occupies some other position, such as in C. trachomatis, where trpL is the second gene of the trpRLBA operon. In some lower Gammaproteobacteria, such as E. coli, trpL is a member of the entire set of trp genes, whereas in other organisms, transcription attenuation is used to regulate only a subset of the trp genes, as is the case for the trpLE and trpLGDC operons of P. aeruginosa, the trpLEG operon of some Alphaproteobacteria (e.g., Agrobacterium tumefaciens) and Actinobacteridae (e.g., Streptomyces coelicolor), as well as the trpLE operon of Deinococci (e.g., Deinococcus radiodurans) (Figure 1). Considering that some intermediates used in tryptophan biosynthesis function in other metabolic pathways, it is not surprising that some organisms exhibit specific trp operon rear rangements, such as the division of the entire operon into smaller transcriptional units, or the merging of trp genes with genes involved in closely related pathways to form supraoper ons. This is true for the trp operon of the Gram-positive bacterium, B. subtilis, and some of its close relatives. In these, six of the seven trp genes form a trpEDCFBA suboperon, which is transcribed as part of a supraoperon that also contains genes involved in the common aromatic pathway, as well as in phe nylalanine, tyrosine, and histidine biosynthesis. The seventh trp gene, trpG (also functioning as pabA), is located in the unlinked folate operon (Figure 1). Other examples where genes of folate biosynthesis (e.g., truA, accD, or folC) are located in the trp operon are found in different Proteobacteria such as Acidovorax sp., Brucella melitensis, and A. tumefaciens. Finally, in some pathogens, such as Haemophilus ducreyi and various chlamydial species, several or all genes of the trypto phan biosynthetic pathway have been lost or disrupted in a process known as “reductive evolution”. Examples of this type
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trp Operon Organization and Regulation in Different Bacterial Species
of trp gene loss in Haemophilus ducreyl (not shown in Figure 1) that are present in its phylogenetic close relative, H. influenzae (see Figure 1), are trpD, trpC, trpF, trpB, and trpA.
See also: Bacteria; Bacterial Transformation; Tryptophan Operon of Escherichia coli.
Further Reading Calhoun DH, Pierson DL, and Jensen RA (1973) The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa. Molecular and General Genetics 121: 117–132. Gutierrez-Preciado A, Jensen RA, Yanofsky C, and Merino E (2005) New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria. Trends in Genetics 21: 432–436. Gutierrez-Preciado A, Yanofsky C, and Merino E (2007) Comparison of tryptophan biosynthetic operon regulation in different Gram-positive bacterial species. Trends in Genetics 23: 422–426.
Merino E, Jensen RA, and Yanofsky C (2008) Evolution of bacterial trp operons and their regulation. Current Opinion in Microbiology 11: 78–86. Xie G, Bonner CA, and Jensen RA (2002) Dynamic diversity of the tryptophan pathway in chlamydiae: Reductive evolution and a novel operon for tryptophan recapture. Genome Biology 3: research0051. Xie G, Bonner CA, Song J, Keyhani NO, and Jensen RA (2004) Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy. BMC Biology 2: 15. Xie G, Keyhani NO, Bonner CA, and Jensen RA (2003) Ancient origin of the tryptophan operon and the dynamics of evolutionary change. Microbiology and Molecular Biology Reviews 67: 303–342. Yanofsky C (2013) Tryptophan operon of Escherichia coli. In: Maloy S and Hughes K (eds) Encyclopedia of Genetics, 2nd edn., San Diego: Academic Press.
Relevant Websites http://cmgm.stanford.edu – Merino: Stanford University Center for Molecular and Genetic Medicine. http://www.ibt.unam.mx – RibEx: Riboswitch Explorer.
Glossary Operator A DNA segment with a specific nucleotide sequence recognized by a DNA-binding regulatory protein. Operon A transcribed unit of genetic material, generally containing one or more structural genes. Riboswitch An RNA element, commonly located in the 5′ leader segment of a transcript, that is used to regulate expression of the genes within its transcription unit, in response to the presence or absence of a specific metabolite. Recognition of these metabolites by riboswitches can be performed without the participation of any protein.
Tryptophan is one of the key amino acids influencing the structure and function of many proteins. The metabolic path way responsible for tryptophan biosynthesis from chorismate, the common precursor of the three aromatic amino acids, requires the enzymatic activities of seven enzyme functional domains encoded by the trp genes. The organization of these trp genes within operons and the regulatory mechanisms used to control trp operon expression vary appreciably, undoubt edly reflecting organismal divergence in response to different metabolic demands. This article describes trp operon organiza tional and regulatory differences in diverse bacterial species, illustrating the flexible range of choices that organisms must have had during evolution of their tryptophan biosynthetic capability.
Regulation of the Biosynthetic trp Operon of Bacteria Tryptophan biosynthesis is complex and remarkably costly. Accordingly, expression of the genes encoding the proteins responsible for this process is generally highly regulated. The elements and factors responsible for trp gene regulation vary significantly from species to species, and include DNA and RNA elements, DNA- and RNA-binding proteins, protein-binding proteins, and translating ribosomes. It is evident that many of these regulatory features were under evolutionary selection to allow an organism to respond to changes in the intracellular levels of free tryptophan and/or uncharged transfer RNATrp (tRNATrp). Some of these ele ments influence the action of RNA polymerase, while others affect premature termination of transcription. In any event, clear phylogenetic preferences can be seen in the types of regulatory strategies used in controlling trp operon expression. In Gram-negative bacteria, such as Escherichia coli, expres sion of the trp operon is finely adjusted through recognition
208
Transcription attenuation A regulatory mechanism used by bacteria to sense – and respond – to a specific metabolic signal, by regulating premature termination of transcription of the transcribed operon. Attenuation is based on cell selection of one of two mutually exclusive secondary structures, one of which is commonly a Rhoindependent transcription terminator. TrpR An aporepressor, which, upon binding two L-tryptophan molecules, undergoes conformational changes to become an active repressor. The active repressor can bind at specific operator sites in its target promoters, blocking initiation of downstream gene transcription.
of the intracellular levels of both free tryptophan and uncharged tRNATrp as signaling cues. The tryptophan concentration is assessed by the trp repressor protein, TrpR, which regulates transcription initiation (Figure 1; Boxes 1(ia) and 1(ib)). When intracellular levels of trypto phan are low, TrpR exists in an inactive conformation that cannot bind to trp operator DNA. Consequently, RNA poly merase is able to initiate trp operon transcription (Box 1 (ia)). As the intracellular level of tryptophan rises, it binds to the TrpR aporepressor, altering its conformation, and activating its DNA-binding ability. In this active form, TrpR binds to specific DNA operator sequences that overlap the trp promoter, preventing initiation of trp operon transcrip tion (Box 1(ib)). In addition, transcription of the trp genes of this bacterial group is regulated by transcriptional attenuation, in response to the level of uncharged tRNATrp. Using this regulatory mechanism, under growth conditions where the charged tRNATrp level is low, the ribosome trans lating the trp operon’s leader peptide coding sequence, trpL, stalls at either of its two adjacent trp codons. The position of the stalled ribosome favors the formation of an RNA antiterminator structure, and, hence, transcription con tinues into the structural gene region of the operon (Box 1(iia)). When the level of charged tRNATrp is high, transla tion of trpL messenger RNA (mRNA) is completed without ribosome pausing, allowing the leader RNA to fold and form a Rho-independent terminator, promoting premature transcription termination (Box 1(iib)). Interestingly, the trp operons of some members of the Chlamydiales, which are phylogenetically remote from Gammaproteobacteria, are also regulated by TrpR repression and ribosome-mediated transcriptional attenuation (Figure 1). A member of the Gammaproteobacteria, Pseudomonas aeru ginosa, and its close relatives possess a trpBA operon, encoding the two subunits of the tryptophan synthase enzyme complex. This operon is positively regulated by the product of the
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 7
doi:10.1016/B978-0-12-374984-0.01591-6
trp Operon Organization and Regulation in Different Bacterial Species
Escherichia coli K12 Haemophilus influenzae
trpL
TrpR TrpI
Pseudomonas aeruginosa
trpL
TrpR
Psychrobacter cryohalolentis
TrpR
Proteobacteria
Gamma
Beta Alpha Epsilon Epsilon Delta
trpR
Thermotogae Thermotogae Actino bacte teria ria tinobac
Plan Planctomy omycetes Bacteroi detes s Bacteroidete TrpR
AT
Bacillales
trpC
trpG
trpD
trpC
trpE
trpG
trpD
trpC
trpE
aroH
trpG
trpD
trpL
trpE G
Brucella melitensis Gluconobacter oxydans Campylobacter jejuni Helicobacter pylori
trpL
trpE G
ppiD trpE
trpL
truA
trpF truA
ppiD
trpD
trpC
moaC
trpG D
trpF
trpG
trpE
trpG
trpB
trpC F
trpD
trpC
trpF aroA trpB
trpD
trpF
trpE
trpG
trpD
trpC
trpF aroA trpB
trpE
trpG
trpD
trpC
trpF
trpB
trpA aroH aroA
Thermotoga maritima
trpE
trpC
trpF
trpB
trpA
Mycobacterium tuberculosis Streptomyces coelicolor
trpE
trpL
trpE
trpE
trpG
Leptospira interrogans
trpE
trpG
Pirellula sp.
trpE trpB
Chlamydia trachomatis mtrB Bacillus subtilis Bacillus thuringiensis
trpE trpL
trpR aroF aroB aroH
Staphylococcus aureus Streptococcus pneumoniae Leuconostoc mesenteroides
trpC
trpD
priA
trpD trpA
trpG
trpD
trpB
trpC
trpA
trpA
trpC
trpF
trpB
trpA
hisC
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
ltbR
trpC
phzE
) trpB
(17 trpB
trpA trpA
trpC
trpD
tyrA
aroE
TrpI mtrB
trpA
trpB trpA
trpF
purH
trpF trpF
trpA
trpC
trpC
trpD trpD
trpB trpB
trpD
trpG
trpB
trpA
trpA
trpG
trpE
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
aroA
trpE
trpG
trpD
trpC
trpF
trpB
trpA
trpE
trpG
trpD
trpC
trpF
aroF
trpE
trpG
trpD
trpC
trpF
trpB
trpA
Clostridium thermocellum Clostridium hydrogenoformans
trpD
trpA
trpC F
trpB trpA
trpC
trpB
trpA accD folC
trpB trpA
trpB
trpF
trpG
hisI
trpA accD
trpC
Dehalococcoides sp. Sphaerobacter thermophilus
trpE G
trpA accD
trpB
trpA
trpE
trpL
trpB trpF
trpF trpB
moeA
trpA trpB
trpC
trpC
trpA trpA
trpF trpB trpA accD folC
Synechococcus sp.
trpG D
trpB trpB
trpF
trpD
trpD
trpI
trpF
trpC moaC moeA
trpE
trpR
trpA
trpF
trpC
trpE
trpG
trpB
trpD
trpC
trpE
trpR
trpA
ppiD
trpG trpD
trpE G
Oceanobacillus iheyensis Lactobaci llales cillales
trpG
trpD
trpL
Bacteroides fragilis
Chla Chlamydiae
Firmicutes Firmicutes
trpE
trpB
trpC F
trpG
trpL
Corynebacterium glutamicum Spirochaetes Spirochaetes
trpL
Acidovorax sp. Agrobacterium tumefaciens
Deinococcus radiodurans
Chlorofle Chloroflexi
trpC F trpD
Bordetella parapertussis
Solibacte rusitatus
Deino coc ccus Deinoco Cyanobacter obacteriia
trpG D trpG
trpE
Pelobacter carbinolicus
Aci Acidobacteria
Clostridia
trpE trpE
209
trpB
TrpR
pabB
trpG ( 7
rtpA
)
TrpI activator and its binding site TrpR and its DNA-binding sites TRAP and its RNA-binding sites trpL
trpA mtrB 14
( )
trpL leader peptide gene Transcription attenuator T-box element AT
Anti-TRAP
Figure 1 Operon organization and transcription regulation of trp genes. The dendogram shown is based on the phylogenetic classification of the different bacterial species. Selected species are color coded according to major phylogenetic groupings. The different trp genes are color coded. trp gene fusions are indicated by single arrows with more than one color. For large operons with more than five consecutive non-trp genes (white arrows), the number of these genes is indicated. mtrB, rtpA, and trpI are, respectively, the structural genes for the TRAP, AT, and TrpI regulatory proteins.
trpI gene in response to the accumulation of indoleglycerol phosphate (InGP), a tryptophan biosynthetic intermediate (Figure 1). In these organisms, the trpI and trpBA operons are divergently transcribed from overlapping promoters. When the intracellular level of tryptophan is low, InGP is overproduced and it binds to TrpI, inducing a conformational change that favors its binding to DNA operator sequences located in the trpI–trpBA intergenic region. TrpI binding simultaneously represses transcription of its own gene, trpI, and activates tran scription of the trpBA operon, either by direct interaction with RNA polymerase or by inducing DNA bending (Box 1(iiia)). Under growth conditions where there is excess tryptophan, the InGP concentration is low and TrpI can only bind to its highaffinity binding site; thus, transcription of trpBA cannot be activated (Box 1(iiib)). Although the biosynthetic trp operons of Gram-positive bacteria are also regulated in response to the intracellular level of free tryptophan and the availability of charged tRNATrp, quite different molecular mechanisms are used by
these organisms (Figure 1). For example, in the vast majority of Gram-positive bacteria, transcription initiation of the trp operon is regulated in response to uncharged tRNATrp by the T-box riboswitch mechanism (Figure 1). This element regulates expression of a large number of amino acid-related genes, such as those specifying aminoacyl-tRNA synthetases, amino acid transporters, and biosynthetic proteins, including those involved in tryptophan biosynthesis. For these genes, the RNA leader region of the trp operon can be folded into a set of highly conserved RNA secondary structures that are able to interact, with great specificity and affinity, with uncharged tRNATrp. This interaction stabilizes an RNA antiterminator structure, prevent ing transcription termination in the respective operon, thereby promoting transcription of the remaining segment of the operon (Box 1(iva)). When the pool of charged tRNATrp becomes elevated, this charged tRNA is incapable of stabilizing the antiterminator structure. Thus, the more energetically favored secondary structure – the transcription terminator – forms, and transcription of the trp operon is terminated
Box 1 Molecular mechanisms used in regulating transcription of the trp biosynthetic operons of bacteria. The variety of factors and elements participating in the regulation of transcription of trp biosynthetic operons are shown. These factors include the DNA-binding proteins TrpR and TrpI; translating ribosomes; the RNA-binding protein TRAP; protein–protein interactions by the anti-TRAP (AT) protein; and the riboswitch T-box. (i) Transcription regulation based on tryptophan activation of the TrpR aporepressor. The TrpR protein is found mainly in Gammaproteobacteria and in some Chlamydiales (Figure 1). (a) When intracellular levels of tryptophan are low, TrpR exists in its aporepressor conformation and thus transcription of the trp operon proceeds. (b) As the intracellular level of tryptophan rises, the binding of free tryptophan to TrpR converts it to an active conformation, which allows it to bind specific operator DNA sequences that overlap the trp promoter. As a result of this binding, transcription of the trp operon is repressed. (ii) Ribosome-mediated transcription attenuation. Regulation of the trp operon by transcriptional attenuation is observed in different Proteobacteria, as well as in some Chlamydiales, Actinobacteridae, and Deinococci (Figure 1). The intracellular level of charged tRNATrp determines whether a ribosome pauses while translating the trpL leader peptide coding region. (a) At low intracellular levels of charged tRNATrp, the ribosome translating trpL mRNA stalls at one of its adjacent Trp codons. This pausing favors formation of the RNA antiterminator structure (B:C). (b) At high intracellular levels of charged tRNATrp, the translating ribosome does not stall, allowing formation of the transcription terminator structure (C:D). (iii) Regulation based on the activation of the TrpI protein. The TrpI protein appears to be restricted to Pseudomonas aeruginosa, P. putida, and P. syringae (Figure 1). The corresponding structural gene, trpI, is adjacent to and transcribed divergently from the trpBA operon (Figure 1). (a) Whenever the intracellular level of tryptophan is low, InGP is overproduced and TrpI assumes its active conformation, which is able to bind at two operator sites in the trpI–trpBA intergenic region. TrpI binding activates transcription of the trpBA operon. (b) When the tryptophan concentration is high, InGP is not overproduced and TrpI is only able to bind at its high-affinity binding site in the trpI–trpBA intergenic region. This binding represses transcription of trpI but is insufficient to activate transcription of the trpBA operon. (iv) Regulation based on the T-box riboswitch. Regulation of biosynthetic trp operons by the T-box riboswitch is observed in most of the Bacillales, Lactobacillales, and some members of the Clostridia group (Figure 1). (a) Uncharged tRNA can pair with the trp leader RNA stabilizing an antiterminator structure (B:C). (b) When tRNATrp is charged, it is no longer able to interact with the trp RNA leader, thereby favoring formation of the terminator structure (C:D), causing Rho-independent transcription termination. (v) Regulation based on the action of the TRAP protein. TRAP is an RNA-binding protein found in some Bacillales, including Bacillus subtilis, and in a few Clostridia (Figure 1). (a) Whenever tryptophan is growth limiting, TRAP is inactive. Since TRAP is inactive, it cannot bind to leader RNA and the antiterminator structure (B:C) is formed. (b) When there is an excess of tryptophan, TRAP is activated, and it binds to the leader RNA disrupting the antiterminator structure, allowing the terminator structure (C:D) to be formed. (vi) Regulation based on the action of the anti-TRAP protein AT. (a) In organisms with an at gene, whenever the uncharged tRNATrp level is high, AT is synthesized and it binds to tryptophan-activated TRAP, inhibiting its activity. (b) As the level of uncharged tRNATrp decreases, AT synthesis is arrested and thus active TRAP is able to regulate the trp operon.
(ia)
(ib)
(iia)
(iib) B C D
A
C D uuuu
A
(iiia)
B
(iiib)
trpI
trpI
trpBA
(iva)
trpBA
(ivb) B
C
C D uuuu B
D
(va)
(vb) B C C D
A (via)
A
B
D trpEDCBA uuuu
(vib) B C A
D
A
B
C D trpEDCBA uuuu
trp Operon Organization and Regulation in Different Bacterial Species (Box 1(ivb)). Interestingly, T-box elements regulating trp bio synthetic genes are often arranged in tandem in the upstreamtranscribed region of the trp operon. However, in some species, only a single T-box element is present (Figure 1). It has been postulated that the existence of tandem T-boxes may expand the regulatory range of these elements for the respective operon because pairing of two uncharged tRNATrp molecules, instead of one, would be required to stabilize the two antiterminator structures of these T-boxes. This regulatory advantage might compensate for the lack of a separate regulatory mechanism sensing free tryptophan. In some Bacillales, including Bacillus subtilis, and in a few Clostridia, regulation of the trp genes is not mediated by the T-box mechanism; instead, the trp operon is regulated by an attenuation mechanism mediated by the RNA-binding pro tein, trp RNA-binding attenuation protein (TRAP) (Figure 1; Boxes 1(va) and 1(vb)). In these organisms, the leader RNA sequence of the trp operon can be folded into two mutually exclusive secondary structures, one corresponding to a Rhoindependent transcription terminator, and the other corre sponding to an antiterminator. Whenever tryptophan is growth limiting, TRAP is inactive and cannot bind to RNA. In this case, the leader RNA of the trp operon folds to form the energetically favored antiterminator secondary structure. This structure prevents the formation of the transcription termina tor, and consequently, transcription proceeds into the structural gene region of the operon (Box 1(va)). When there is an excess of tryptophan, TRAP is activated and it binds to leader RNA, disrupting the antiterminator structure and favor ing formation of the terminator structure. This leads to premature termination of transcription in the leader region of the operon (Box 1(vb)). In addition to this mechanism of regulation, it has been demonstrated that the few transcripts that escape transcription termination are subject to transla tional regulation via formation of a secondary structure that sequesters the ribosome-binding site of the first gene of the operon, trpE. Also, in B. subtilis, expression of trpG, located in the unlinked folate operon, is coordinately regulated along with the other trp genes by TRAP, which, in its tryptophanactivated conformation, binds at the trpG mRNA ribosomebinding site, inhibiting trpG translation. In B. subtilis, the accumulation of uncharged tRNATrp is also sensed in regulating trp operon and trpG expression. This regulation is due to the action of an additional regulatory protein, anti-TRAP, or AT. Expression of the structural gene for AT, rtpA, is regulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. Transcription of the at operon is regulated by a T-box element, and transcription proceeds whenever the level of uncharged tRNATrp is high. The at operon also contains a leader peptide coding region. Whenever the intracellular level of uncharged tRNATrp is high, translation of the leader peptide coding region stalls at one of its Trp codons. AT is then synthesized, and it is capable of binding to tryptophan-activated TRAP, inhibiting its function (Box 1(via)). AT appears to have evolved very recently because it has been identified only in B. subtilis and very closely related organisms (Figure 1). In the other TRAP-containing organisms, it is not known whether the accumulation of uncharged tRNATrp is sensed by some other regulatory mechanism.
211
Operon Organization of the trp Genes All organisms capable of synthesizing tryptophan from choris mate share a similar metabolic pathway that involves the activities of the seven enzyme functional domains encoded by the trp genes. Genome organization of these genes appears to have diverged significantly between different bacterial groupings, although spe cific gene arrangements are observed among phylogenetically related organisms, as described for the following groups. In the lower Gammaproteobacteria, including E. coli, the seven trp biosynthetic genes are organized within a single tran scriptional unit, the trp operon. In this phylogenetic group, the trp operon always contains a trpCF gene fusion, and, less often, a trpGD fusion (Figure 1). Interestingly, differences in gene arrangements are not restricted to the genes encoding the trp enzymes. Genome organizational plasticity has also played an important role in determining the location of trp genes coding for regulatory proteins, such as trpR. For example, in the Gammaproteobacteria group, trpR is commonly transcribed as a monocistronic operon. However, it is also found within larger operons containing other trp genes, allowing coordinate expression of trpR and biosynthetic genes. Examples of this type are found in Psychrobacter cryohalolentis and Chlamydia trachomatis, where trpR is located on the trpREG and trpRLBA operons, respectively (Figure 1). As previously mentioned, in some organisms, the intracellu lar level of charged tRNATrp is sensed during ribosome translation of a small leader peptide coding region, specified by the gene trpL. This small coding region is located almost invariably as the first gene of the trp operon, although, on occa sion, trpL occupies some other position, such as in C. trachomatis, where trpL is the second gene of the trpRLBA operon. In some lower Gammaproteobacteria, such as E. coli, trpL is a member of the entire set of trp genes, whereas in other organisms, transcription attenuation is used to regulate only a subset of the trp genes, as is the case for the trpLE and trpLGDC operons of P. aeruginosa, the trpLEG operon of some Alphaproteobacteria (e.g., Agrobacterium tumefaciens) and Actinobacteridae (e.g., Streptomyces coelicolor), as well as the trpLE operon of Deinococci (e.g., Deinococcus radiodurans) (Figure 1). Considering that some intermediates used in tryptophan biosynthesis function in other metabolic pathways, it is not surprising that some organisms exhibit specific trp operon rear rangements, such as the division of the entire operon into smaller transcriptional units, or the merging of trp genes with genes involved in closely related pathways to form supraoper ons. This is true for the trp operon of the Gram-positive bacterium, B. subtilis, and some of its close relatives. In these, six of the seven trp genes form a trpEDCFBA suboperon, which is transcribed as part of a supraoperon that also contains genes involved in the common aromatic pathway, as well as in phe nylalanine, tyrosine, and histidine biosynthesis. The seventh trp gene, trpG (also functioning as pabA), is located in the unlinked folate operon (Figure 1). Other examples where genes of folate biosynthesis (e.g., truA, accD, or folC) are located in the trp operon are found in different Proteobacteria such as Acidovorax sp., Brucella melitensis, and A. tumefaciens. Finally, in some pathogens, such as Haemophilus ducreyi and various chlamydial species, several or all genes of the trypto phan biosynthetic pathway have been lost or disrupted in a process known as “reductive evolution”. Examples of this type
212
trp Operon Organization and Regulation in Different Bacterial Species
of trp gene loss in Haemophilus ducreyl (not shown in Figure 1) that are present in its phylogenetic close relative, H. influenzae (see Figure 1), are trpD, trpC, trpF, trpB, and trpA.
See also: Bacteria; Bacterial Transformation; Tryptophan Operon of Escherichia coli.
Further Reading Calhoun DH, Pierson DL, and Jensen RA (1973) The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa. Molecular and General Genetics 121: 117–132. Gutierrez-Preciado A, Jensen RA, Yanofsky C, and Merino E (2005) New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria. Trends in Genetics 21: 432–436. Gutierrez-Preciado A, Yanofsky C, and Merino E (2007) Comparison of tryptophan biosynthetic operon regulation in different Gram-positive bacterial species. Trends in Genetics 23: 422–426.
Merino E, Jensen RA, and Yanofsky C (2008) Evolution of bacterial trp operons and their regulation. Current Opinion in Microbiology 11: 78–86. Xie G, Bonner CA, and Jensen RA (2002) Dynamic diversity of the tryptophan pathway in chlamydiae: Reductive evolution and a novel operon for tryptophan recapture. Genome Biology 3: research0051. Xie G, Bonner CA, Song J, Keyhani NO, and Jensen RA (2004) Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy. BMC Biology 2: 15. Xie G, Keyhani NO, Bonner CA, and Jensen RA (2003) Ancient origin of the tryptophan operon and the dynamics of evolutionary change. Microbiology and Molecular Biology Reviews 67: 303–342. Yanofsky C (2013) Tryptophan operon of Escherichia coli. In: Maloy S and Hughes K (eds) Encyclopedia of Genetics, 2nd edn., San Diego: Academic Press.
Relevant Websites http://cmgm.stanford.edu – Merino: Stanford University Center for Molecular and Genetic Medicine. http://www.ibt.unam.mx – RibEx: Riboswitch Explorer.