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Journal of Bacteriology, September 2005, p. 6419-6429, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6419-6429.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus

Yunwei Xie{dagger} and John N. Reeve*

Department of Microbiology, Ohio State University, Columbus, Ohio 43210

Received 24 May 2005/ Accepted 7 July 2005


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ABSTRACT
 
Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNATrp available to translate the second codon of the trpY mRNA.


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INTRODUCTION
 
The biosynthesis of tryptophan from chorismate is catalyzed by enzymes with a common ancestry in Bacteria, Archaea, eukaryotic microorganisms, and plants (14, 26, 31, 48). This pathway is metabolically very expensive, and expression of the trp genes that encode the enzymes that catalyze tryptophan biosynthesis is therefore tightly regulated (48). An interesting evolutionary issue is that although the trp genes themselves are conserved in all three biological domains, a variety of very different mechanisms regulate their expression, with regulation often imposed at more than one level (26, 48, 53). In Escherichia coli, for example, when the tryptophan pool is sufficient, TrpR, a tryptophan-activated repressor, binds upstream of the trpEGCBA operon blocking RNA polymerase (RNAP) access to the promoter (35, 43, 52, 53). If initiation nevertheless does occur, regulation is then imposed during translation of a short leader peptide (TrpL). With the availability of charged tRNATrp adequate for efficient TrpL translation, transcription is terminated by attenuation upstream of trpE (54). Similarly, in Bacillus subtilis, two sequential events in trp gene expression are regulated by the availability of tryptophan. With an adequate supply of tryptophan, transcription of the trpEDCFBA genes is prevented by attenuation but not as a consequence of efficient leader-peptide translation. Instead, the trp transcript is bound by a tryptophan-activated RNA-binding protein, TRAP, preventing the formation of an antiterminator and allowing attenuation. If trp operon transcription escapes attenuation, tryptophan-activated TRAP binds to the mRNA, and this sequesters the trpE ribosome binding site and prevents translation (5, 54). Yet a third attenuation mechanism, the T-box system (24), regulates transcription of the trp genes in Lactococcus lactis (38). In the absence of tryptophan, uncharged tRNATrp hybridizes to the leader region of the transcript, preventing attenuation and so promoting tryptophan biosynthesis. The regulation of trp gene expression has also been studied in eukaryotic systems (26, 48), most extensively in fungi in which starvation for many different nutrients, including tryptophan, activates the cross-pathway response. Starvation results in increased translation of transcripts that encode a global activator, designated GCN4 in Saccharomyces cerevisiae (34) and CPCA in Aspergillus species (17, 28), and also increases the stability of this regulator (27) that binds to conserved sequences located upstream of many genes, including the trp genes. Binding of the activator results in chromatin reorganization, RNA polymerase II (RNAPII) recruitment, and activation of transcription of the downstream genes (34).

Homologues of the trp genes in Bacteria and eukaryotes are also present in Archaea, but the only evidence for their regulation is that trp gene transcripts are more abundant in cells of Methanothermobacter marburgensis and Pyrococcus kodakaraenis grown in the absence than in the presence of tryptophan (19, 44). M. marburgensis mutants have been isolated that are resistant to 5-methyltryptophan (5MT) which were shown to constitutively express the trpEGCFBAD operon and to have sequence changes in the gene (here designated trpY) located immediately upstream and transcribed divergently from the trp operon or between trpY and the trp operon (20) (Fig. 1A). A subsequent bioinformatics study predicted that TrpY could be a tryptophan-binding regulator that might bind to one or more TGTACA-related sequences (designated TRP boxes) identified between trpY and trpEGCFBAD (21)



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  		FIG. 1. Organization and transcription of the M. thermautotrophicus trpY-trpEGCFBAD genes. (A) The trp operon and intergenic region are drawn to scale, with the region used as template T1 indicated by the black bar. Potential TRP boxes (21) are labeled 1 through 6, above the sequence of the intergenic region, with nucleotides that conform to the TRP box consensus (TGTACA) identified by asterisks. The TATAE and TATAY sequences are identified, and the TATA box sequence changes in templates T2 and T3 are highlighted. The sites of transcription initiation are identified by the arrows labeled Y and E. As illustrated, the G:C base pair at which Y is initiated is deleted ({blacktriangleup}) in a 5MT-resistant mutant (MWR1) of M. marburgensis (20). This base pair was deleted to generate template T7 and was replaced by A:T, T:A, and C:G base pairs to generate templates T4, T5, and T6, respectively. (B) 32P-labeled transcripts synthesized in vitro from templates T1, T2, and T3 separated by polyacrylamide gel electrophoresis and visualized by phosphorimaging (16, 49-51). The control lane (S) contained size standards with the lengths indicated in nucleotides (n). (C) Electrophoresis of the primer extension products, identified by asterisks, generated from transcript E (upper gel) and Y (lower gel) templates in lanes (P) adjacent to dideoxy sequencing products (lanes T, A, C, and G) generated from the intergenic region using the same primers. The symmetry in the organization of the two divergent promoters separated by one helical turn (11 bp) is indicated. (D) E and Y transcripts synthesized in vitro from the templates listed with the nucleotide at the site of initiation noted below the template designation. The control lanes (S) contained size standards. (E) DNase I footprints of the complexes formed by TBP plus TFB binding to the T1, T2, and T3 BRE-TATA box sequences. Aliquots of DNA (25 ng) with (+) and without (–) TBP (100 ng) plus TFB (300 ng) were subjected to DNase I digestion, and the products were separated by electrophoresis. The DNase I-protected regions are indicated by gray boxes between the lanes in the gel and below the corresponding intergenic sequence. The control lane (S) contained size standards.

In Archaea, transcription initiation closely resembles the eukaryotic RNAPII system with archaeal TATA box binding protein (TBP) and transcription factor B (TFB, the archaeal homologue of eukaryotic general transcription factor TFIIB) binding to TATA box and BRE (B-responsive element) sequences located ~25 bp upstream of the site of transcription initiation. These general transcription factors then recruit and orient the archaeal RNAP appropriately for initiation (10, 36, 39). Regulators of archaeal transcription initiation have been shown to repress initiation by preventing TFB and TBP access to the BRE-TATA box region (9, 29, 33) or RNAP access to the site of transcription initiation (7, 12, 15, 46) and to activate initiation by stimulating TBP binding to the TATA box region (37). We have established an in vitro transcription system using TBP, TFB, and RNAP from Methanothermobacter thermautotrophicus (16, 49-51), and, given the predictions for trp gene regulation in M. marburgensis (19, 20), a close relative of M. thermautotrophicus, we have used this system to investigate the regulation of expression of the archaeal trp genes in M. thermautotrophicus. The trpY-trpEGCFBAD operon organization is conserved (32, 41), and a second trpB homologue (trpB2) is present at a remote location in the M. thermautotrophicus genome (41, 47). The results obtained demonstrate that TrpY is a tryptophan-sensing trp gene regulator that binds, as predicted (3, 21), to TRP box sequences. TrpY binding autorepresses trpY transcription in the absence of tryptophan and represses trpY, trpEGCFBAD, and trpB2 transcription in the presence of tryptophan. As TrpY contains only one tryptophan residue (W2) and there is only one tryptophan codon in the entire trpEGCFBAD operon (32, 41), it seems likely that the ability to translate trpY mRNA also plays a pivotal role in regulating the expression of this archaeal trp operon.


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MATERIALS AND METHODS
 
Reagents and enzymes. Except where otherwise noted, all chemicals were purchased from Sigma Chemicals (St. Louis, MO). DNA-modifying and restriction enzymes and TOPO-TA cloning kits were from Invitrogen (Carlsbad, CA). Nickel-nitrilotriacetic acid spin columns, plasmids, and PCR product purification kits were from QIAGEN Inc. (Valencia, CA). 32P-labeled reagents were from MP Biomedicals (Irvine, CA). Table 1 provides the sequences and identifies the DNA molecules generated and uses of the oligonucleotides obtained from Ransom Hill Biosciences (Ramona, CA).


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  		TABLE 1. Oligonucleotides used in this study

Transcription templates. A DNA molecule (533 bp) that contains the intergenic region (102 bp) between trpY (MTH1654) and trpE (MTH1655) and ~200 bp of the 5' regions of trpY and trpE was PCR amplified from M. thermautotrophicus genomic DNA (41) using oligonucleotides MX75 and MX57 as primers (Table 1). The PCR product was cloned into pCR21-TOPO using a TOPO-TA cloning kit, resulting in plasmid p7557, and the cloned DNA was sequenced to confirm accuracy. Template T1 (Fig. 1A) and T11 DNAs were PCR products amplified from p7557 DNA using primer pair MX75 and MX57 and primer pair MX56 and MX64, respectively. All PCR-generated DNA molecules were purified using QIAGEN purification kits before use in in vitro transcription, electrophoretic mobility shift, or DNase I protection assays.

The sequence of the M. thermautotrophicus DNA cloned in p7557 was changed by site-directed mutagenesis to obtain the plasmids from which templates T2 through T9 were generated by PCR amplification using primers MX75 and MX57. Mutagenic reaction mixtures (50 µl) were assembled that contained 20 ng p7557 DNA, 200 µM deoxynucleoside triphosphates, 2.5 U Turbo Pfu DNA polymerase (Stratagene, La Jolla, CA), and 50 pmol of the pairs of mutagenic primers (MX80/MX81, MX82/MX83, MX84/MX85, MX103/MX104, MX113/MX114, MX115/MX116, MX117/MX118, MX124/MX141; Table 1) in Pfu buffer [200 mM Tris-HCl (pH 8.8), 100 mM (NH4)2SO4, 100 mM KCl, 1% Triton X-100, 1 mg bovine serum albumin (BSA)/ml, 20 mM MgSO4]. They were incubated at 94°C for 1 min and then cycled 18 times through incubations at 45°C for 1 min, 68°C for 8 min, and 94°C for 15 s before DpnI (5 U) was added, and incubation was continued at 37°C for 1 h. Aliquots (1 µl) of the reaction products were used to transform E. coli DH5{alpha} cells, and transformants were selected by growth on Luria-Bertani (LB) plates containing 20 µg kanamycin/ml. Plasmids, designated p8081, p8283, etc., based on the mutagenic primers used (Table 1), were isolated from kanamycin-resistant transformants, and the mutagenized region was sequenced to confirm the presence of the desired mutation(s).

Template T10 (438 bp; see Fig. 5A) containing the MTH1477-MTH1476 (trpB2) intergenic region was PCR amplified using MX111 and MX138 as primers from M. thermautotrophicus genomic DNA. The T10 sequence contained the entire intergenic region (133 bp) and ~150 bp of the 5' regions of both MTH1477 and trpB2 (see Fig. 5A).



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  		FIG. 5. TrpY inhibition of trpB2 transcription. (A) The MTH1477 (conserved protein with unknown function)-MTH1476 (trpB2) region of the M. thermautotrophicus genome (41) is shown with the region PCR amplified and used as template T10, indicated by the black bar. The site of B2 transcription initiation in vitro was determined by primer extension (results not shown). The leader region of the B2 transcript could direct the synthesis of the 15-residue boxed peptide. (B) B2 transcripts synthesized in vitro from T10 (30 ng) in the presence of 0 (–), 25, 50, 100, 150, and 200 ng TrpY and in the absence (–trp) or presence (+trp) of 800 µM tryptophan.

Transcription in vitro. Preparation of recombinant TBP and TFB, RNAP from M. thermautotrophicus, assembly of in vitro transcription reaction mixtures, and purification, electrophoretic separation, detection, and quantification of 32P-labeled transcripts synthesized in vitro by phosphorimaging have been described previously (16, 49-51). Each in vitro transcription reaction mixture (25 µl) contained 30 ng of template DNA, 250 ng RNAP, 25 ng TBP, 150 ng TFB, 200 µM each ATP, UTP, and GTP, 20 µM CTP (Roche Applied Science, Indianapolis, IN, or Amersham Pharmacia, Piscataway, NJ), and 5 µCi of [{alpha}-32P]CTP (3 kCi/mmol) in transcription buffer (20 mM Tris [pH 8], 120 mM KCl, 10 mM MgCl2, 2 mM dithiothreitol). Reaction mixtures were incubated at 58°C for 30 min before the synthesized 32P-labeled transcripts were separated by polyacrylamide gel electrophoresis, detected, and quantified by phosphorimaging (Storm 840; Amersham Pharmacia). When TrpY, tryptophan, or other amino acids were added, their concentrations are stated in the relevant figure legends.

Mapping of transcription initiation sites by primer extension. In vitro transcription reaction mixtures (scaled to 200 µl) were incubated for 30 min at 58°C. DNase I (5 U) was added, incubation continued at 37°C for 30 min, and 2 µl of glycogen (200 mg/ml), 100 µl 1 M sodium acetate and 900 µl ethanol were then added, and the mixture was placed at –20°C for 10 min. After centrifugation in an Eppendorf microcentrifuge at 14,000 rpm for 15 min at 4°C, the supernatant was discarded and the pellet was dissolved in 30 µl of TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA). Primer extension reaction mixtures (20 µl) contained 2 µl of in vitro-transcribed RNA solution, 500 µM deoxynucleoside triphosphates, 0.25 pmol of 32P-end-labeled oligonucleotide primer DNA (MX77, MX64, or MX133), and OmniScript reverse transcriptase (4 U) in reverse transcriptase buffer (QIAGEN). They were incubated at 37°C for 60 min before the reaction was terminated by addition of 12 µl of 96% (vol/vol) formamide containing 25 mM EDTA. The 32P-labeled products were separated by electrophoresis through denaturing DNA sequencing gels adjacent to the products of DNA sequence reactions, generated as size standards, using the same 32P-labeled primer with T1 or T10 DNA.

Cloning of trpY (MTH1654) in E. coli. MX86 and MX87 were used as primers to PCR amplify the trpY (MTH1654) gene from M. thermautotrophicus genomic DNA. The amplified product was purified, digested with NdeI and HindIII, and ligated with NdeI- plus-HindIII-digested pET30a DNA (Novagen, Madison, WI). An aliquot of the ligation products was used to transform E. coli DH5{alpha} cells, and transformants were selected on LB plates that contained 50 µg kanamycin/ml. The sequence of the cloned DNA was confirmed, and the resulting plasmid (p8687) was transformed into E. coli Rosetta (DE3) (Novagen) with transformants selected by resistance to both kanamycin (50 µg/ml) and chloramphenicol (25 µg/ml).

Expression and purification of recombinant TrpY. Cultures (50 ml) of E. coli Rosetta (DE3; p8687) were grown in LB medium containing kanamycin (50 µg/ml) and chloramphenicol (25 µg/ml) at 37°C to an optical density at 600 nm of ~0.6, isopropyl-1-thio-ß-D-galactopyranoside was added (1 mM final concentration), and incubation was continued at 37°C for 2 h. The cells were harvested by centrifugation, washed, resuspended in 1.2 ml of lysis buffer (50 mM Na3PO4 [pH 8], 300 mM NaCl), and lysed by passage twice through a French pressure cell, and the lysate was centrifuged for 20 min in an Eppendorf microcentrifuge at 14,000 rpm. The resulting supernatant was loaded on a QIAGEN nickel-nitrilotriacetic acid column, and bound proteins were washed twice using 0.6 ml of 50 mM Na3PO4, 300 mM NaCl, 50 mM imidazole (pH 6) and then eluted using two 0.2-ml aliquots of 50 mM Na3PO4, 300 mM NaCl, 500 mM imidazole (pH 6). The eluates were pooled and dialyzed against 500 ml of 50 mM Tris-HCl, 300 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 8), and 20% (vol/vol) glycerol. The presence and >95% purity of a protein with the electrophoretic mobility predicted for TrpY with a C-terminal LAAALQHHHHHH extension (His tag) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GelCode blue staining (Pierce Biotechnology, Rockford, IL). The concentration of the TrpY solution was determined by a Bradford assay (Bio-Rad, Hercules, CA) using BSA as the standard before glycerol was added to a final concentration to 35% (vol/vol), and the solution was stored at –20°C.

Preparation of 32P-labeled TRP boxes containing DNA molecules and use in electrophoretic mobility shift assays (EMSA) of TrpY binding. Pairs of complementary oligonucleotides (Table 1) were mixed at equimolar concentrations, incubated at 95°C for 3 min, and allowed to cool to room temperature. The double-stranded DNA molecules generated were designated A12 through A19 (see Fig. 3 for sequences). DNA molecules designated A56, B12, B13, B15, and B16 (see Fig. 3) were PCR amplified from p7557 DNA using primer pairs MX64/MX97, MX91/MX96, MX103/MX96, MX92/MX124, and MX91/MX125 (Table 1), respectively. Preparations of these DNAs were 32P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 µl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 µCi [{gamma}-32P]ATP (~7 kCi/mmol). The labeled DNA molecules were separated from unincorporated nucleotides by passage through a Sephadex G-50 column, and aliquots (1 ng) were mixed with 50 ng of unlabeled poly(dI-dC) and incubated in transcription buffer (10 µl) with increasing amounts of TrpY and/or L-tryptophan, as stated in the figure legends. The products were mixed with 1 µl of gel loading buffer (40% sucrose, 0.4% bromophenol blue, 0.4% xylene cyanol), subjected to electrophoresis through nondenaturing 6% polyacrylamide gels run in 90 mM Tris-borate, 2 mM EDTA (pH 8) buffer at 8 V/cm, and visualized by phosphorimaging.



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  		FIG. 3. EMSA of TrpY binding to TRP boxes. The DNA molecules A12, A23, A34, A50, A56, and B12 have the intergenic sequences underlined. The sequences of A12 through A19 and B12, B15, and B16 are shown with the TRP boxes highlighted and differences from wild-type A12 and B12 sequences identified. (A) Aliquots (1 ng) of 32P-labeled A12 through A19 were incubated with 50 ng of poly(dI-dC) and 200 ng of TrpY in the absence (–) and presence (+) of 800 µM tryptophan. The complexes formed were separated by electrophoresis and visualized by phosphorimaging. (B) Aliquots (1 ng) of 32P-labeled B12, B13, B15, and B16 were incubated with 50 ng of poly(dI-dC) and 0 (–), 25, 50, 100, 150, and 200 ng of TrpY with (+) or without (–) 800 µM tryptophan. The complexes formed were separated and visualized as in panel A. Larger complexes formed by TrpY binding to B12 in the presence of tryptophan that migrated as a diffuse band are indicated by an asterisk. (C) Aliquots (1 ng) of 32P-labeled T11 were incubated with 200 ng of TrpY in the absence (–) or presence of the amino acid (800 µM) listed above the corresponding gel lane. Control lanes contained aliquots of T11 incubated without (c) and with tryptophan (c+trp) in the absence of TrpY. As in panel B, the asterisk indicates the larger complexes that migrated as a diffuse band, formed when TrpY bound in the presence of tryptophan. These complexes were not formed in the absence of tryptophan or in the presence of other amino acids.

DNase I footprinting. DNA molecules were generated by PCR amplification from plasmids p7557, p8283 (mutation in TATAE), and p8485 (mutation in TATAY) using 32P-end-labeled MX64 and unlabeled MX75 as primers. The DNase I footprinting reaction mixtures (10 µl) contained 25 ng of 32P-labeled DNA, the transcription factors TFB, TBP, and/or TrpY, tryptophan as listed in figure legends, and DNase I (1 U) in transcription buffer. DNase I digestion was allowed for 7 min at 20°C and then stopped by addition of 10 µl of 96% formamide containing 25 mM EDTA. The digestion products were separated by electrophoresis through DNA sequencing gels, and 32P-labeled molecules were visualized by phosphorimaging.


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RESULTS
 
trpY and trpEGCFBAD are transcribed from divergent overlapping promoters. The organization of the trpY-trpEGCFBAD region in the M. thermautotrophicus genome (32, 41), the region amplified and used as template T1, and the sequence of the intergenic region between trpY and trpE are shown in Fig. 1A. Two transcripts were synthesized from T1 in vitro (designated E and Y) with lengths consistent with divergent runoff transcripts (262 and 169 nucleotides, respectively) initiated within the intergenic region (Fig. 1B). The precise sites of initiation were determined by using in vitro-synthesized transcripts as the templates in primer extension reactions. The lengths of the primer extension products demonstrated that E and Y were initiated between trpY and trpE, with initiation occurring on opposite strands but in both cases at the G residue in the sequence 5' TTGT (Fig. 1C). E initiation occurred at the site established for trpEGCFBAD initiation in vivo (19, 20) (results not shown), and Y initiation occurred at the G that was deleted in a 5MT-resistant mutant (MWR1) of M. marburgensis (20). Given this coincidence, templates T4 through T7 were constructed with the G replaced by A, C, or T or deleted ({blacktriangleup}). Y transcription occurred only with the wild-type template T1 and, to a lesser extent, with T4 with A at the site of initiation. There was no Y transcription from T5 and T6 with pyrimidines (T and C, respectively) at this location or from T7 with the G deleted (Fig. 1D) that resulted in an extended pyrimidine-only sequence, 5'TCTT{blacktriangleup}TCT. These mutations also changed the BREE sequence (see below and Fig. 1C) but at the most variable position in the BRE consensus sequence (8) and did not reduce E synthesis.

Y and E transcription was initiated 23 bp downstream from potential BRE-TATA box sequences, designated BREE-TATAE (5'ACAAGA-TTTAAATA) and BREY-TATAY (5'ACAAAG-TACATATA), respectively, and templates T2 and T3 were generated with mutations in the putative TATA box sequences to test these predictions. In T2 and T3, the AA and AT dinucleotides at positions 4 and 5 in TATAE and TATAY, respectively, were replaced by GG (Fig. 1A). When used as the template DNA, T2 directed only Y synthesis and T3 directed only E synthesis, confirming the transcript-specific roles and requirements of TATAE and TATAY for E and Y synthesis, respectively (Fig. 1B). DNase I footprinting confirmed that TBP plus TFB bound to the BREE-TATAE and BREY-TATAY regions. Complexes formed by TBP plus TFB on the T2 and T3 sequences protected ~25 bp from DNase I digestion that included the BREY-TATAY and BREE-TATAE sequences, respectively. With the T1 sequence, TBP plus TFB binding protected an extended ~45-bp region from DNase I digestion that included both BRE-TATA box sequences (Fig. 1E). As previously reported (8, 9, 23), TBP plus TFB binding also generated a DNase I hypersensitive site immediately upstream of the TATA box.

TrpY represses trpY and trpEGCFBAD transcription. The amino acid sequence of TrpY is shown in Fig. 2A. As illustrated, the N-terminal region is predicted to fold to form several {alpha}-helices (2, 21), with the sequences of helices 3 and 4 and their configuration consistent with a helix-turn-helix DNA-binding motif (11). The C-terminal region is predicted to fold to form an ACT domain (2, 18) and therefore to bind an allosteric effector (4, 13). When TrpY, generated and purified from E. coli, was added to in vitro transcription reaction mixtures containing template T1, Y synthesis was inhibited, but there was no effect on E synthesis. When tryptophan was also added, both Y and E syntheses were inhibited (Fig. 2B and C). E synthesis was 50% inhibited by the presence of 8 µM tryptophan and completely inhibited above 250 µM tryptophan (Fig. 2D). For reference, the intracellular pool contained ~10 µM tryptophan in M. marburgensis cells grown in the absence of tryptophan (19). When TrpY was added, with and without tryptophan, to in vitro transcription reaction mixtures with templates that carried unrelated M. thermautotrophicus promoters (22, 50), there was no effect on transcription (results not shown)



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  		FIG. 2. TrpY sequence and predicted structure, inhibition of transcription, and DNase I footprints. (A) The TrpY amino acid sequence with gray bars identifying predicted {alpha}-helices and a potential helix-turn-helix (HTH) DNA binding motif (3, 11, 21). The residues predicted to form an ACT domain are boxed (2). The oval representing tryptophan is positioned to contact residues located where serine is bound in the ACT domain of 3-phosphoglycerate dehydrogenase (13). trpY homologues are present in many euryarchaeal genomes (COG2150), and homologues with amino acid sequences >40% identical to that of TrpY (MTH1654) are encoded by AF1020 in Archaeoglobus fulgidus, PH1554 in Pyrococcus horikoshii, MA1396 in Methanosarcina acetivorans, Mbu0904 in Methanococcoides burtoni, and PT0908 in Picrophilus torroidus. Residues conserved in all of these TrpY homologues are circled. (B) E and Y synthesis from template T1 (30 ng) in the presence of 0 (–), 25, 50, 100, 150, and 200 ng of TrpY in the absence (–trp) and presence (+trp) of 800 µM tryptophan. (C) The transcripts in panel B (identified as •, {circ}, {blacksquare}, and {square}) graphed as percentages of the amount of that transcript synthesized in the absence of TrpY. (D) E and Y synthesis from T1 in the absence (–) and presence of 200 ng TrpY without (0) and with 2, 8, 24, 80, 160, 240, 480, and 800 µM tryptophan added. A very similar concentration-dependent inhibition of E synthesis was observed when 5MT was substituted for tryptophan (results not shown). (E) Aliquots of 32P-labeled DNA (25 ng) were incubated with 0 (–), 25, 50, 100, and 150 ng of TrpY in the absence (–trp) and presence (+trp) of 800 µM tryptophan, and the complexes formed were subjected to DNase I digestion. The products were separated by electrophoresis in lanes adjacent to size standards (S10, S50). DNase I-hypersensitive sites introduced by TrpY binding are indicated by asterisks, and DNase I-protected regions are identified by shaded boxes.

TrpY binds to TRP box sequences. Gelfand et al. (21) noted the presence of several sequences between trpY and trpE with the consensus sequence 5'TGTACA and suggested that these could be "TRP box" binding sites for a trp operon regulator. Several of these occur in pairs, separated by 4 bp (Fig. 1A), suggesting binding by a polypeptide dimer and exposure of TrpY in solution to formaldehyde resulted in covalently cross-linked TrpY dimers (results not shown). To determine if TrpY bound to the TRP box sequences, DNase I protection experiments were undertaken. The results confirmed that TrpY bound and protected the TRP box 1 and 2 regions in the absence of tryptophan and also introduced DNase I-hypersensitive sites into the TRP box sequences (Fig. 2E). When tryptophan was also present, TrpY binding protected TRP boxes 1 and 2 and also TRP boxes 3 and 4 and introduced DNase I-hypersensitive sites into all of these sequences and between TRP boxes 2 and 3. A DNase I-hypersensitive site was also introduced into TRP box 5 by TrpY in the presence and absence of tryptophan, but this interaction did not result in a detectable DNase I footprint (Fig. 2E).

To identify the sequences specifically required for TrpY binding, electrophoretic mobility shift assays (EMSA) were undertaken with and without added tryptophan, using the DNA molecules listed in Fig. 3. Based on the gel shifts, TrpY bound and formed stable complexes with DNA molecules that contained TRP boxes 1 and 2 (A12 and B12; Fig. 3A and B) but not with DNA molecules that contained TRP boxes 2 and 3 (A23), 3 and 4 (A34), or 5 and 6 (A56). Increasing the distance to 6 bp between TRP boxes 1 and 2 (A13), decreasing it to 2 bp (A14), or changing the sequences of TRP box 1 (A17 and B13) or 2 (A19) eliminated TrpY binding, but changing the sequence between the TRP boxes (A18) did not effect binding (Fig. 3A and B). Addition of tryptophan had no effect on the gel shifts observed with A12, A15, A16, or A18, but with B12 (TRP boxes 1, 2, 3, and 4) the complexes formed in the presence of tryptophan migrated more slowly than those formed in the absence of tryptophan and also migrated as a diffuse rather than a discrete band (indicated by an asterisk in Fig. 3B). These apparently larger but less stable complexes were not formed by TrpY in the presence of other amino acids (Fig. 3C) and were not formed with B15 and B16 that had mutations in the TRP box 3 and 4 sequences, respectively (Fig. 3B). They were also not formed with B13 that had wild-type TRP box 3 and 4 sequences but mutations in TRP box 1. To assemble these larger complexes with sufficient stability in vitro for detection by an EMSA apparently required the presence of TrpY, tryptophan, and TRP boxes 1, 2, 3, and 4.

Effects on TrpY repression of mutations in the TRP box sequences. Templates T8 and T9 were generated from T1 with the sequence changes in TRP boxes 1 and 3, respectively (Fig. 4), that when introduced into A12 (generating A17) and B12 (generating B13) eliminated TrpY binding (Fig. 3). In in vitro reaction mixtures containing T8 (mutation in TRP box 1), both transcripts Y and E were synthesized, but Y synthesis was no longer inhibited by TrpY in the absence of tryptophan (see the asterisk in Fig. 4). Both Y and E syntheses were still sensitive to TrpY addition in the presence of tryptophan. In reaction mixtures with T9 (mutation in TRP box 3) as the template, both transcripts were synthesized. Y synthesis was still sensitive to TrpY addition in both the absence and presence of tryptophan, but E synthesis was only marginally inhibited, even at very high concentrations of TrpY, in the presence of tryptophan (see the double asterisks in Fig. 4).



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  		FIG. 4. E and Y transcription from templates with mutations in TRP boxes 1 and 3. The changes introduced into the T1 sequence resulting in templates T8 and T9 are illustrated. E and Y transcripts synthesized from these templates in the absence (–) and presence of 25, 50, 100, 150, and 200 ng of TrpY, with (+) and without 800 µM tryptophan, are shown. Y synthesis from T1 was inhibited by TrpY in the absence of tryptophan but not, as indicated (*), from T8. E synthesis from T1 was fully inhibited in the presence of tryptophan but not, as indicated (**), from T9.

trpY regulates trpB2 transcription. MTH1476 encodes a protein related to the ß-subunit of tryptophan synthases (47), as encoded by trpB in the trpEGCFBAD operon, and this gene has been designated trpB2. The intergenic region upstream of trpB2 contains a sequence identical to TATAE and two consensus TRP box sequences separated by 4 bp (Fig. 5A). In in vitro transcription reaction mixtures with T10 as the template, only one transcript was synthesized and designated B2, despite the expected presence of a promoter for the divergently oriented MTH1477. When B2 was used as the template in primer extension reactions, the product generated confirmed that this was a 201-nucleotide runoff transcript of the trpB2 gene initiated 23 bp downstream from the TATAB2/E sequence (results not shown). Synthesis of the B2 transcript was completely inhibited by TrpY addition in the presence of tryptophan and was also partially inhibited at very high concentrations of TrpY in the absence of tryptophan (Fig. 5B). As illustrated (Fig. 5A), the transcribed region upstream of trpB2 could encode a serine-rich 15-residue, potentially regulatory leader peptide (see Discussion).


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DISCUSSION
 
The trpY-trpEGCFBAD intergenic region contains overlapping and divergent promoters. M. thermautotrophicus has a very small ~1.7-Mbp genome (41) but nevertheless has the capacity to synthesize all of its cellular components and generate energy from only CO2, H2, N2, and salts (45). To accomplish this, the genome must be used very efficiently for enzyme coding and the lengths of noncoding regions minimized. As a consequence, transcription initiation and termination and their regulation have to occur within very short, presumably information-rich intergenic regions. The presence of multiple, adjacent, and overlapping regulatory signals, as documented here for the intergenic region separating trpY and trpE, is probably more typical than exceptional. The experimental challenge is to determine how transcription and transcription regulators can function within such a confined space. For the trpY-trpE region, the results obtained argue for the organization and regulation modeled in Fig. 6B. TATAE and TATAY are separated by one helical turn (11 bp) and are therefore located, as illustrated, on opposite faces of the DNA. Based on DNase I footprints, M. thermautotrophicus TBP plus TFB can bind coincidentally to both BRE-TATA box regions, and TrpY can also bind to the TRP box 1 and 2 region in the presence or absence of TBP and TFB (Fig. 1E and 6A). Binding of the C-terminal region of TFB to the BRE regions determines the orientation of the preinitiation complexes and the direction of transcription. The details of TBP binding to TATA box sequences and TFB to BRE sequences are well documented (8, 30), and these do not obviously conflict with the coincident binding, as illustrated, of TBP and TFB to the two divergent BRE-TATA box regions. However, TFB also makes contacts with DNA downstream of the TATA box, reaching to the site of transcription initiation (6, 37, 40, 42), and these could pose steric hindrance problems for two overlapping TFB-TBP complexes, but this remains to be determined. Regardless of the ability to assemble two TBP plus TFB complexes on the intergenic region, it seems impossible that both could recruit RNAP and direct initiation concurrently. Footprinting and cross-linking results indicate that an archaeal RNAP in a preinitiation complex contacts DNA that extends at least 15 bp both upstream and downstream from the site of initiation (0 bp) and melts a DNA region that extends from –11 to +5 bp during initiation (6, 23, 42). Given the organization of the trpY and trpE promoters (Fig. 1A and C and 6B), the site of initiation from one promoter is within the BRE of the second promoter, and the single-stranded initiation bubble includes the TATA box sequence of the second promoter. It seems therefore impossible that both promoters could direct initiation at the same time, and initiation from one promoter may, in fact, be a mechanism that dislodges the transcription factors from the second promoter. Archaeal TBP does not dissociate spontaneously from the promoter DNA after directing transcription initiation in vitro (51), but a mechanism to achieve this must exist in vivo.



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  		FIG. 6. DNase I footprints and model of transcription factor assembly on the trpY-trpEGCFBAD intergenic region. Aliquots of 32P-labeled DNA (25 ng) with the T1 sequence were incubated with TrpY (200 ng) and/or TBP (100 ng) and TFB (300 ng) as indicated, in the absence or presence (W) of 800 µM tryptophan, and the complexes formed were subjected to DNase I digestion. The 32P-labeled products were separated by electrophoresis and visualized by phosphorimaging. The regions protected by the transcription factors are indicated and boxed, with the broken-line box noting the extended region protected by TrpY, TBP, and TFB binding in the absence of tryptophan. TrpY binding generated a consistent pattern of DNase I-hypersensitive sites. (B) TrpY, TBP, and TFB are shown assembled on the trpY-trpEGCFBAD intergenic sequence in an organization that conforms with the footprinting and EMSA results and with TBP, TFB, and RNAP positioning data obtained by footprinting and cross-linking of other archaeal preinitiation complexes (6, 8, 23, 30, 37, 40, 42). In this arrangement, with tryptophan (W) bound, TrpY must compete with TBP for binding to the TATA box regions.

TrpY synthesis as a sensor of charged tRNATrp availability. As discussed below, TrpY is a tryptophan-responsive regulator of transcription of trp genes and, in this regard, functions in a manner superficially similar to TrpR in E. coli (14, 35, 43, 52, 53). There is, however, no evidence for a common ancestry, and the archaeal gene and protein were designated trpY and TrpY to avoid any inference of homology in ancestry or detailed mechanism of function with TrpR. TrpY homologues are encoded in many euryarchaeal genomes (COG2150) but have not been detected in crenarchaeal, bacterial, or eukaryotic genomes. Bioinformatics predicts that these proteins have an N-terminal DNA binding region and a C-terminal ACT domain for ligand binding (2, 3, 13, 18). For M. thermautotrophicus TrpY, these predictions have been confirmed experimentally by mutagenesis, EMSA, and [14C]tryptophan binding (L. Cubonova, K. Sandman, and J. N. Reeve, unpublished data). Most of the euryarchaeal TrpY homologues are not encoded by genes physically adjacent to trp genes, but they almost all have only one tryptophan residue, specifically W2, which could well function as a second-level sensor for trp gene regulation. When the intracellular concentration of charged tRNATrp is sufficient to translate the second codon of the trpY mRNA, TrpY will be synthesized and will repress trpEGCFBAD transcription. If the level of charged tRNATrp is insufficient to translate this codon, then with no TrpY synthesized, transcription of the trp operon will proceed. This would be wasteful if the enzymes needed for tryptophan biosynthesis could not themselves be synthesized in the absence of tryptophan, but this is apparently not the case. Strikingly, there is only one tryptophan codon in the entire trpEGCFBAD operon (32, 41) that encodes W175 in TrpB (Fig. 1A), a position occupied by a tryptophan residue in almost all TrpB proteins (47, 48). This lack of tryptophan residues in the tryptophan biosynthetic enzymes in M. thermautotrophicus is a compelling example for the argument that minimizing the use of an amino acid in enzymes required to synthesize that amino acid provides a biological advantage (1).

TrpY represses trpY and trpEGCFBAD transcription by different mechanisms. DNase I footprinting demonstrated that TrpY binds to the TRP box 1 and 2 region in the absence or presence of tryptophan (Fig. 2E and 6A), and EMSA confirmed that binding requires the TRP box sequences separated by 4 bp (Fig. 3). Given that this is a palindromic binding sequence and that TrpY is a dimer in solution, it seems probable that TrpY binds to this region as a dimer. TrpY binding to this region does not prevent TBP plus TFB binding (Fig. 6A) and therefore most likely inhibits Y transcription by preventing RNAP binding and/or RNAP access to the site of trpY initiation. The need for the TRP box 1 sequence for TrpY regulation of trpY transcription was directly confirmed by transcription in vitro using template T8 (Fig. 4). The changes in TRP box 1 that eliminated TrpY binding (Fig. 3) also eliminated the sensitivity of Y transcription to the presence of TrpY. This mechanism of transcription repression, preventing RNAP access to the site of initiation, has also been documented for MRD1 in Archaeoglobus fulgidus (7) and for LrpA and Phr from Pyrococcus furiosus (12, 15, 46).

In contrast to the tryptophan-independent binding of TrpY to the TRP box 1 and 2 region, TrpY binding to the TRP box 3 and 4 region could only be detected in the presence of tryptophan (Fig. 2E, 3, and 6A). Possibly, tryptophan binding to TrpY results in a conformational change that increases the affinity of TrpY for the nonconsensus TRP box 3 (AGTACC) and 4 (TGTATA) sequences while retaining affinity for the near-consensus TRP box 1 and 2 sequences. An alternative but not mutually exclusive explanation would be that tryptophan binding to TrpY facilitates higher order oligomerization, and this stabilizes binding to the TRP box 3 and 4 region, possibly with a DNA loop facilitating oligomerization with TrpY molecules bound to TRP boxes 1 and 2. In support of this concept, the ability to form a tryptophan-dependent complex that could be detected by EMSA required not only the presence of the TRP box 3 and 4 region but also coincident TrpY binding to TRP boxes 1 and 2 (Fig. 3B), and a DNase I-hypersensitive site was introduced between TRP boxes 2 and 3 when TrpY was bound to all four TRP boxes (Fig. 2E). Regardless of how TrpY binding to TRP boxes 3 and 4 is stabilized, the overlap of these sequences with TATAE and TATAY (Fig. 1A and 6B) must mean that TrpY and TBP compete for binding sites, and, in vitro, TrpY binding dominates. The DNase I footprints obtained with TrpY plus tryptophan were the same in the presence or absence of TBP and TFB (Fig. 6A) and regardless of the order of addition of the transcription factors. Inhibition of transcription by preventing TBP access to the TATA box region has also been documented for the Lrs14 repressor from Sulfolobus solfataricus (9, 33) and TrmB repressor in Thermococcus litoralis (29). This mechanism of repression seems formally analogous to operator binding by bacterial repressors, including TrpR (35, 43, 52), preventing sigma factor-directed promoter binding by bacterial RNAPs. But sigma factors dissociate from the DNA following departure of the RNAP, resetting the competition between repressor and RNAP, whereas archaeal TBP may remain bound to the TATA box region after initiation (51). This would seem to preclude a continued competition between an archaeal repressor and TBP for TATA box binding unless, as seen for TrpY in vitro, the archaeal repressor binds with an affinity that outcompetes and therefore effectively removes TBP from the TATA box.

TrpY regulation of trpB2 transcription. Gelfand et al. (21) identified TRP box sequences associated with additional genes related to tryptophan metabolism in the M. thermautotrophicus genome that were not linked to the trpEGCFBAD operon, most notably two consensus TRP box sequences located upstream of MTH1476 (Fig. 5A). MTH1476 encodes a protein closely related to TrpB, the ß subunit of tryptophan synthase, and has been designated trpB2. Consistent with participating in tryptophan biosynthesis and membership in a TrpY regulon, trpB2 transcription was inhibited in vitro by TrpY in the presence of tryptophan (Fig. 5B). It has been suggested that TrpB2 proteins are tryptophan synthases that function as indole scavengers (25), but it has also been alternatively proposed that they are serine deaminases rather than tryptophan synthases (47). Intriguingly, with this latter proposal in mind, as illustrated in Fig. 5A, the site of trpB2 transcription initiation is unusually distant from the initiation codon for TrpB2 translation, and the resulting 53-nucleotide leader RNA could direct the synthesis of a peptide with 15 amino acid residues, 5 of which would be serines. This hints at a leader peptide whose synthesis might sense the availability of charged tRNASer and therefore serine, but, despite the conservation of the TRP boxes and TATA box, this open reading frame is not conserved upstream of the trpB2 gene PCR amplified from M. marburgensis genomic DNA (results not shown).


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ACKNOWLEDGMENTS
 
This research was supported by grants DE-FG02-87ER13731 and GM53185 from the Department of Energy and National Institutes of Health, respectively.

We thank K. Sandman for her analysis and predictions for the TrpY structure and all of our OSU colleagues for many helpful discussions and for providing details of their research with TrpY before publication.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Ohio State University, Columbus, OH 43210-1292. Phone: (614) 292-2301. Fax: (614) 292-8120. E-mail: reeve.2{at}osu.edu. Back

{dagger} Present address: Lombardi Cancer Center, Georgetown University, Washington, DC 20057. Back


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Journal of Bacteriology, September 2005, p. 6419-6429, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6419-6429.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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