INTRODUCTION

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LysR-type proteins are the most common class of transcriptional regulatory proteins in prokaryotes (1). Regulation by LysR-type proteins is distinguished by several highly conserved unique properties. LysR-type proteins typically activate transcription of a target gene(s) and autoregulate their own synthesis from a single regulatory locus. In most cases, the target gene is divergently transcribed from the structural gene encoding the LysR-type protein. Activation of the target gene requires the binding of a metabolically important small molecule inducer, whereas autoregulation of the LysR gene is inducer-independent. Inducer binding, however, does not significantly alter the DNA binding affinity of these proteins (1). Thus, unlike other well characterized inducer-responsive activator proteins, LysR-type proteins are unusual in that the binding of inducer activates transcription by affecting an activator property of the protein distinct from its DNA binding activity. However, the molecular basis of this regulation has not been determined for any member of this important class of transcriptional regulatory proteins.

IlvY protein is a prototypic member of the LysR family of transcriptional regulatory proteins and is required for the regulation of ilvC gene expression in Escherichia coli (2-5). The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.86), an enzyme involved in the biosynthesis of the branched chain amino acids, L-isoleucine, L-valine, and L-leucine (5). Together with IlvY protein, either substrate of this enzyme, -acetolactate or -acetohydroxybutyrate, induces the activation of ilvC gene expression. Like other LysR-type proteins, IlvY protein also represses transcription of its own divergently transcribed structural gene in an inducer-independent manner (3, 4, 6).

In this report, we describe the first molecular mechanism for inducer-mediated activation by a LysR-type protein. Specifically, we show that IlvY protein binds cooperatively to the tandem operator sites in the divergent-overlapping promoter region of the ilvYC operon (Fig. 1) in an inducer-independent manner and autoregulates its own gene expression by an RNA polymerase occlusion mechanism. We further show that the binding of IlvY protein to the tandem operators is necessary but not sufficient to activate transcription from the ilvC promoter. Activation of transcription from the divergent ilvC promoter requires the additional binding of an inducer molecule to an IlvY protein-operator DNA complex. Inducer binding effects the partial relief of an IlvY protein-induced DNA bend centered around the 35 hexanucleotide element of the ilvC promoter. This ligand-induced structural change in the DNA helix enhances the binding affinity of RNA polymerase at the ilvC promoter. Thus, IlvY protein activates transcription from the ilvC promoter by the binding of an inducer molecule that directs a conformational change in the structure of an IlvY protein-operator DNA complex and enhances the recruitment of RNA polymerase to the ilvC promoter without affecting the occupancy of IlvY protein at the tandem operator sites.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Organization of the ilvYC operon. A, planar representation of the DNA helix in the divergent overlapping ilvYC promoter region. The position of each nucleotide residue is projected onto the surface of a cylinder, which is then unrolled onto a flat surface. A continuous ramp connects each phosphate residue. The position of the plane of each base pair is indicated by a vertical line drawn only across the major groove. The strand designated by a stippled gray line has its 5'-end at the left end of the diagram; thus, this strand should be read left to right, top to bottom. Nucleotide positions relative to the start of ilvC transcription are indicated on the top of the diagram, and nucleotide positions relative to the start of ilvY transcription are indicated on the bottom of the diagram. The open circles denote guanine residues protected from dimethyl sulfate methylation by IlvY protein. The shaded regions denote the major grooves of the 35 and 10 hexanucleotide regions of the ilvC (dark gray) and ilvY (stippled gray) promoters. The symbols O1 and O2 denote the tandem operator sequences recognized by IlvY protein. The in vivo transcriptional initiation sites for the ilvC and ilvY genes are indicated by arrows. B, nucleotide sequence of the region depicted above (ilvC bp positions 81 to +12). The in vivo transcriptional initiation sites for the ilvC and ilvY genes are indicated by arrows and the 10 and 35 hexanucleotide sequences of each promoter are underlined (3).

	MATERIALS AND METHODS

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Chemicals and Reagents-- Polyethyleneimine was purchased from Miles Laboratories. Restriction endonucleases, T4 DNA ligase, and T4 polynucleotide kinase were purchased from New England Biolabs. E. coli RNA polymerase, pancreatic RNasin, and DNase I were purchased from Boehringer Mannheim. Shrimp alkaline phosphatase was purchased from United States Biochemicals. Radiolabeled nucleotides were purchased from NEN Life Science Products. DNA oligonucleotides were synthesized by Operon Technologies. Site-directed mutagenesis was performed using the oligonucleotide directed in vitro mutagenesis kit version 2.0 from Amersham Pharmacia Biotech. DNA sequencing was performed using the Sequenase^TM kit from United States Biochemicals.

Plasmids-- Plasmid DNA isolation and all recombinant DNA manipulations were carried out using standard methods (7). Plasmid pET3cY, used for the overexpression of the ilvY gene product, was created by site-directed mutagenesis of the ilvY gene, contained on a 1400-bp BamHI-PvuII DNA fragment (derived from plasmid pRW1Y) (3). An NdeI restriction site was created at the beginning of the ilvY protein coding sequence. This site replaced the natural GTG translation initiation codon with the more common ATG codon. The 1225-bp NdeI-BamHI restriction fragment from the resulting plasmid, containing the ilvY gene, was ligated into the compatible NdeI-BamHI sites of the Studier vector pET3c (8) to yield the plasmid pET3cY. Plasmid pRWSR-2, used to generate the DNA fragments (containing ilvYC sequence from bp position 111 to +1 cloned into the BamHI and EcoRI sites of pUC19) described in the binding and chemical footprinting experiments, has been previously described (6). Plasmid pBENDY, used to create the isometric O₁O₂-containing DNA fragments for circular permutation assays, was created by ligating an end-filled 132-bp EcoRI-HindIII DNA fragment containing the tandem O₁O₂ operator sites into the unique SalI restriction site of plasmid pBEND2 (9) Plasmid pDD3Y, used for the in vitro transcription reactions, was constructed by ligating an end-filled 220-bp BglII-EcoRI DNA restriction fragment (containing ilvYC sequence from bp 220 to +1 relative to the transcription start site of ilvC) into the BamHI site (end-filled) of the plasmid pDD3 (10). The BamHI site in pDD3 is flanked on both sides by tandem rrnBT₁T₂ transcriptional terminator sequences (10).

Purification and Characterization of IlvY Protein-- A 4-liter culture of strain BL21DE3 (8) containing plasmid pET3cY was grown to an A₆₀₀ = 0.8 at 20 °C, induced with the addition of isopropyl-1-thio--D-galactopyranoside to a final concentration of 0.75 mM, and allowed to continue growing for an additional 6-8 h. Cells were harvested by centrifugation and resuspended in 40 ml of an ice-cold solution containing 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 5% glycerol, and 0.5 mM dithiothreitol (Buffer A). Cells were disrupted by sonication in a Rosett cell using a Tekmar Sonic Disruptor set at 100 W. Sonication of cells was performed on ice with 12 × 20 s bursts interrupted by 2-min cooling intervals. To avoid partial proteolysis owing to the high arginine/lysine ratio of the IlvY protein (11), phenylmethylsulfonyl fluoride was added to the cell suspension at a final concentration of 1 mM immediately before and after sonication. All subsequent steps were performed at 4 °C or on ice. The crude cell lysate was cleared by centrifugation at 12,000 rpm in a Beckman JA20 rotor for 40 min at 4 °C. The resulting supernatant solution was transferred to an Erlenmeyer flask, and a 10% (v/v) solution of polyethyleneimine was slowly added dropwise to a final concentration of 0.6% (v/v) with gentle stirring and incubated for an additional 20 min. This mixture was centrifuged for 30 min at 10,000 rpm in a Beckman JA20 rotor. The resulting precipitate was resuspended in 10 ml of Buffer A containing 0.3 M NaCl and gently stirred for 15 min. This suspension was centrifuged for 20 min at 12,000 rpm in a Beckman JA20 rotor. The resulting supernatant solution was slowly loaded onto a 4 ml phosphocellulose column pre-equilibrated in Buffer A. After loading the sample, the column was washed with one column volume of Buffer A. IlvY protein was eluted from the column with an 80-ml linear KCl gradient (0.1-0.5 M) in Buffer A. Typically, the protein eluted at a [KCl] = 0.4 M. 1-ml fractions were collected and analyzed by SDS-polyacrylamide gel electrophoresis. 2 µl of each fraction were assayed in a 20-µl reaction volume using the mobility shift binding protocol described below. Appropriate fractions were pooled and concentrated in an Amicon Microcon-10 ultrafiltration device. Protein concentrations were measured according to the method of Bradford (12) using the Bio-Rad DC Protein Assay Kit. A final yield of about 0.5 mg of greater than 95% purified IlvY protein is routinely obtained from 1 liter of cells.

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Mobility Shift Binding Assays-- Mobility shift binding assays were performed according to the methods of Fried and Crothers (13) as described by Wek and Hatfield (6) with the exception that gels were run in 1× TAE buffer (40 mM Tris acetate (pH 8.5), 2 mM EDTA·2H₂O). Radiolabeled DNA used in these experiments was present at final concentrations not exceeding 2 × 10¹¹ M to ensure that the free concentration of IlvY protein was equivalent to the total IlvY protein concentration. Binding reactions were performed in a final volume of 20 µl containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 6 mM -mercaptoethanol, 100 µg of bovine serum albumin/ml, and 2.5 µg of sonicated herring sperm DNA/ml. Free and bound DNA fragments were visualized by autoradiography following the exposure of Kodak XAR-5 film to the dried gels overnight at 25 °C. The autoradiograms were digitized with a Hewlett-Packard ScanJet IIcx digital scanner. Band intensities were measured using the public domain NIH IMAGE gel quantitation software. The intensity of each band was corrected for background differences and normalized to the total intensity of all bands in each lane. The equilibrium binding data were analyzed by nonlinear least squares estimations. The algorithm for this analysis (14) uses a variation of the Gauss-Newton procedure to determine the best fit model-dependent parameter values corresponding to a minimum in the variance of each data point (15). The confidence levels for the curve fits reported correspond to approximately one standard deviation (65% confidence).

DNase I Footprinting Experiments-- DNase I footprinting reactions were performed according to the methods of Galas and Schmitz (16) as described by Wek and Hatfield (6) using uniquely 5'-³²P-end-labeled DNA fragments containing ilvYC DNA from bp positions 111 to +1, derived from plasmid pRWSR-2. Binding reactions contained 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 6 mM -mercaptoethanol, 100 mg of bovine serum albumin/ml, and 2.5 mg of sonicated herring sperm DNA/ml in a final volume of 40 µl. Operator DNA was present at a final concentration of less than 10¹⁰ M. Purified IlvY protein was present at a final protein dimer concentration of 5 × 10⁸ M. -Acetohydroxybutyrate was present as specified in the figure legends. DNase I exposure was for 1 min at 25 °C. This yielded less than 30% nicked DNA fragments, determined by Cerenkov counting of the band corresponding to uncleaved DNA. This low nicking frequency is not expected to affect the intrinsic protein-DNA binding (17). Samples were resolved by electrophoresis on an 8% denaturing polyacrylamide gel (7.6% acrylamide, 0.4% N,N'-methylenebisacrylamide) containing 8 M urea in TBE buffer (7) and visualized by autoradiography following the exposure of Kodak XAR-5 film to the gels at 70 °C in the presence of a Cronex Quanta III intensifying screen (DuPont). The autoradiograms were digitized with a Hewlett-Packard ScanJet IIcx digital scanner. The band intensity in each lane corresponding to the DNase I-hypersensitive site located at bp 37 in the ilvC promoter region was measured using the public domain NIH IMAGE gel quantitation software. The intensity of each band was corrected for background differences and normalized to the intensity of the unprotected band in the same lane at bp 90.

Hydroxyl Radical Footprinting Experiments-- Hydroxyl radical footprinting was performed as described by Tullius and Dombroski (18) using the same DNA fragments (<1 × 10¹⁰ M) used in the DNase I footprinting reactions. Binding reactions were performed under equilibrium binding conditions as described above with the exception that glycerol was omitted from the reaction mixture. Samples were treated with hydroxyl radical for exactly 2 min at 25 °C and quenched with the addition of 20 µl of 0.2 M thiourea to ensure conditions of single-hit kinetics as described above. Reaction products were resolved by electrophoresis on a 10% denaturing polyacrylamide gel (9.5% acrylamide, 0.5% N,N'-methylenebisacrylamide) containing 8 M urea in TBE buffer (7) and visualized by autoradiography as described above. Data were analyzed by utilizing the public domain NIH IMAGE gel quantitation software. Regions of protection were determined by comparing the relative peak heights of each integrated band obtained from an integration of the bands of a digital image of each lane.

In Vitro Transcription Reactions-- In vitro transcription reactions were conducted using the closed circular supercoiled (>80%) plasmid pDD3Y (described above), in the absence and presence of purified IlvY protein and/or inducer, according to the procedures of Hauser et al. (19). RNA polymerase-plasmid DNA complexes were formed by preincubating 1 unit (2.4 pmol) of RNA polymerase and 100 ng of plasmid DNA (0.2 pmol) in a 20-µl reaction mixture (0.04 M Tris-HCl (pH 8.0), 0.1 M KCl, 0.01 M MgCl₂, 1.0 mM dithiothreitol, 0.1 mM EDTA, 200 mM CTP, 20 mM UTP, 10 mCi of [-³²P]UTP (3000 Ci/mmol), 0.1 mg/ml bovine serum albumin and 40 units of RNasin for 10 min at 25 °C. Transcription reactions were initiated by the addition of 2 µl of 2 mM ATP, 2 mM GTP solution. Reactions were terminated after 4 min with the addition of 20 µl of stop solution (95% formamide, 0.025% bromphenol blue, 0.025% xylene cyanol). Transcription under these conditions was determined to be linear with respect to time for at least 6 min and was proportional to the amount of plasmid DNA template used. To correct for differences in the total amount of transcription between reactions, the amount of ilv-specific transcription was determined by normalizing the relative signal of ilv-specific transcription to the relative signal of transcription arising from the RNA-I promoter present on each plasmid DNA template in the transcription reaction. Reaction products were separated by electrophoresis on an 8% denaturing polyacrylamide gel (7.6% acrylamide, 0.4% N,N'-methylenebisacrylamide) containing 8 M urea in TBE buffer (7) and visualized by autoradiography as described above.

Circular Permutation Assays-- Binding reactions were performed as described above using isometric DNA fragments containing the tandem O₁O₂ operators derived from the plasmid pBENDY. Products were resolved by electrophoresis through an 8% nondenaturing polyacrylamide gel (7.92% acrylamide, 0.079% N,N'-methylenebisacrylamide) in 1× TAE (20) buffer (7). In reactions performed with inducer, 1 mM -acetolactate was included both in the gel and in the running buffer. Analysis of bend mapping was performed as described by Wu and Crothers (21). Estimates of bend angles were obtained as described by Thompson and Landy (22) using the empirical relationship cos(/2) = R_f (slowest)/R_f (fastest).

Abortive Transcription Assays-- Abortive transcription initiation assays were performed in the presence or absence of 2 mM inducer (-acetohydroxybutyrate) according to the methods of Hawley et al. (23) as described by Parekh and Hatfield (24). Briefly, reactions were initiated by the addition of RNA polymerase at final concentrations of 20, 40, 60, 80, and 100 nM to a preformed IlvY protein-DNA complex. IlvY protein-DNA complexes were preformed by incubating 30 nM IlvY protein dimer with 1 nM supercoiled DNA plasmid template, pDD3Y, for 20 min at 37 °C in reaction buffer A (0.04 M Tris-HCl (pH = 8.0), 0.1 M KCl, 0.01 M MgCl₂, 1.0 mM dithiothreitol, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin), 40 mM UTP, 100 mCi [-³²P]UTP, and 0.5 mM ApA. The inclusion of the dinucleotide ApA in the reaction mixture restricts transcription initiation to the ilvC promoter of the plasmid DNA template, pDD3Y, yielding the abortive product ApApUpU. Abortive transcription products were isolated by nondenaturing electrophoresis in a 25% polyacrylamide gel (23.75% acrylamide, 1.25% N,N'-methylenebisacrylamide) in 1× TBE buffer (7), and quantitated by Phosphor-Imager analysis.  is a measure of the lag time required for open complex formation observed in a product versus time plot. _obs values were determined by a nonlinear least-squares analysis of the kinetic data using the program LAGPLOT described by Goodrich (25). The apparent binding affinity of RNA polymerase (K_B) and the first-order isomerization rate constant (k₂) were determined by plotting _obs as a function of 1/(RNA polymerase) on the basis of a least-squares fit to the equation _obs = (1/k₂) + (1/k₂K_B(RNA polymerase)) using the TAUPLOT program of Goodrich (25).

	RESULTS

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Operator Binding by Purified IlvY Protein-- Previous analyses with IlvY protein-enriched cell-free extracts indicated that IlvY protein binds to tandem operator sites (O₁O₂) in the divergent-overlapping ilvYC promoter region and that this binding is cooperative (6). To explore the quantitative nature of this cooperativity, binding titrations of purified IlvY protein to DNA fragments containing the tandem operators (O₁O₂) and to DNA fragments with either O₁ or O₂ deleted were performed (Fig. 2). These assays were conducted using concentrations of operator sites well below the effective binding dissociation constant for the tandem operator sites to evaluate the microscopic binding constants governing the assembly of IlvY protein-operator DNA complexes. The gel mobility shift of the tandem operator-containing DNA fragment showed only a single liganded complex, which we interpret to be O₁O₂ with both sites bound by IlvY protein dimer. The half-saturation point for IlvY binding to the DNA fragment containing O₁ and O₂ corresponds to an effective affinity of 2.2 nM, consistent with a previous estimate (2 nM) obtained with IlvY protein enriched cell free extracts (6). The lack of an intermediate band corresponding to IlvY protein bound to either O₁ or O₂ alone in the gel mobility shift experiment performed to generate the data in Fig. 2 is indicative of highly cooperative binding (17). In fact, the IlvY protein concentration required to half-saturate O₁ alone is 8-fold greater than the concentration required to half-saturate the tandem operator sites (Fig. 2). We found previously that it was not possible to saturate the O₂ site alone; at 8 × 10⁸ M IlvY dimer, the highest concentration used in the binding experiments, less than 15% of the DNA migrated as bound in gel shift assays (6). Based on this measurement, the affinity of IlvY protein for the O₂ site alone is calculated to be K₂ < 0.45 µM. When this experiment was repeated with the highly purified IlvY protein reported here, the same result was obtained. Therefore, the affinity for O₂ alone is at least 200-fold weaker than for sites O₁O₂ together (6). The observation that the apparent binding affinity for either operator site is reduced when the other site is removed indicates cooperative binding to the adjacent sites.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   IlvY protein-DNA binding isotherm curves as a function of increasing IlvY protein concentration in the presence of a limiting amount of 32P-end-labeled DNA fragment containing either the tandem operators (O1O2) () or the O1 operator alone (). The data presented are the average values obtained from three separate experiments ± S.E. The solid curves represent the best fit to both the O1 alone and O1O2 data shown. The interaction free energy changes used to simulate these curves are: G1 = 10.4 kcal/mol; G2 = 9.8 kcal/mol; Gc = 3.0 kcal/mol.

DNA Binding Site Specificity of Purified IlvY Protein-- DNase I footprinting experiments were conducted to demonstrate that the purified IlvY protein exhibits site-specific binding to the tandem operator sites in the divergent promoter region of the ilvYC operon (Fig. 1). The results in Fig. 3A show that purified IlvY protein protects 2 adjacent 27-bp regions of DNA (ilvC bp 76 to 50 and 44 to 18) separated by 4 intervening DNase I-sensitive nucleotides on a 240-bp EcoRI-HindIII DNA fragment containing the tandem operator sites. These observed regions of protection on the nontranscribed strand of the ilvC gene correspond to those previously defined as O₁ and O₂ with partially purified IlvY protein (6). The results in Fig. 3B demonstrate that the purified protein protects three regions of DNA on the transcribed strand of the ilvC gene (ilvC bp 78 to 55, 51 to 43, and 41 to 19). These sequences are also contained in the DNA regions of dyad symmetry previously defined as O₁ and O₂.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   DNase I footprint of IlvY protein-operator DNA interactions in the ilvYC promoter region. Autoradiogram of DNase I protection patterns obtained in the absence and presence of a minimally saturating amount of purified IlvY protein (4 × 108 M dimer) bound to a 240-bp EcoRI-HindIII DNA fragment containing ilv bp positions 111 to +1 (relative to ilvC transcription). DNA fragments were uniquely 32P-end-labeled on either the transcribed strand of the ilvY gene (A) or the transcribed strand of the ilvC gene (B). Regions of protection are designated with brackets, and the exact nucleotide positions of protection are indicated adjacent to the brackets. Base pair position numbering is relative to the start of ilvC transcription (3). Lanes 1 and 3, DNA probe only; lanes 2 and 4, IlvY protein bound-DNA.

Effect of IlvY Protein Binding to O₁O₂ on Transcription from the ilvY Promoter-- To investigate how IlvY protein binding to the tandem operators, O₁O₂, affects transcription from the ilvY promoter, transcription from the ilvY promoter was assayed in vitro using a negatively supercoiled plasmid DNA template, pDD3Y. This plasmid contains the divergent ilvC and ilvY promoters flanked by Rho-independent ribosomal, rnnB T₁ T₂, terminators. Transcription from the ilvY and ilvC promoters generates 369 nucleotide and 154 nucleotide products, respectively. The results in Fig. 4 demonstrate that transcription from the ilvY promoter is repressed as a function of increasing concentration of IlvY protein. The fractional repression calculated from the data in Fig. 4 and the fractional saturation of O₁O₂ taken from Fig. 2 have similar dependences on IlvY protein concentration (Fig. 5). Both repression of transcription from the ilvY promoter and binding of the IlvY protein to O₁O₂ are unaffected by the addition of inducer (6).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   In vitro transcription reactions of the divergent overlapping ilvYC promoter region in the absence and presence of increasing concentrations of purified IlvY protein. Autoradiogram of the RNA products of in vitro transcription reactions using the supercoiled DNA template, pDD3Y (see "Materials and Methods"). ilvY and ilvC identify the 369- and 154-nucleotide RNA transcripts that originate from the respective promoters. Lane 1, no IlvY protein; lanes 2-9, [IlvY protein] = 2.0, 4.0, 8.0, 12, 16, 20, 32, and 40 nM, respectively. The 108-nucleotide transcript designated ori originates from the RNA-I promoter.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Comparison of IlvY protein binding () to the tandem operators (O1O2) and repression of transcription originating from the ilvY promoter (). IlvY protein DNA binding was determined by mobility shift experiments of the type described under "Materials and Methods." Transcription levels were determined from autoradiographic band intensities of ilvY promoter-specific transcripts obtained from experiments of the type illustrated in Fig. 4. All measurements were obtained from autoradiogram exposures where band intensities were linear with respect to time. The solid curves show the results of the analysis of these data as described in the text. For this analysis, the free energy changes used for binding of IvY protein to O1O2 were: G1 = 10.4 kcal/mol; G2 = 9.8 kcal/mol; Gc = 3.0 kcal/mol.

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Effects of Inducer on IlvY Protein-ilvC Promoter Interactions-- The binding of inducer to IlvY protein does not significantly increase the affinity of IlvY protein for the tandem operators. Yet, it results in the appearance of a DNase I hypersensitive band in the region of the O₂ operator sequence that overlaps the 35 region of the ilvC promoter (6). These results suggested that the binding of inducer might direct a conformational change of the IlvY protein-operator DNA complex. In addition, the binding of RNA polymerase to the ilvC promoter is detectable by DNase I footprinting only when both IlvY protein and inducer are present (6). We proposed, therefore, that the role of inducer is to facilitate a conformational change in the IlvY protein-O₁O₂ complex, which recruits RNA polymerase to the ilvC promoter (6). To test this hypothesis, in vitro transcription and DNase I footprinting assays were conducted to quantitatively compare the effects of inducer on the DNase I hypersensitivity in the ilvC 35 hexanucleotide region and on transcription from the ilvC promoter.

View larger version (55K): [in this window] [in a new window]   Fig. 6.   Effects of co-inducer binding to the IlvY protein-DNA complex on DNA structure. Autoradiogram of DNase I protection patterns of a 240-bp EcoRI-HindIII DNA fragment (containing ilv bp positions 111 to +1 relative to ilvC transcription; Ref. 10) obtained in the presence of a minimally saturating amount of purified IlvY protein (4 × 108 M dimer) and increasing concentrations of -acetohydroxybutyrate (AHB). DNA fragments were uniquely 32P-end-labeled on either the nontranscribed (panel A, lanes 1-5) or transcribed (panel B, lanes 6-8) strands. The regions of protection corresponding to the tandem operators O1O2 are identified. The positions of the DNase I hypersensitive sites on both strands that are induced by the addition of AHB are indicated with arrows. Lane 1, DNA fragment only; lanes 2-5, [AHB] = 0, 0.15, 0.30, and 1.25 mM, respectively; lane 6, DNA fragment only; lanes 7-8, [AHB] = 0 and 1.25 mM, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Comparison of inducer-directed activation of transcription originating from the ilvC promoter () and inducer-directed induction of the DNase I hypersensitive site at bp 37 (). Transcription levels were determined from autoradiographic band intensities of ilvC promoter-specific transcripts obtained from experiments of the type illustrated in Fig. 8 and normalized to the transcription level obtained at the highest inducer (AHB) level (1.25 mM). The band intensity in each lane corresponding to the DNase I hypersensitive site located at bp 37 in the ilvC promoter region was corrected for background differences and normalized to the intensity of an unprotected band in the same lane. All measurements were obtained from autoradiogram exposures where band intensities were linear with respect to time.

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View larger version (51K): [in this window] [in a new window]   Fig. 8.   In vitro transcription reactions of the divergent overlapping ilvYC promoter region in the presence of a minimally saturating concentration of purified IlvY protein and increasing concentrations of the co-inducer -acetohydroxybutyrate (AHB). Autoradiogram of the RNA products of in vitro transcription reactions using the supercoiled DNA template, pDD3Y (see "Materials and Methods"). ilvY and ilvC identify the 369- and 154-nucleotide RNA transcripts that originate from their respective promoters. Lane 1, no IlvY protein; lanes 2-11, [IlvY protein] = 4.0 × 108 M dimer and [AHB] = 4.7 × 103 mM; 9.4 × 103 mM; 1.8 × 102 mM; 3.75 × 102 mM; 7.5 × 102 mM; 1.5 × 101 mM; 3.0 × 101 mM; 1.25 mM; 2.5 mM; 5 mM, respectively. The 108 nucleotide transcript designated ori originates from the RNA-I promoter.

Effects of Inducer on the Conformation of the IlvY Protein-DNA Complex-- The appearance of DNase I hypersensitive sites in and around the protected regions of DNase I footprints (Fig. 6) are often indicative of protein-induced DNA bends. In fact, other LysR type activator-repressor proteins have been shown to induce DNA bends at their target DNA binding sites; and, in some cases, these bends are modulated by the binding of an effector ligand (26-28). To examine the possibility that IlvY protein might incite a bend in the DNA helix, circular permutation assays were conducted according to the methods of Wu and Crothers (21). A 240-bp EcoRI-HindIII DNA fragment containing the tandem operator sites of the ilvYC operator-promoter region, was ligated into the unique SalI site of the plasmid pBEND2 (9), and isometric (circularly permuted) DNA fragments containing the tandem operators were generated by cleavage at six tandemly repeated restriction endonuclease sites flanking the SalI site. The relative base pair positions of the ilv specific DNA fragment within each of these circularly permuted fragments is identified in Fig. 9B. These DNA fragments were incubated with IlvY protein in the presence and absence of inducer, and the products of these binding reactions were resolved by nondenaturing polyacrylamide gel electrophoresis.

View larger version (35K): [in this window] [in a new window]   Fig. 9.   Circular permutation analysis of IlvY protein binding to the tandem operators. A, autoradiogram of a minimally saturating concentration of IlvY protein (4 × 108 M dimer) bound to isometric "circularly-permuted" 32P-end-labeled DNA fragments containing the tandem operators O1O2. Bound denotes IlvY protein-bound DNA complexes and Free denotes unbound DNA fragments. Relative base pair position of an ilv-specific DNA fragment (bp positions 111 to +1 relative to the start of ilvC transcription) (3) contained within a 270-bp circularly permuted DNA fragment: lanes 1 and 11, 13 bp; lanes 2 and 10, 43 bp; lanes 3 and 9, 61 bp; lanes 4 and 8, 67 bp; lanes 5 and 7, 79 bp; lane 6, 110 bp. Lanes 11-7 are repeats of lanes 1-5, respectively. B, table of relative Rf value as a function of the relative base pair position of an ilv-specific DNA fragment (bp positions 111 to +1 relative to the start of ilvC transcription) (3) contained in a 270-bp DNA fragment and calculated angles of bending for Free, IlvY protein-bound, and IlvY protein--acetolactate-bound DNA fragments. The relative Rf value of each of these species was determined by normalizing the migration of the DNA fragment yielding the fastest migrating species as an Rf value of 1. Calculated angles of DNA bending were determined using the empirical relationship cos (/2) = Rf (slowest)/Rf (fastest).

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Schematic representation of IlvY protein-operator DNA interactions in the ilvYC promoter region. Indicated are the protection patterns of the IlvY protein-bound ilvC promoter region in the absence (A) and presence (B) of inducer. , nucleotide positions protected by IlvY protein from hydroxyl radical cleavage; , nucleotide positions protected by IlvY protein from dimethyl sulfate modification. Solid black arrows indicate nucleotide positions whose sensitivity to DNase I cleavage is enhanced (up arrows) or diminished (down arrows) with the addition of inducer to an IlvY protein-DNA complex. Solid gray arrows indicate nucleotide positions whose sensitivity to hydroxyl radical cleavage is enhanced (up arrows) or diminished (down arrows) with the addition of inducer to an IlvY protein-DNA complex. The dark bars denote the 35 and 10 hexanucleotide regions of the ilvC promoter. The symbols O1 and O2 denote the tandem operator sequences recognized by IlvY protein. The in vivo transcriptional initiation site for the ilvC promoter is indicated with an arrow (3). The DNA helix is oriented to display the open face of the major grooves of the tandem operators O1O2.

View larger version (39K): [in this window] [in a new window]   Fig. 11.   Hydroxyl radical footprint of IlvY protein-operator DNA interactions in the ilvYC promoter region. Plots of the relative band intensities in digitized images of autoradiograms of hydroxyl radical protection patterns obtained in the absence (thick line) and presence (thin black line) of a minimally saturating amount of purified IlvY protein (4 × 108 M dimer) bound to a 240 bp EcoRI-HindIII DNA fragment containing ilv bp positions 111 to +1 (relative to ilvC transcription) (3). DNA fragments were uniquely 32P-end-labeled on either the nontranscribed (A) or the transcribed (B) strand of the ilvC gene. Hydroxyl radical protection patterns obtained in the presence of both IlvY protein (4 × 108 M dimer) and AHB (1.25 mM) are indicated with a thin gray line.

Effects of Substrate Inducer on the Kinetics of the Transcription Initiation Reaction at the ilvC Promoter-- To determine the effects of inducer binding to a preformed IlvY protein-DNA complex on the kinetics of the transcription initiation reaction at the ilvC promoter, abortive transcription assays of the ilvC promoter region complexed with IlvY protein were performed in the presence and absence of inducer (23). An IlvY protein dimer concentration of 4 × 10⁸ M, which yields greater than 99% saturation of O₁O₂ (Fig. 2) was used in each transcription reaction. The results of these assays showed that the addition of 1 mM inducer to an IlvY protein-operator DNA complex increases the binding affinity of the ilvC promoter for RNA polymerase (K_B) nearly 100-fold and decreases the isomerization rate for open complex formation (k₂) approximately 7-fold (Table I). Thus, the principal effect of substrate-inducer binding to an IlvY protein-DNA complex on the transcription initiation reaction at the ilvC promoter is to increase the affinity of ilvC promoter for RNA polymerase.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We previously demonstrated that, in vivo, IlvY protein autoregulates expression of its own structural gene in the divergent-overlapping ilvYC operon in an inducer-independent manner and that both inducer and IlvY protein are required for the activation of ilvC gene expression (6). We further demonstrated that both activation and repression appear to be mediated from the same regulatory locus. Thus, we suggested that the binding of inducer to IlvY protein targets an activator property distinct from its DNA binding activity and that the inducer-directed activation of transcription from the ilvC promoter might functionally be correlated with a conformational change of a preformed IlvY protein-operator DNA complex (6). Previous work also demonstrated that the binding of inducer to IlvY protein does not significantly affect the occupancy of IlvY protein at the tandem operators (6). The results of DNase I and hydroxyl radical footprinting experiments reported here demonstrate that, in the absence or presence of inducer, IlvY protein remains bound to both operator sites. Therefore, these results definitively establish that the role of inducer in the IlvY protein-mediated activation of transcription from the ilvC promoter is not to recruit IlvY protein to the ilvC promoter region.

The binding of inducer to an IlvY protein-DNA complex evokes the induction of a hypersensitive DNase I site in the 35 hexanucleotide region of the ilvC promoter (Fig. 6) and other changes in the DNase I and hydroxyl radical footprinting patterns in the tandem operator region (Figs. 6, 10, and 11). These results suggest a ligand-induced conformational change in the IlvY protein-operator DNA complex. Quantitative analyses of these ligand-induced DNA structural changes further establish that this conformational change is functionally correlated with transcriptional activation. For example, the inducer concentration that results in half-maximal induction of the DNase I hypersensitive site at bp 37 in the 35 hexanucleotide region of the ilvC promoter is the same concentration that half-maximally activates transcription from this promoter (Fig. 7).

The nature of the inducer-directed conformational change in the IlvY protein-operator DNA complex is suggested by the results of circular permutation assays (Fig. 9). These experiments indicate that the binding of IlvY protein to DNA fragments containing the tandem operator sites increases a sequenced-directed DNA bend of approximately 36° to a bend angle of approximately 60°. The mean geometric center of both the sequence-directed and IlvY protein-induced bends is positioned in the 35 hexanucleotide region of the ilvC promoter. The addition of inducer to the IlvY protein-operator DNA complex causes the partial relief of this bend to about 50°. This conclusion is supported by the distribution of hydroxyl radical and DNase I reactive sites on both strands of an IlvY protein-operator DNA complex in the presence and absence of inducer. In fact, these results suggest that the binding of inducer facilitates the conversion of a sharp IlvY protein-induced DNA bend to a smoother bend of a smaller angle. We conclude, therefore, that alterations in the local geometry of the DNA helix around the ilvC promoter region play an important role in the IlvY protein-mediated activation of transcription from the ilvC promoter.

Protein-induced DNA bending is known to influence the rate of transcription initiation at many promoters. In some cases, protein-induced bending of the DNA helix enhances the binding of RNA polymerase to the promoter region (29). In other cases, it enhances the rate of open complex formation (23, 24). The results of abortive transcription assays (Table I) indicate that the principal effect of the inducer-mediated conformational change in the IlvY protein-operator DNA complex is to increase the binding affinity of RNA polymerase for the ilvC promoter nearly 100-fold. This suggests that the functional effect of the inducer-mediated conformational change in the IlvY protein-DNA complex and the associated alterations in DNA bending is to remodel the structure of the poor 35 hexanucleotide region of the ilvC promoter region to make it a better template for RNA polymerase recognition. The results of these abortive transcription assays are also in general agreement with the proposal that the 35 region of a promoter is involved in the initial recognition of a promoter by RNA polymerase (29). The nucleotide sequence of the 35 hexanucleotide region of the ilvC promoter (TTTCCG) exhibits only a 3/6 match to the consensus sequence (TTGACA).

The results reported here also demonstrate that IlvY protein represses the expression of its own structural gene by a simple RNA polymerase occlusion mechanism. The results of in vitro transcription reactions conducted in the presence of increasing concentrations of IlvY protein show that IlvY protein binding to the tandem operators represses transcription from the ilvY promoter and that the fractional repression of ilvY expression and the fractional saturation of the tandem operator sites exhibit the same dependences on IlvY protein concentration (Fig. 5). The simplest interpretation consistent with this set of data is that IlvY protein competitively inhibits the binding of RNA polymerase at the ilvY promoter site. This conclusion is consistent with the results of chemical and enzymatic structural probing experiments, which demonstrate that both IlvY protein and RNA polymerase bind to overlapping DNA sequences on the same face of the DNA helix (6).

It is interesting that the inducer titration curves of ilvC transcription shown in Fig. 7 correspond to a simple noncooperative binding model. This result shows that the activation of transcription from the ilvC promoter is not dependent on cooperative binding of inducer to multiple sites on the IlvY protein at the O₁O₂ sites. These experiments also show that the measured inducer concentration required to half-maximally activate in vitro transcription from the ilvC promoter is nearly the same as that required to half-maximally saturate the enzyme product of the ilvC gene (5). These concentrations are in good agreement with the inducer concentrations required for in vivo activation of ilvC expression (6, 30, 31).

It is interesting to consider the activation mechanism described here in relation to that of the MerR protein (32), a mechanistically similar ligand-responsive activator protein which is not a member of the LysR family of proteins. In this case, the binding of the inducer, mercuric ion, to a MerR-operator DNA complex in the divergent-overlapping merR-merTPCAD operator-promoter region also facilitates the partial relief of a MerR-induced DNA bend. However, in this case the primary effect of the binding of inducer, and relief of protein-induced DNA bending, is the partial unwinding of the spacer region between the  35 and 10 hexanucleotide regions of the merTPCAD promoter. This unwinding facilitates open complex formation and/or promoter clearance by the transcription apparatus rather than the recruitment of RNA polymerase to the promoter site as is the case for IlvY protein. Thus, whereas both of these cases illustrate an important role for ligand-induced conformational changes of preformed protein-DNA complexes in the regulation of gene expression, they also highlight the functionally distinct roles these conformational changes may play. However, both of these examples emphasize the importance of protein-induced conformational changes on the structure of the DNA helix for the regulation of gene expression.

The kinetic effect of the ligand-induced, IlvY protein-directed, structural change in the IlvY protein-ilvC promoter DNA complex reported here is to recruit RNA polymerase to the promoter site. This might be accomplished by remodeling the DNA helix to facilitate improved RNA polymerase-DNA interactions. Alternatively, the primary effect of the inducer-mediated IlvY protein conformational change might be to facilitate protein-protein interactions between IlvY and RNA polymerase. In this case, the relaxation of the bend angle in the ilvC promoter region might be the fortuitous consequence of the inducer-mediated IlvY protein conformational change, and the primary role of IlvY protein might be to enhance RNA polymerase recruitment via facilitated protein-protein interactions with RNA polymerase across opposite faces of the DNA helix. The relative contributions of each of these components awaits further analysis.

Finally, it is interesting to note that inducer-responsive alterations in the angle of protein-induced bending of the DNA helix have also been reported for other LysR-type proteins such as OccR (33), CysB (28), and CatR (27). Thus, ligand-mediated alterations in protein-induced bending of the DNA helix appears to be a general feature among members of the LysR family. However, the functional relationship between ligand-induced conformational changes and the activation mechanism in these cases is uncertain. Thus, the initial elucidation of the activation mechanism employed by the IlvY protein reported here should facilitate our understanding of the regulatory mechanisms of other LysR-type regulatory systems.