INTRODUCTION

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Tyrosine phenol lyase (TPL; EC 4.1.99.2), formerly known as -tyrosinase, catalyzes a reversible pyridoxal phosphate-dependent , -elimination reaction that degrades L-tyrosine to phenol, pyruvate, and ammonia (22, 23, 25). In the reverse direction, TPL can catalyze the synthesis of L-tyrosine or 3,4-dihydroxyphenylalanine from pyruvate, ammonia, and phenol or catechol. Detailed studies, including the cloning and characterization of the structural gene for TPL, have been carried out for Citrobacter freundii (3, 24), Erwinia herbicola (14, 54), and Escherichia intermedia (26).

In E. herbicola, TPL is induced by L-tyrosine and repressed when cells are grown in glucose (13). The regulatory mechanism(s) that controls the expression of the TPL gene (tpl) is not well understood. A previous study on the regulation of transcription from the tpl promoter of C. freundii demonstrated a role for the TyrR protein in regulating the tpl system (50).

The TyrR protein of Escherichia coli regulates the expression of a number of genes involved in the biosynthesis and transport of aromatic amino acids known as the TyrR regulon (for a review, see reference 39). Seven of the genes of the regulon are repressed by the TyrR protein (39), one gene (mtr) is activated (18, 46), and another (tyrP) is regulated either positively or negatively, depending on whether phenylalanine or tyrosine is present (21, 58). In general, tyrosine serves as the effector for repression, while phenylalanine is the cofactor for activation (39). However, in the TyrR-mediated activation of the mtr promoter, either tyrosine or phenylalanine can function as an inducer (18, 46). The detailed mechanisms of TyrR-mediated repression and activation are unknown.

To investigate possible mechanisms for the regulation of TPL, we employed a variety of genetic and biochemical procedures to analyze transcription from the tpl promoter of C. freundii. The present work enlarges our understanding of the TyrR-regulated tpl system. Two proteins, integration host factor (IHF) and cyclic AMP (cAMP) receptor protein (CRP) were shown to participate in the TyrR-mediated activation of the tpl promoter. The most likely role of these two proteins is to bend DNA in the region upstream of the tpl promoter, thereby enhancing the oligomerization of TyrR dimers. We propose that the functional role of the cis-acting sites within the tpl promoter is not merely to tether TyrR at a high local concentration near the tpl promoter, thereby increasing its ability to contact ⁷⁰ RNA polymerase, but also to facilitate the formation of a complex containing at least two TyrR dimers that is required for transcriptional activation. The requirement for additional protein factors in the TyrR-mediated activation of the tpl promoter sets this system apart from other TyrR-activated systems.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, bacteriophages, and oligonucleotides. The biological materials used in this study are described in Table 1, along with the chemically synthesized oligonucleotides used in site-directed mutagenesis.

Media. The liquid minimal medium was salt mix E of Vogel and Bonner (56) containing vitamin B₁ (1 mg/liter), biotin (0.1 mg/liter), and either glucose (0.2%) or glycerol (0.2%). Lurig broth (LB) medium (30) contained Bacto Yeast Extract (5 g/liter), Bacto Tryptone (10 g/liter), sodium chloride (5 g/liter), and glucose (1 g/liter). Solid medium was Bacto Nutrient Agar (Difco) (31 g/liter). The following compounds were included when appropriate: L-tyrosine (50 µg/ml), 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal), 40 µg/ml; ampicillin, 50 µg/ml; chloramphenicol, 25 µg/ml; streptomycin, 30 µg/ml; and kanamycin, 25 µg/ml.

DNA preparation. Plasmid DNA was isolated with the Wizard plus miniprep DNA purification system (Promega). Cultures for plasmid preparation were grown to saturation in L broth supplemented with appropriate antibiotics. The preparation of competent cells and their transformation were carried out by the procedure of Mandel and Higa (32). When thermosensitive lysogens were being transformed, the heat step was omitted.

Chemicals and reagents. Restriction endonucleases, T₄ DNA ligase, and DNA polymerase I large (Klenow) fragment were purchased from New England Biolabs. [-³⁵S]dATP and [-³²P]dATP were purchased from Amersham. Special-purpose oligonucleotides for use in PCR and site-directed mutagenesis were synthesized in the Laboratory for Macromolecular Structure, Purdue University. The protein markers and reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting) were purchased from Bio-Rad. Anti-TyrR antibodies were prepared as previously described (9). Dimethyl pimelimidate (DMP) was purchased from Pierce. o-Nitrophenyl--D-galactopyranoside (ONPG) was purchased from Sigma. All other chemicals were of the highest quality that was commercially available.

Strain construction. The construction of tyrR⁺/tyrR isogenic strains and  lysogens containing a lacZ reporter system specific for the tpl promoter was described in reference 50. High-titer lysates of Ptpl-lacZ⁺  phages were used to lysogenize the Lac strains SP1312 (tyrR⁺) and SP1313 (tyrR) to generate SP1312 Ptpl-lacZ⁺ and SP1313 Ptpl-lacZ⁺. Strains SP1626 (tyrR⁺) and SP1627 (tyrR) are crp derivatives of the aforementioned lysogens that were constructed by standard P1 transduction methods with CA8439 as the donor (50). These strains were  resistant, streptomycin resistant, and temperature sensitive. Strains SP1628 (tyrR⁺) and SP1629 (tyrR) are IHF-negative derivatives of SP1312 Ptpl-lacZ⁺ and SP1313 Ptpl-lacZ⁺ that were constructed with P1 grown on BW12848. This P1 lysate was also used to construct himD mutants of SP1626 (Ptpl-lacZ⁺) and SP1627 (Ptpl-lacZ⁺). tpl promoter variants containing mutations in either the IHF site, box C, or boxes B and C (Fig. 1) were generated in derivatives of pUC19 as described below. Segments of tpl promoter DNA were inserted as EcoRI-BamHI fragments into pMLB1034. The resulting pMLB1034 derivatives were transformed into CSH26(RZ11). Lac⁺  recombinants were isolated as described in reference 50, with NK5031 as the plating indicator.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   The tpl promoter and associated regulatory elements. Only the nucleotide sequence of the messenger-equivalent strand is shown. The coordinate system has been changed from the system in the original publication (3) by assigning +1 to the start point of transcription. DNA fragments used in the functional analysis of the tpl promoter were synthesized by PCR, with pRVT1 (3) as the template. Cleavage sites for restriction endonucleases EcoRI and BamHI were installed at the indicated locations (coordinates 329 and +180) in order to facilitate the construction of single-copy reporter systems. The EcoRI-BamHI fragment was cloned into pUC19, which contains a PstI site downstream of the BamHI site. The fragment from pUC19-tpl generated by EcoRI-PstI digestion was used for DNase I footprinting experiments. The 5' end points of three truncated derivatives of the tpl promoter are shown as broad arrowheads labeled 2, 3, and 4 at coordinates 262, 193, and 144. The 10 and 35 recognition elements are underlined with heavy black lines. The target sites for cAMP-CRP (CRP1 and CRP2) are outlined with broken lines. TyrR boxes, named A, B, and C, are shaded. Residues marked by asterisks (280, 166, 55 and 40) are the locations of TyrR operator mutations or IHF mutations (see text). The IHF binding site is presented in white letters on black.

Enzyme assays. -Galactosidase was assayed by the method of Miller (35). Cells were grown to early mid-log phase at 30°C in liquid minimal medium containing L-tyrosine (50 µg/ml). Assays, carried out in triplicate, had standard errors of <10%. The enzyme assay values are reported in Miller units.

Proteins used in footprinting studies. TyrR protein was purified as previously described (10). CRP was purified by a modification of a method developed by R. Ebright, Rutgers University, using cAMP-Sepharose affinity chromatography. An affinity column was prepared by coupling 8-(6-aminohexyl)amino-adenosine 3',5'-cyclic monophosphate (AHAcAMP; Sigma) to CNBr-activated Sepharose 4B gel (Pharmacia). E. coli CA8445/pXZCRP, which carries a multicopy plasmid encoding the CRP protein, was grown in L-broth medium at 37°C for 15 h and harvested. Crude extracts were prepared using a French pressure cell (Aminco) operated at 1,000 lbs/in² and then loaded on a 6-ml cAMP affinity column. The column was washed with 2 bed volumes of wash buffer (20 mM sodium phosphate [pH 7.0], 2 mM EDTA [pH 8.0], 5 mM -mercaptoethanol, 300 mM NaCl, 6% glycerol) and then with 2 bed volumes of wash buffer plus AMP (5 mM 5' AMP and 5 mM 3' AMP). After the washes, CRP was eluted with buffer containing 500 mM NaCl and 5 mM cAMP. About 90% pure CRP was obtained. A further purification step was carried out by fast protein liquid chromatography with a Mono S column (Pharmacia). The purity of CRP, by SDS-PAGE, was at least 99%. The IHF protein was a gift from Steven D. Goodman, University of Southern California. All protein concentrations were determined by Bio-Rad protein assay reagent with bovine serum albumin as a standard.

DNase I footprinting. A DNA fragment of 527 bp carrying the tpl promoter was released from pUC19-tpl by digestion with EcoRI plus PstI, isolated by electrophoresis on 1% agarose, followed by purification with a QIAEX II gel extraction kit (Qiagen). Elution was carried out with TE buffer (20 mM Tris [pH 8.0], 0.1 mM EDTA). This fragment (0.5 µg) was selectively labeled at the EcoRI site by treatment with DNA polymerase I (Klenow fragment) in the presence of [-³²P]dATP by the method of Brenowitz et al. (7). The unincorporated radiolabel was removed with a G-25 spin column (Boehringer Mannheim), and residual protein was removed with a QIA spin column (Qiagen). The optimal concentration of DNase I in footprinting analyses was established to be 1.5 to 2.0 µg/ml. For CRP footprinting analyses, radiolabeled DNA (1 nmol/tube) was incubated with CRP (10 to 100 nM) at room temperature in assay buffer containing 10 mM Tris-Cl (pH 8.0), 5 mM MgCl₂, 1 mM CaCl₂, 2 mM dithiothreitol, 100 mM KCl, 50 µg of bovine serum albumin per ml, 2 µg of calf thymus DNA per ml, and 100 µM cAMP. The total volume of each binding reaction mixture was 200 µl. After 30 min, each tube was treated with 5 µl of DNase I (1.5 µg/ml) for 2 min. Each reaction was stopped by the addition of 40 µl of 50 mM EDTA (pH 8.0), followed by a single treatment with 200 µl of phenol-chloroform-isoamyl alcohol (24:25:1). The DNA was precipitated with ethanol containing 5 µg of tRNA per ml and 0.1 volume of 3 M sodium acetate. The DNA was resuspended in 5 µl of loading dye, heated for 5 min at 90°C, and loaded onto an 8% acrylamide-6 M urea gel. The products of the A+G cleavage reactions (33) were coelectrophoresed with sample to identify protected nucleotides. After electrophoresis, the gel was exposed overnight at 70°C to Kodak XAR-5 film. For the TyrR and IHF footprinting reactions, the procedure was the same as described as above. Unless otherwise stated, the assay buffer for TyrR also contained 0.2 mM L-tyrosine and 0.2 mM ATP. DNA fragments containing G-to-A changes in box A were prepared by EcoRI-PstI digestion of pUC19-tplmut1. DNA fragments from the deletion mutant 64 or 179 were generated by EcoRI-PstI cleavage of pUC19-tpl64 or pUC19-tpl179, respectively.

Mutagenesis of TyrR and IHF binding sites. The introduction of the G-to-A changes in box A or box B and the deletion of segments of the tpl promoter is described in reference 50. Mutations in box C (G-to-A and C-to-T) were introduced by using a Quick-change site-directed mutagenesis kit (Stratagene) with the oligonucleotide pair 351K and 352K (Table 1). The template DNA was either pUC19-tpl or pUC19-tplmut2. The consensus sequence of the TyrR target is TGTAAAN₆TTTACA (N is any nucleotide). Box C DNA(TGATTTGCATCACCTACA) was replaced by the box A sequence (TGTACATTTGCTTTACA), except for the six nonconserved central nucleotides. These mutational changes were introduced into either pUC19-tpl64 or pUC19-tpl179 using the oligonucleotide pair 488K and 489K. Mutations in the IHF site (A₆CTTGTTGAATATGAACA₆CTTGTCGACTATGAAC) were introduced in similar fashion with the oligonucleotide pair IHFc and IHFd (Table 1). The template DNA was pUC19 tpl. Each mutational change was verified by DNA sequencing.

Chemical cross-linking. Reactions were allowed to proceed at room temperature (25°C) in 0.2 M triethanolamine buffer at pH 8.0 containing 1 mg of TyrR protein and 2 mg of DMP per ml. Samples were removed at different times. Each reaction was stopped by the addition of an equal volume of 2× loading buffer (10) and then frozen at 20°C. Circular DNA from pUC19-tpl was prepared with a mega kit from Qiagen. In the presence of DNA, cross-linking was carried out with TyrR and CRP in a buffer containing 200 µM cAMP, 200 µM L-tyrosine, 5 mM MgCl₂, 1 mM CaCl₂, and 200 µM ATP. The molar ratio of DNA to TyrR to CRP was 1:3:1. After incubation for 20 min, 2 mg of DMP per ml was added. Successive steps were performed as described above. Cross-linking mixtures were analyzed either by 0.7% SDS-PAGE or Western blotting. The primary polyclonal anti-TyrR antibody was a 1:3,000 dilution of high-titer antiserum (9). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad) was employed as the second antibody (1:3,000 dilution). Staining was carried out with H₂O₂ and 4-chloronaphthol by standard procedures (17).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Regulation of the tpl promoter by TyrR and cAMP-CRP. SP1312 (Ptpl-lacZ⁺) and SP1313 (Ptpl-lacZ⁺) are isogenic tyrR⁺/tyrR strains that carry single-copy lacZ reporter genes driven by the tpl promoter of C. freundii (50). When these strains were grown in the presence of L-tyrosine, the -galactosidase levels in the tyrR⁺ strain were at least 20-fold higher than in the (tyrR)strain (Table 2, lines 1 and 2). When SP1313 (Ptpl-lacZ⁺) was transformed with a tyrR⁺ plasmid (pJC100 [51]) that led to haploid level expression of a functional TyrR protein, the reporter enzyme levels were restored to control values. These results confirm that the tpl promoter, like the mtr and tyrP promoters, is subject to positive control by the TyrR protein (Table 2, lines 1 to 3).

Chemical identification of TyrR and CRP binding sites in the tpl promoter. DNase I footprinting of tpl promoter DNA was carried out to define with precision the locations of the TyrR binding sites. A fragment of DNA identical to the one that had been used to construct the tpl reporter systems was isolated from pUC19-tpl and ³²P labeled at one end as described in Materials and Methods. In DNase I footprinting studies, three TyrR binding sites, designated A, B, and C were identified (Fig. 2). Boxes A and B, centered at coordinates 272.5 and 158.5 respectively, are very similar in sequence to the consensus TyrR operator sequence (TGTAAAN₆TTTACA) (39). Box C, centered at coordinate 49.5, bore little resemblance to the consensus TyrR targets other than the two invariant G and C residues (GN₁₄C). According to the classification scheme of Pittard (39), boxes A and B are likely to be strong TyrR boxes, while box C would appear to be a weak TyrR box. The approximate affinity of TyrR for each operator target was estimated. Box A was fully protected by 1 to 5 nM TyrR (Fig. 2, lane 3 of left gel). Under the same conditions, 20 nM (lane 5 of right gel) or 80 nM (lane 7 of right gel) TyrR were required to fully protect box B or C. The apparent affinity of the TyrR protein for each target site decreased in relation to the proximity of the site to the transcription start point of the tpl promoter.

View larger version (87K): [in this window] [in a new window]   FIG. 2.   DNase I footprints of TyrR bound to the promoter regions of tpl or tpl-mut1. The DNA fragments were generated by EcoRI-PstI digestion of pUC19-tpl (lanes 2 to 7, both panels), or pUC19-tplmut1 (lanes 8 and 9, both panels). The latter construct contains a G-to-A change in box A. Both fragments were labeled with 32P at the 5' ends as described in Materials and Methods. Treatment with DNase I was carried out in the presence (lanes 3 to 9, both gels) or absence (lanes 2, both gels) of TyrR. All reaction mixtures contained 0.2 mM L-tyrosine, 0.2 mM ATP, and 0.7 nM DNA. The concentrations of TyrR used were as follows: lanes 3, 5 nM; lanes 4, 10 nM; lanes 5 and 8, 20 nM; lanes 6, 40 nM; lanes 7 and 9, 80 nM. Lanes 1 contain the A+G sequence of tpl DNA. The regions protected by TyrR are indicated. Box A is shown in the left gel, and boxes B and C are shown in the right gel. Each of the DNA sequences shown reads from the 5' end (bottom) toward the 3' end (top) as shown in Fig. 1.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   DNase I footprints of CRP bound to the tpl promoter region. The DNA fragment used was generated by EcoRI-PstI digestion of pUC19-tpl and labeled with 32P at the 5' end. The DNA was treated with DNase I in the presence (lanes 3 to 7) or absence (lane 2) of CRP and cAMP. The concentrations of DNA and cAMP were 1 nM and 100 µM, respectively. The concentrations of CRP were as follows: lane 3, 10 nM; lane 4, 25 nM; lane 5, 50 nM; lane 6, 75 nM; lane 7, 100 nM. Lane 1 contains the A+G sequence of the DNA. Segments protected by CRP are indicated to the right. The sequences read from the 5' end (bottom) toward the 3' end (top) as shown in Fig. 1.

View larger version (70K): [in this window] [in a new window]   FIG. 4.   Requirement for cAMP in DNase I footprinting of tpl promoter DNA by CRP. The DNA fragment used in lanes 1 to 5 was an EcoRI-PstI fragment from pUC19-tpl. The fragment used in lanes 6 to 8 was an EcoRI-PstI fragment from pUC19-tplmut1 which contains a critical G-to-A change in box A. Each DNA fragment was labeled with 32P at the 5' end. The concentrations of CRP and cAMP are shown at the bottom. The segments protected by CRP are indicated to the right. A+G standards are in the leftmost lane. TyrR (50 nM), L-tyrosine (0.2 mM), and ATP (0.2 mM) were present in each tube.

Effects of ATP and tyrosine on DNase I protection. In order to assess the roles of ligands in the binding of TyrR to its respective target sites, a series of DNase I footprinting assays were carried out under a variety of different conditions (Fig. 5). Box A was protected by unliganded TyrR. The pattern of protection was unaltered by the addition of ATP and tyrosine, alone or in combination. Box B was partially protected by unliganded TyrR; full protection was observed when ATP or ATP plus tyrosine were present. Box C was protected only when TyrR, ATP, and tyrosine were present. When cAMP-CRP was present, there were no changes in the protection of the TyrR boxes.

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Effects of ATP and tyrosine on DNase I footprinting by TyrR and cAMP-CRP. The DNA used in each case was 1 nmol of a 32P-labeled EcoRI-PstI fragment from pUC19-tpl. The label was at the 5' end (see Materials and Methods). The presence (+) or absence () of other compounds added to each tube are shown at the top of the figure. When present, TyrR was included at a final concentration of 100 nM, except in lane 7, when the TyrR concentration was 50 nM. The concentrations of other additives were as follows: ATP, 0.2 mM; L-tyrosine, 0.2 mM; CRP, 50 nM; cAMP, 100 µM. The segments protected by TyrR (boxes A, B and C) and cAMP-CRP are indicated to the right. Lane M, A+G standards.

Regulation of the tpl promoter by IHF. The center-to-center distance between boxes A and B is almost the same as the distance between boxes B and C (Fig. 1). Two CRP binding sites are located between boxes A and B. The binding of cAMP-CRP to its target site may serve to bend DNA in this region, thereby mediating interactions between TyrR dimers bound to boxes A and B. This raised the possibility that a binding site for an unknown factor lay between boxes B and C and that its role was to facilitate interactions between TyrR dimers bound to boxes A, B, and C. A putative IHF binding site was detected by a computer search of the region upstream of the tpl promoter. An approach similar to that used in the investigation of the role of cAMP-CRP was chosen to explore a possible role for IHF. The himD gene, encoding the  subunit of IHF, was knocked out in a pair of tyrR⁺ and (tyrR) strains [SP1628 (Ptpl-lacZ⁺) and SP1629 (Ptpl-lacZ⁺)]. The ability of TyrR to activate the tpl promoter was severely affected in IHF-negative strains. An IHF-specific reduction of about 35-fold in reporter enzyme levels was detected (Table 2, compare lines 1 and 9). The utilization of the tpl promoter was also studied in strains with knockouts of both the himD and crp genes [SP1630 (Ptpl-lacZ⁺) and SP1631 (Ptpl-lacZ⁺)]. In glycerol-grown cultures, severe reductions in enzyme level were observed. Reporter enzyme levels were approximately 500-fold lower than in the tyrR⁺ reference strain SP1312 (Ptpl-lacZ⁺) grown in the same medium (Table 2, compare lines 1 and 12). On the other hand, about a 10-fold reduction below that of the (tyrR) host SP1313(Ptpl-lacZ⁺) was observed in glycerol-grown cells (compare Table 2, lines 2 and 13), indicating that IHF can slightly activate the tpl promoter in the absence of TyrR. This activation was strongly enhanced to about 150-fold when TyrR was present (Table 2, lines 4 and 13). Introduction of pHN₂, which encodes two copies of the  subunit of IHF and one copy of the  subunit, fully restored the activation function of the TyrR protein (Table 2, lines 1, 8, and 14). The results indicated that IHF is not only involved in the regulation of tpl but also plays a critical supporting role in enabling TyrR to function as an activator.

View larger version (68K): [in this window] [in a new window]   FIG. 6.   DNase I footprint of the IHF binding site within the tpl promoter region. The EcoRI-PstI fragment from pUC19-tpl was labeled with 32P at the 5' end as described in Materials and Methods. The DNA was treated with DNase I in the presence (lanes 3 to 7) or absence (lane 2) of IHF. The concentration of DNA was 0.7 nM in each reaction mixture. The concentrations of IHF were as follows: lane 3, 9 nM; lane 4, 18 nM; lane 5, 36 nM; lane 6, 72 nM; lane 7, 144 nM. Lane 1 contains the A+G sequence. The region protected by IHF is indicated to the right. The protected sequence reads from the 5' end (bottom) toward the 3' end (top) as shown in Fig. 1.

View larger version (106K): [in this window] [in a new window]   FIG. 7.   Effects of IHF and cAMP-CRP on DNase I footprinting of boxes B and C. The DNA used was 0.7 nmol of an EcoRI-PstI fragment from pUC19-tpl that had been 32P labeled at the 5' end (see Materials and Methods). The tubes alone were used (lane 1), or CRP (20 nM) and IHF (145 nM) (lanes 2, 4, 6, 8, 10, and 12) were added to the tubes. The TyrR concentrations were as follows: lanes 2 and 3, 10 nM; lanes 4 and 5, 20 nM; lanes 6 and 7, 40 nM; lanes 8 and 9, 60 nM; lanes 10 and 11, 80 nM; lane 12, 100 nM. Each reaction mixture contained ATP and tyrosine (0.2 mM each). Tubes containing CRP also contained cAMP (100 µM).

Effects of mutations within the TyrR boxes of the tpl promoter. Previous comparisons (39) of TyrR binding sites have shown that there are only two absolutely conserved nucleotides (GN₁₄C) within this class of operators. Alteration of either residue severely reduced the TyrR-mediated regulation of promoter activity. To examine the roles of boxes A, B, and C in the regulation of the tpl promoter, G-to-A changes were introduced at the first invariant position in each of the TyrR boxes. When box A of the tpl promoter was mutated, the -galactosidase activity of glucose-grown cells fell to a level characteristic of (tyrR) host strains, i.e., there was a reduction of approximately 13-fold in reporter enzyme level from that in tpl promoter systems where box A was intact (compare line 1 of Table 3 to line 1 of Table 2). The effect of a G-to-A change in box B was not as severe as that in box A. In glucose-grown cells, a sevenfold reduction in reporter enzyme levels was observed (compare line 2 of Table 3 to line 1 of Table 2). Considering only glycerol-grown cells, where the cAMP-CRP effect is maximal, the -galactosidase activities were reduced about 4.6-fold when either box A or box B was mutated (compare lines 1 and 2 of Table 3 to line 1 of Table 2). These data are consistent with the results of Table 2 (lines 1 and 4), where the contribution of cAMP-CRP to tpl promoter activity was found to be about threefold. There was no detectable TyrR or cAMP-CRP-mediated activation when box C was mutationally inactivated. Even the stimulatory effect of cAMP-CRP was lacking (Table 3, lines 3 and 4). This was unexpected, since box C showed little sequence homology to other TyrR boxes and fits the criteria for a weak box. Evidently cAMP-CRP can stimulate transcription from the tpl promoter when either box A or box B is disabled, but not when box C is incapable of engaging the TyrR protein.

Interaction between TyrR dimers at the tpl promoter. Mutational studies on DNA carrying TyrR boxes suggested that the DNA-mediated association of TyrR dimers plays a role in activating transcription at the tpl promoter. Earlier studies demonstrated that TyrR exists as a dimer in solution (9) and that dimers could interact to give rise to hexamers (59). To further investigate the range of aggregation states available to TyrR, a series of chemical cross-linking studies were conducted. Upon incubation with DMP, the subunits of TyrR protein were readily converted, in a time-dependent manner, to species with increasing multiples of the molecular weight of the monomer (Fig. 8). DMP mediates cross-linking via the  amino groups of lysine residues, provided that these functional groups are separated by no more than 9.2 Å. In the absence of DNA, complexes corresponding to the dimeric, tetrameric, hexameric, and octameric forms of TyrR were detected both on stained SDS-polyacrylamide gels or on Western blots developed with anti-TyrR antibodies (Fig. 8, lanes 7 and 8). When tpl DNA, bearing operator targets for TyrR, was present, the percentage of hexamer was dramatically increased, while the octamer form of TyrR became almost undetectable (data not shown). When cAMP-CRP was included in the cross-linking reaction mixtures with TyrR and DNA, two major complexes likely to contain TyrR-CRP cross-linked species were demonstrated by immunoblotting (Fig. 8). The molecular mass of each complex was estimated to be 83 to 95 kDa (broad) and 200 kDa. The 83- to 95-kDa complexes consist of two species. One is likely to contain one subunit of TyrR and one subunit of CRP; the other is likely to contain one subunit of TyrR and two subunits of CRP. The 200-kDa protein complex is likely to have originated from cross-linking between one TyrR dimer and two CRP dimers. This is reasonable because there are two CRP binding sites near TyrR boxA (Fig. 1).

View larger version (66K): [in this window] [in a new window]   FIG. 8.   DNA dependence of chemical cross-linking of TyrR demonstrated by immunoblotting. The cross-linking reagent was DMP. The reaction was carried out with 3.6 µM TyrR, 1.2 µM CRP, 100 µM ATP, 50 µM cAMP, and 200 µM L-tyrosine in the presence (lanes 1 to 6) or absence (lanes 7 and 8) of pUC19-tpl DNA. Lane 1 is a control (no DMP). The concentration of DMP was 4 mg/ml. The incubation times for each reaction mixture were 10 min (lane 2), 20 min (lane 3), 40 min (lanes 4 and 7), 80 min (lanes 5 and 8), and 160 min (lane 6). The locations of molecular weight markers (in thousands) are indicated to the right. The positions of TyrR monomer, dimer, and hexamer are indicated with arrows to the right. The positions of presumptive TyrR-CRP complexes are indicated with arrows to the left. For details, see Materials and Methods.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The TyrR protein of E. coli is known to regulate the expression of a number of genes involved in the biosynthesis and transport of aromatic amino acids (39). The present results, together with previous data (50), enlarge our understanding of TyrR-regulated systems. TPL, an enzyme of C. freundii that degrades tyrosine to phenol, pyruvate, and ammonia is clearly a member of the TyrR regulon. The regulation of tpl requires L-tyrosine as a cofactor. Like mtr and tyrP (18, 21, 46, 58), tpl is positively regulated by TyrR. In tester strains bearing a deletion of the tyrR gene, there was a reduction of at least 20-fold in the production of -galactosidase from a single-copy tpl-lacZ reporter system from that of tyrR⁺ controls. Promoter function was completely restored when a plasmid expressing the tyrR⁺ gene was introduced into the (tyrR) strain. As revealed by DNase I footprinting experiments, TyrR binds to three operator targets, named A, B, and C, within the tpl promoter region. The binding sites are centered at positions 49.5, 158.5, and 272.5 relative to the transcriptional start point. Mutational alteration of any of the three operators abolished or severely reduced transcription.

Inspection of the sequence of each TyrR binding site (Fig. 1) reveals that boxes A and B are quite symmetrical and closely resemble the TyrR consensus sequence (TGTAAAN₆TTTACA). This was not the case for box C. Unliganded TyrR readily bound to the box A region of tpl. The DNase I footprint of Tyr bound to box A was unaffected by L-tyrosine and ATP. Box B was also protected by unliganded TyrR, but binding was enhanced when ATP was present. Under the same conditions, the box C region was protected by TyrR only when ATP and L-tyrosine were present (Fig. 5). Thus, box C conforms to the general criteria for a weak TyrR box first enunciated by Pittard and coworkers (39). The inability of TyrR to bind to box C in the absence of tyrosine and ATP probably reflects a poor fit between the operator recognition site of TyrR and the DNA of box C. TyrR binds to the three targets within the tpl promoter with progressively diminishing affinity, in the order box A > box B > box C. We assume that these different affinities for TyrR are functionally related to the regulatory response that is observed during the expression of tpl. It appears that transcription from the tpl promoter occurs only when TyrR occupies the lower-affinity site. Our data are consistent with the notion that TyrR binds cooperatively to the three boxes. When box A or boxes A and B were deleted, the utilization of the tpl promoter fell to a very low level. The interdependence of TyrR targets observed in vivo was supported by DNase I footprinting. The protection of box C was significantly diminished when box A or boxes A and B were deleted. In the 64 version of the tpl promoter (box A deleted), the DNase I protection of box C required >80 nM TyrR compared to a requirement of 80 nM TyrR for wild-type tpl DNA. With the 179 version of the tpl promoter (boxes A and B deleted), the protection of box C required >120 nM TyrR compared to a requirement of 80 nM for wild-type tpl DNA (data not shown). This apparently cooperative binding of the TyrR protein to its multiple binding sites is likely to offer a physiological advantage by increasing the overall effectiveness and specificity of occupancy by TyrR of binding sites within the tpl promoter. Similar situations exist for other systems, including the Lrp-regulated ilvIH promoter (57), the simian virus 40 early promoter (5), Drosophila heat shock promoters (62), and yeast promoters that are subject to general amino acid control (4, 20).

In contrast to the situation that prevails in several other TyrR-regulated promoters, two of the operator targets (boxes A and B) of the tpl promoter lie far upstream of the transcriptional start point, while box C is immediately adjacent to the RNA polymerase-binding site (Fig. 1). The three targets are separated from each other by 10 helical turns of B-form DNA. In the mtr and tyrP promoters, there are only two TyrR binding sites within each promoter whose centers are separated by no more than 30 bp. Neither mtr nor tyrP has TyrR target sites further upstream than coordinate 66 (21). The importance to transcriptional activation of the spacing between TyrR boxes has been studied in the tyrP system. Activation of tyrP can be detected if the boxes are separated by one turn of the helix but not if the separation involves three turns of the helix (2). In contrast, we found that even TyrR binding sites situated far upstream from the promoter were essential for the regulation of the tpl promoter. It is uncommon to find such remote regulatory elements in association with ⁷⁰ promoters, where activators tend to bind predominantly to targets between coordinates 80 and 30 (16). The ⁷⁰ tpl promoter is an exception (50). The regulatory function of boxes A and B appears to be obligatorily linked with box C, located near the 35 recognition element of the tpl promoter. Inactivation of box C essentially eliminates the function of the other two sites, even when boxes A and B are fully intact. Since boxes A and B are far from the tpl promoter region, only looping of DNA would allow interactions between TyrR proteins, thus generating higher-order multimers of TyrR. Our cross-linking study of TyrR in the presence of tpl promoter DNA (Fig. 6) provides supporting evidence for multimerization of the TyrR protein.

Effects of global transcriptional regulatory proteins. Two DNA-binding proteins, IHF and CRP, were shown to participate in the regulation of tpl expression. Although IHF and CRP are generally considered to be global regulators, the involvement of both factors in the activation of a single promoter has been reported only for the tdc promoter (60). Here we explored a transcriptional regulatory system in which IHF and CRP are both required for full TyrR-mediated activation of tpl. Initially, this was studied with appropriately constructed background strains carrying a tpl reporter system. When a (himD) mutation was introduced into host strains that contained a tpl-lacZ reporter system, the transcriptional activity of the tpl promoter became virtually undetectable, even when TyrR was available. On the other hand, a (crp) mutation lowered the expression of tpl only about threefold. Although both IHF and CRP were shown to interact directly with the tpl promoter region, the IHF appears to be more critical in tpl regulation than CRP. In the absence of TyrR, IHF showed about a 10-fold effect on induction of the tpl promoter. This induction was dramatically increased when TyrR was present, indicating that IHF acts as a coactivator of the tpl system. Given what is known about the interaction of IHF with DNA (1), a plausible role for IHF is to bend tpl DNA in the region between boxes B and C. The bend angle, predicted to be 140° or greater, would alter the shape of DNA from approximately a straight line to something resembling a hairpin (42). IHF is involved in numerous processes in E. coli and some of its bacteriophages and plasmids, including site-specific recombination, DNA replication, and gene expression (12, 15). Most IHF-specific transcriptional regulatory events involve ⁵⁴-dependent promoters, which become fully functional only when activator proteins bind to remote upstream sites. In NtrC-responsive ⁵⁴ promoters such as nifA, IHF binds to a site midway between an upstream activation site and the promoter, where it mediates the formation of a DNA loop that brings these elements into close proximity (19, 27). In contrast, ⁷⁰-dependent promoters are rarely regulated via remote upstream activator binding sites.

Model for activation of transcription from the tpl promoter. The in vivo and in vitro results presented herein, viewed within the framework of the established properties of the IHF and CRP proteins, suggest a model for the regulation of tpl expression (Fig. 9). This model is based on the hypothesis that the occupancy of the promoter-proximal TyrR binding site (box C) is a prerequisite for transcription. Box C binds TyrR weakly. The cooperative binding of TyrR to the tpl promoter via boxes A and B, with the assistance of IHF and cAMP-CRP, is proposed to facilitate the binding of TyrR to box C. At low tyrosine concentrations, it is proposed that TyrR, bound to box A, can become positioned near box B of the tpl promoter region, provided that there is sufficient cAMP-CRP available to bend tpl DNA between boxes A and B. This would happen readily in glycerol-grown cells but would not occur if the production of cAMP or CRP were blocked by mutation. A TyrR dimer bound at box B is presumed to interact with TyrR at box A, forming a stable complex. When the concentration of tyrosine increases, TyrR acquires the ability to bind to box C; meanwhile, the tetrameric TyrR-box A-box B complex could approach the tpl promoter near box C by virtue of the DNA bending activity of IHF. It is proposed that this gives rise to a very stable TyrR hexamer-DNA structure that can interact with RNA polymerase to initiate transcription. The nonavailability of any of the three proteins or relevant target sites would impair transcription.

View larger version (20K): [in this window] [in a new window]   FIG. 9.   Model for involvement of IHF and CRP in TyrR-mediated activation of tpl. TyrR boxes A, B, and C are shown as hatched bars. Two CRP binding sites are shown as black bars. The 70 promoter region is indicated by white bars. See Fig. 1 for the sequences. (A) Activation of the wild-type tpl promoter. (I) Resting state. At low tyrosine concentrations, TyrR dimers occupy strong boxes A and B, while weak box C is unoccupied. The DNA between boxes B and C is bent by IHF. (II) Catabolite repression lifted. cAMP-CRP binds to target sites, introducing a bend between boxes A and B. Interaction between TyrR dimers bound to boxes A and B, induced by DNA bending, stabilizes DNA-TyrR interaction. In states I and II, the transcriptional activity, induced by IHF, is at a very low level (indicated by a broken arrow). The mechanism of activation by IHF is not clear. (III) Tyrosine induction. At increased concentrations of tyrosine, TyrR dimers are able to occupy weak box C. Interactions between TyrR dimers bound to boxes A, B, and C become possible. The interactions between TyrR dimers are indicated by shaded regions. This gives rise to a very stable TyrR-hexamer DNA structure. This higher-order nucleoprotein complex is proposed to fully activate the tpl promoter either by inducing a conformational change within promoter DNA or by interacting directly with 70 RNA polymerase. Activation of the tpl promoter is indicated by a heavy black arrow. (B) Model for activation of 179 mutant tpl promoter. A DNA segment of 179 nucleotides that included boxes A and B was deleted from the region upstream of the tpl promoter region. In a separate operation, weak box C, centered at coordinate 49.5, was replaced by a strong box essentially identical to box A (see Materials and Methods). This construct is designated box A. (I) Promoter inactivity. The 179 tpl promoter cannot be utilized when wild-type box C is the only TyrR target, probably because the affinity of TyrR for box C is too low to enable higher-order structures required for promoter activity to form. (II) Weak promoter activity. When box C is replaced by strong box A, TyrR dimers can attach to this region of the promoter. Occasional formation of higher-order structures would allow 70 RNA polymerase to launch transcription. A low level of promoter activity is observed (indicated by a solid arrow). (III) Promoter activation. When the cellular content of TyrR dimers is elevated, there is an increased chance for the formation of complexes between TyrR dimers bound at box A and free TyrR dimers in solution. The formation of such higher-order complexes between TyrR dimers permits launching of 70 RNA polymerase. Activation of the tpl promoter is indicated by a heavy black arrow.

Distinctions in mode of action of TyrR at tpl, mtr, and tyrP. It is likely that the mechanism of TyrR-regulated tpl expression is quite different from the role of TyrR as a positive regulator of mtr and tyrP. In the case of tyrP, there is dual regulation, namely, tyrosine-mediated repression or phenylalanine-mediated activation, at the same promoter. Mutational studies (2) of two tyrP targets (boxes 1 and 2) showed that both TyrR boxes were required for repression but that only the upstream box (box 2) was required for activation. The degree of activation of the tyrP promoter was critically related to the location of box 2. Maximal activation was observed when box 2 was moved 3 or 12 to 14 residues upstream, while no activation was seen at intermediate positions such as +7 and 4 (2). This positional restriction of box 2 in tyrP was attributed to the dual role of box 2 in both repression and activation. We predict that the relative positions of the TyrR targets (boxes A and B) in the tpl promoter will be less critical owing to their remote distance from the promoter. In mtr, mutational experiments were used to demonstrate that the strong box plays an important role in activation by phenylalanine and tyrosine. Mutations in the weak box had no effect on activation by phenylalanine but decreased activation by tyrosine (45). Comparing these results with our data obtained from mutational studies of the tpl promoter, one is forced to conclude that phenylalanine- and tyrosine-mediated TyrR activation involves distinct regulatory mechanisms. Whether a multimeric TyrR complex is involved in tyrosine-mediated regulation of mtr remains to be determined. Further studies will be necessary to establish the precise mechanistic roles of IHF and CRP in tpl expression and how the formation of hexameric TyrR is regulated by tyrosine.