From the Departments of Microbiology and Molecular
Genetics and ¶ Molecular Biology and Biochemistry, University of
California, Irvine, California 92697
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IlvY protein binds cooperatively to tandem operator sites in the divergent, overlapping, promoter-regulatory region of the ilvYC operon of Escherichia coli. IlvY positively regulates the expression of the ilvC gene in an inducer-dependent manner and negatively regulates the transcription of its own divergently transcribed structural gene in an inducer-independent manner. Although binding of IlvY protein to the tandem operators is sufficient to repress ilvY promoter-specific transcription, it is not sufficient to activate transcription from the ilvC promoter. Activation of ilvC promoter-specific transcription requires the additional binding of a small molecule inducer to the IlvY protein-DNA complex. The binding of inducer to IlvY protein does not affect the affinity of IlvY protein for the tandem operator sites. It does, however, cause a conformational change of the IlvY protein-DNA complex, which is correlated with the partial relief of an IlvY protein-induced bend of the DNA helix in the ilvC promoter region. This structural change in the IlvY protein-DNA complex results in a 100-fold increase in the affinity of RNA polymerase binding at the ilvC promoter site. The ability of a protein to regulate gene expression by ligand-responsive modulation of a protein-DNA structure is an emerging theme in gene regulation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 SequenaseTM 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.
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 10
10 M.
Purified IlvY protein was present at a final protein dimer concentration of 5 × 10
8 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) = Rf (slowest)/Rf (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 (KB) 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/k2KB(RNA polymerase))
using the TAUPLOT program of Goodrich (25).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
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).
|
|
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.
|
|
|
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.
|
|
|
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 (KB)
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Elaine Ito for technical assistance, Stuart Arfin, Steve Sheridan, and She-Pin Hung for helpful discussions, and J. Goodrich for supplying the LAGPLOT and TAUPLOT computer programs.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9723452 (to G. W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a University of California Irvine Medical Scientist Training Program Fellowship.
To whom correspondence should be addressed. Tel.:
714-824-5858; Fax: 714-824-8598; E-mail: gwhatfie{at}uci.edu.
1
The abbreviations used are: bp, base pair(s);
TBE, Tris-borate-EDTA; AHB, -acetohydroxybutyrate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|