Journal of Bacteriology, April 1999, p. 2338-2345, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specific Contacts between Residues in the DNA-Binding Domain
of the TyrR Protein and Bases in the Operator of the
tyrP Gene of Escherichia coli
J. S.
Hwang,
J.
Yang, and
A. J.
Pittard*
Department of Microbiology and Immunology,
University of Melbourne, Parkville, Victoria 3052, Australia
Received 24 November 1998/Accepted 9 February 1999
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ABSTRACT |
In the presence of tyrosine, the TyrR protein of Escherichia
coli represses the expression of the tyrP gene by
binding to the double TyrR boxes which overlap the promoter.
Previously, we have carried out methylation, uracil, and ethylation
interference experiments and have identified both guanine and thymine
bases and phosphates within the TyrR box sequences that are
contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have
used missing contact probing to test the involvement
of all of the bases within the tyrP operator in the binding
of TyrR. Our results indicate that nearly all the bases within the
palindromic arms of the strong and weak boxes are important for the
binding of the TyrR protein. Two alanine-substituted mutant TyrR
proteins, HA494 and TA495, were purified, and their binding
affinities for the tyrP operator were measured by a gel
shift assay. HA494 was shown to be completely defective in
binding to the tyrP operator in vitro, while, in comparison
with wild-Type TyrR, TA495 had only a small reduction in DNA binding.
Missing contact probing was performed by using the purified TA495
protein, and the results suggest that T495 makes specific contacts with
adenine and thymine bases at the ±5 positions in the TyrR boxes.
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INTRODUCTION |
Regulation of gene expression in
bacteria requires the binding of the regulatory protein to a specific
DNA target which is termed an operator. In Escherichia coli
K-12, the transcriptional expression of the tyrP gene, which
codes for the tyrosine-specific transporter, is repressed by the TyrR
regulatory protein in the presence of the cofactor tyrosine (21,
24). The tyrP operator partially overlaps the 
35
region of the tyrP promoter and contains two
adjacent TyrR binding sites, which are termed TyrR boxes
(13). Each of the TyrR boxes is related to the palindrome
TGTAAAN6TTTACA (consensus sequence). The
upstream box of tyrP, which has a high degree of homology
with the TyrR box consensus sequence and which binds TyrR in the
absence of cofactors in vitro, has been referred to as the strong box
(4, 13). The downstream TyrR box, which is less homologous
to the TyrR box consensus sequence and binds TyrR only in the presence
of the cofactors tyrosine and ATP and an adjacent strong TyrR box, has
been referred to as a weak box (4, 13). The TyrR-mediated
repression of tyrP expression requires the binding of
TyrR protein to both the strong and weak TyrR boxes
(4).
Although extensive studies have been carried out to elucidate the
mechanism of TyrR-mediated repression and activation at tyrP
as well as other promoters of the TyrR regulon, relatively little is
known about the specific interactions between the TyrR protein and its
DNA targets. The TyrR system differs from those of other classic
repressors like TrpR in a number of ways: (i) the binding of the
cofactor tyrosine causes the protein to self-associate to form a
hexamer rather than directly modulating the tertiary structure of the
DNA-binding domain; (ii) the hexamer but not the dimer is able to bind
to a combination of strong and weak boxes; and (iii) the differences
between a strong and a weak TyrR box reside in the central six bases
(AT rich in strong boxes) and in the overall agreement with the
consensus sequence. Therefore it is of considerable interest to map the
important contact sites in both boxes. In a previous study, we have
carried out in vitro methylation, uracil, and
ethylation interference experiments and in vivo mutational
studies to probe the bases and phosphates of the tyrP
operator which are important for TyrR binding (11). We have
shown that the guanine residues at the ±8 positions, the thymine
residues at the ±7 positions, and the thymine residues at the +5
positions (in the top strand) in both TyrR boxes play a key role
in making contacts with the TyrR protein (11). A number of contacts with the phosphate backbone have also been found at
the end of and within the central regions of the TyrR boxes
(11).
The DNA-binding domain of TyrR is located near the carboxyl-terminal
end of the protein and is composed of a classic helix-turn-helix (HTH)
motif (10, 27). An alanine scan of the HTH motif of the TyrR
protein has identified amino acid residues in both helices, whose
side chains are believed to be involved either in maintaining the
correct conformation of the DNA-binding motif or in binding to the TyrR
boxes (27). The residue arginine-484 in the first helix and
the residues histidine-494, threonine-495, asparagine-499, and
arginine-502 in the second helix are important for TyrR-mediated repression of tyrP expression (11, 27), and by
using the coordinates of the lambda Cro and catabolite gene activator
protein (CAP), they have been predicted to face DNA (2, 7, 16,
19). The side chains of these amino acid residues have thus been
assumed to make contacts with the tyrP operator DNA.
The previous modification interference experiments did not allow the
examination of any involvement of cytosine or adenine residues exposed
in the major groove of the tyrP operator in the interactions
with the TyrR protein. In this paper, we describe the use of missing
contact probing (9) to test the involvement of all of the
bases in the tyrP operator in TyrR binding. We also report
an analysis of specific amino acid-base contacts between the TyrR
protein and the tyrP operator, using missing contact probing
with the mutant TyrR protein TyrR-TA495.
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MATERIALS AND METHODS |
Chemicals and Enzymes.
The enzymes used in this study were
all purchased commercially. Dimethyl sulfate, formic acid (88%),
hydrazine, and piperidine were purchased from Dupont-NEN.
[
-32P]dATP and [-32P]dGTP (3000 Ci/mmol; 10 mCi/ml) were purchased from Amersham.
Preparation of the mutant TyrR proteins.
Plasmids which
allow expression of the mutant TyrR proteins TyrR-TA495 and TyrR-HA494
from the T7 promoter were constructed as described below. The M13tg131
derivatives which carry the mutant tyrR genes coding for
TyrR-TA495 and TyrR-HA494 (11) were each mutagenized by
using the oligonucleotide 5'CGCATGGGATCCTTCACC3' to
create a BamHI site immediately upstream of the start
codon of tyrR. The resultant phagemids were each
digested with BamHI, and the 2.3-kb DNA fragments containing
the mutant tyrR genes were purified and ligated into
the expression vector pET15b (Novagen, Madison, Wis.). The
resultant pET15b derivatives were each transformed into E. coli BL21(DE3) (Novagen). The mutant TyrR proteins were overexpressed and purified as N-terminal fusions with an oligohistidine tag. The conditions and procedures for overexpression and purification of the mutant TyrR proteins were set up as described in the technical manual supplied by Novagen.
Preparation of labelled DNA fragments.
The 151-bp
BamHI-HindIII fragment containing the
tyrP regulatory region was generated by PCR using mpMU330 as
a template. Following sequence verification, this PCR fragment was
cloned into the BamHI and HindIII sites of
pUC19. The tyrP fragment was labelled at the top (or the
bottom) strand by digesting the pUC19 derivative with BamHI
(or HindIII) and by filling in the restriction end with
the Klenow fragment of DNA polymerase in the presence of
[-32P]dGTP (or [-32P]dATP). The
labelled DNA was precipitated with ethanol and digested with
HindIII (or BamHI). The labelled
tyrP fragment was then purified on a 5% polyacrylamide gel.
Missing contact probing.
Depurination (A+G) and
depyrimidation (C+T) reactions were carried out as described by
Brunelle and Schleif (9). Each reaction mixture contained
approximately 105 cpm of the end-labelled tyrP
fragment. The modified DNA fragments were each incubated with the
wild-type or the mutant TyrR protein (TA494) in a footprinting buffer
containing 5 mM Tris-HCl (pH 7.6), 80 mM KCl, 8 mM MgCl2, 1 mM dithiothreitol, 0.2 mM ATP, 1 mM tyrosine, and 4% (vol/vol)
glycerol. The concentration of the TyrR protein was determined
empirically such that the amounts of bound and unbound fractions of the
modified DNA were approximately equal. Following incubation for 20 min
at 37°C, the TyrR-DNA complexes were separated from the free DNA by
electrophoresis on a 5% polyacrylamide gel in 50 mM Tris-borate (pH
7.0)-1 mM MgCl2-0.2 mM ATP-1 mM tyrosine at 4°C. The
gel was autoradiographed for 2 h at 4°C, and DNA bands corresponding to the bound and unbound fractions were eluted from the
gel by a gel eluter (The Australian Chromatography Company-Hoefer). About 3 × 104 cpm of the DNA sample recovered from
the bound or unbound fraction was resuspended in 30 µl of 0.5 M
piperidine and heated for 20 min at 95°C. After freeze-drying, the
piperidine-treated DNA samples were electrophoresed on a 6%
polyacrylamide sequencing gel.
Gel shift assay.
The end-labelled tyrP fragment
described above was used in a gel shift assay to determine the binding
affinities of the wild-type and mutant TyrR proteins for the
tyrP operator. The binding reactions were carried out at
37°C for 20 min in the footprinting buffer (as described above) in
the presence of 0.5 pmol of the tyrP fragment and various
nanomolar amounts of the wild-type and mutant TyrR proteins. The
samples were then analyzed on a 5% polyacrylamide gel.
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RESULTS |
Effects of depurination and depyrimidation of the tyrP
operator on binding by the wild-type TyrR protein.
The missing
contact experiments were performed by using a 151-bp
BamHI-HindIII fragment carrying the
tyrP operator region. The 3' end of either the top or the
bottom strand of this DNA fragment was labelled with
32P by filling in the restriction end with Klenow enzyme in
the presence of [-32P]dGTP or
[-32P]dATP. Each of the labelled fragments
was then subjected to A+G depurination or T+C depyrimidation under
conditions where, on average, each molecule contained less than one
missing base (see Materials and Methods). The modified DNA fragments
were each mixed with wild-type TyrR protein in the presence of ATP and
tyrosine to allow the formation of protein-DNA complexes, and the
reaction mixtures were then subjected to a mobility gel shift assay.
The bound and free DNA fractions were recovered from the gel, cleaved at the phosphate backbone where a base had been eliminated, and examined on a sequencing gel.
The important base contacts for TyrR on the top and bottom strands are
shown in Fig. 1, and the data are also
quantitatively summarized below the autoradiographs. Data from the top
strand (Fig. 1A) indicate that depurination or depyrimidation at any one of the positions of the palindromic sequences, 8G, 7T,
6A, 4A, +4T, +5T, +7A, +8C, and +9A of the strong box and 9T,
8G, 7T, 6A, 5A, 3G, +6G, +7A, and +8C of the weak box,
significantly interferes with the binding of the TyrR protein. In
addition, weak but detectable signals were also identified from the
bases at the palindromic positions 9T, 5C, and +6T of the strong
box and at positions 4C, +4T, and +5T of the weak box. By examining the pattern of the bottom strand (Fig. 1B), it can be seen that removal of any base in the palindromic sequence of either
the strong or the weak box can interfere with the binding of the
TyrR protein. The depurination in the central region of the strong box
also had a significant effect on the binding of the TyrR protein. At
the center of the strong box, effects of missing contact were identified at positions 1T, +1A, and +2T in the top strand and positions 3A, 2A, 1A, and +2A in the bottom strand. In contrast, at the center of the weak box, only one effect was found at position 3G in the top strand. Several sites showing stronger bands in the
bound rather than the free DNA fractions were observed at positions
which flank the palindromic sequences of each of the two TyrR boxes,
and therefore it appears that the removal of a base at any of these
positions favors the formation of a protein-DNA complex.
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FIG. 1.
Effects of depurination or depyrimidation on binding of
the wild-type TyrR protein to the tyrP operator. Boxes
indicate the regions corresponding to the strong and weak TyrR boxes.
The first two lanes in each autoradiogram display the Maxam-Gilbert
sequencing ladders. A+G and T+C, depurination and depyrimidation
reactions, respectively; B, bound lane derived from the
TyrR-DNA complex; UB, unbound lane derived from the free DNA.
Results shown are from experiments using the DNA fragment labelled in
the top (A) and bottom (B) strands. The sequence shown at the
bottom corresponds to the sequence shown in the autoradiograms. The
relative strength of interference due to the removal of any base is
illustrated by the length of the respective bar. The palindromic
sequences of the strong and the weak boxes are shown in boldface, and
the positions in the boxes are indicated by the numbers between the two
strands.
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Determination of binding affinities of the mutant TyrR proteins
HA494 and TA495 for the tyrP operator by mobility gel shift
assay.
The amino acid residues histidine-494 and threonine-495 are
situated at positions 1 and 2 in the second helix of the HTH
DNA-binding motif of TyrR (27). These residues have been
studied by mutational analysis and are believed to interact with
specific bases within the TyrR boxes (11, 27). The
alanine-substituted mutants HA494 and TA495 have previously been
constructed, and it has been shown that HA494 was completely unable to
repress the tyrP promoter, while TA495 was only partially
defective in repression (11). In order to study the effects
of these substitutions in vitro, TyrR proteins with these changes were
purified as N-terminal fusions with oligohistidine domains by using
pET15b as the expression vector (for details, see Materials and
Methods). As the N-terminal domain of TyrR is primarily involved in
interactions with RNA polymerase in transcription activation (25,
26), the histidine tag at the N terminus of TyrR is not expected
to affect the DNA-binding function of the protein. The mutant proteins
with oligohistidine domains were therefore used directly in mobility
gel shift assays without protease cleavage.
In this experiment, the 151-bp tyrP operator fragment
described in the previous section was used. Approximately 0.5 pmol of the end-labelled fragment was incubated with various amounts of each of
the mutant TyrR proteins as well as the wild-type TyrR protein in the
presence of tyrosine and ATP. Under this assay condition, the TyrR
proteins are expected to form hexamers in solution (14, 22).
With the wild-type TyrR protein, the amount of the retarded band
representing the TyrR-DNA complex increases as the TyrR concentration
is increased (Fig. 2). Judged by the concentration of the wild-type TyrR, at which half of the total DNA is
present in the retarded complex, the dissociation constant is estimated
to be approximately 200 nM. For the mutant protein HA494, no retarded
band was observed at a concentration of 700 nM (Fig. 2). Increasing the
protein concentration to 2 µM did not improve the specific binding,
indicating that the affinity of TyrR protein for the tyrP
operator was lost when histidine-494 was replaced by alanine. For the
mutant protein TA495, the specific binding affinity for the
tyrP operator was only slightly decreased (Fig. 2). The
alanine substitution at position 495 caused an increase in the
dissociation constant of TyrR from 200 nM to slightly less than 300 nM.
These in vitro results are in agreement with the previous in vivo
observation that histidine-494 plays a critical role in DNA binding
whereas the involvement of threonine-495 in DNA binding is less
important (11).
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FIG. 2.
Gel shift assay. The gel shift assay was carried out by
using the 151-bp tyrP fragment in the presence of the
wild-type TyrR protein, mutant protein HA494, or mutant protein TA495.
Various concentrations of the proteins are shown above the
autoradiograms.
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Rationale for missing contact probing with the mutant TyrR protein
TA495.
Missing contact probing has been successfully used in
determining specific amino acid-base contacts (8, 9). In
this method, an amino acid residue which is known to contact DNA is replaced with a smaller amino acid such as alanine. Unlike the wild-type protein, the alanine-substituted protein is unable to discriminate between the wild-type DNA and the DNA with a base missing
at the site contacted by the original amino acid residue. Therefore a
comparison of the footprinting patterns generated in the presence of
the wild-type protein and the mutant protein may allow the
identification of an individual interaction between a specific base and
an amino acid residue.
A critical aspect of this method is that the alanine mutant must still
retain its ability to bind DNA even though the formation of the
protein-DNA complex requires an increased concentration of this
protein. As shown in the previous section, unlike the mutant protein
HA494, which was completely inactive in DNA binding, the mutant protein
TA495, in spite of a weakened binding affinity for the tyrP
operator, was still able to form a protein-DNA complex. Therefore TA495
was chosen for use in the missing contact probing experiments.
Depurination probing.
The missing contact experiments
were carried out as described above. Results from the
depurination (A+G) analysis are shown in Fig.
3. A visual comparison of the
footprinting patterns generated in the presence of the wild-type and
mutant proteins identified three possible contact sites. These are the
adenine residues at the +5 positions of both the strong and weak TyrR
boxes in the bottom strand as well as the adenine residue at the 5
position of the weak box in the top strand.
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FIG. 3.
Depurination (A+G) analysis using the wild-type TyrR
protein and the mutant TyrR protein TA495. Designations are as
described in the legend to Fig. 1. Arrows indicate positions where
interference is significant with the wild-type TyrR protein but is
weaker or has disappeared with the mutant protein TA495. A summary of
the data is shown below the autoradiograms.
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To obtain a better resolution of the results, densitometric scans of
the autoradiographs were performed. The scan data shown in the boxes in
Fig. 4A and B are focused
on a small region including positions +9 and +3 of the bottom strand in
the weak box. In this region, only the purine residues G--AAA--G can be
seen. The fragment derived from the deletion of guanine at position 8 shows a similar displacement to the unbound fraction in both the
wild-type (Fig. 4A) and mutant (Fig. 4B) proteins. Although the
displacement is less, the same consistent effect is seen with the
fragment arising from depurination at position +4. The adenine at
position +3 has been shown not to be important for TyrR binding
(11), and this is confirmed by inspection of the results
involving both the wild-type and mutant proteins, in which there is an
almost equal distribution of DNA in both the bound and free fractions.
If one now considers the adenine at position +5, a quite different
picture emerges. Whereas the binding of the wild-type protein to the
DNA is severely affected by depurination at this position, in the case
of the mutant, the same fragment is equally distributed between the
bound and free fractions. These results suggest a possible specific interaction between threonine-495 of the TyrR protein and the adenine
at position +5 of in the weak box.
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FIG. 4.
Densitometric scans of the data shown in Fig. 3. Dark
and light lines, scans of the protein-DNA complex from the B lanes and
of the free DNA from the UB lanes, respectively. Arrows indicate
positions where interference is seen for wild-type TyrR but is weaker
or has disappeared for TA495. The locations of the strong and weak TyrR
boxes are indicated. (A) Samples from the depurination reactions
carried out in the presence of wild-type TyrR protein and the
tyrP fragment labelled in the bottom strand. (B) Samples
from the depurination reactions carried out in the presence of the
TA495 protein and the tyrP fragment labelled in the bottom
strand. (C) Samples from the depurination reactions carried out in the
presence of wild-type TyrR protein and the tyrP fragment
labelled in the top strand. (D) Samples from the depurination reactions
carried out in the presence of the TA495 protein and the
tyrP fragment labelled in the top strand.
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The data from this scan also show significant but less-marked changes
at position +5 in the bottom strand of the strong box (Fig. 4A and B)
and at position 5 in the top strand of the weak box (Fig. 4C and D).
The lesser effects might be considered a result of the involvement of
these particular bases in either making more than one contact with the
protein or directing a local DNA distortion. The position 5 in the
bottom strand of the strong box is occupied by a guanine residue and
shows no effect (Fig. 4A and B).
Depyrimidation probing.
The results of depyrimidation
probing and the data from densitometric scans of the
autoradiographs are shown in Fig. 5 and 6, respectively. In the bottom strand,
depyrimidation of the thymine residue at position 5 of the weak box
has a greater effect on the binding of the wild-type protein than on
the binding of the mutant protein TA495 (Fig. 6A and B). This indicates
that unlike the wild-type protein, the mutant TA495 protein cannot
discriminate between the presence and absence of a thymine at this
position. In the top strand, deletion of thymine at the +5 positions of both the strong and weak boxes also resulted in a similar effect, but
the effect of missing contact is shown to be more remarkable in the
weak box than in the strong box (Fig. 6C and D).
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FIG. 5.
Depyrimidation (C+T) analysis using the wild-type TyrR
protein and the mutant TyrR protein TA495. Designations are as
described in the legend to Fig. 1.
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FIG. 6.
Densitometric scans of protein-DNA complexes (B lanes)
and free DNA (UB lanes) in Fig. 5. (A) Samples from the depyrimidation
reactions carried out in the presence of the wild-type TyrR protein and
the tyrP fragment labelled in the bottom strand. (B) Samples
from the depyrimidation reactions carried out in the presence of the
TA495 protein and the tyrP fragment labelled in the bottom
strand. (C) Samples from the depyrimidation reactions carried out in
the presence of the wild-type TyrR protein and the tyrP
fragment labelled in the top strand. (D) Samples from the
depyrimidation reactions carried out in the presence of the TA495
protein and the tyrP fragment labelled in the top strand.
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DISCUSSION |
Implications of the results from missing contact footprinting using
wild-type TyrR protein.
Previous studies of the tyrP
operator have involved site-directed mutagenesis of the strong TyrR box
and methylation and ethylation interference and
uracil substitution experiments to investigate the binding of the TyrR
protein to both boxes. The mutagenesis studies have implicated the
invariant (G · C)(C · G)8 symmetrical base pairs as
playing a critical role (11) (the nomenclature for the
symmetrical base pairs is as follows: the letters in the first set of
parentheses denote the base pair in the left arm of the palindrome, and
the letters in the second set of parentheses represent the base pair in
the right arm of the palindrome; the number indicates the symmetrical
positions). Results from these studies have also shown that the (T
· A)(A · T)7 symmetrical base pairs are of major importance in
TyrR binding (11). Methylation interference
studies have confirmed the importance of the guanine residues at the
±8 positions in both boxes and have indicated that the guanine
residues at the 5 position in the strong box and the 4 position in
the weak box are also necessary for TyrR binding (11).
Uracil substitution experiments have revealed important van der Waals
interactions with thymines at positions 7 and +5 of both boxes and
weaker interactions at the +7 positions of both boxes and at the 5
position of the weak box (11). Ethylation interference
experiments have indicated that contacts between the TyrR protein and
the phosphate backbone occur both at the ends and in the central
regions of the two boxes (11). The limitation of these
methods was their inability to detect interactions with adenine
residues via the major groove and to identify any interactions with
cytosine residues. The missing contact studies reported in this paper
overcome these difficulties. Although we cannot rule out the
possibility that some structural changes caused by the removal of
certain bases are responsible for differences in TyrR binding, it seems
likely, taking into account our previous genetic results, that the
majority of the bases in the palindromic arms of each of the TyrR boxes
are involved in contacts with the TyrR protein. In addition to
confirming previous observations, this work also shows that elimination
of cytosine residues at the ±8 positions from either box has a
major effect on binding between the TyrR protein and the DNA
(Fig. 1). Similarly, removal of adenine residues from their
positions within the palindromic arms of each of the boxes causes major
shifts of the tyrP fragments towards the unbound fraction
(Fig. 1). Since these effects were not seen with the previously
reported methylation interference studies, we must assume
that these interactions with adenine occur via the major groove at the
N7 or the NH6 functional group. Removal of the nonconsensus base C at
the 5 position of the strong box and at the 4 position of the weak
box has only slight effects on binding, as does the removal of the
consensus base T at the +6 position of the strong box (Fig. 1). At
first, the failure to observe a more significant effect at the 5
position in the top strand of the strong box was unexpected, since
mutational studies had suggested that the (C · G)(G · C)5
base pairs were an efficient replacement for the (A · T)(T
· A)5 base pairs at these symmetrical positions (11).
However, a strong effect is seen at 5 in the bottom strand. This may
imply that the importance of (C · G)(G · C)5 base pairs
shown by mutational studies could be due to a critical role that is
played by G at this position. Methylation interference
showed that the G at 5 in the strong box is a contact site
(11). The results from missing contact probing with the
mutant protein TA495 described in this paper suggest that the 5G in
the strong box is contacted by an amino acid residue(s) other than T495.
The strong TyrR boxes of various genes of the TyrR regulon contain
AT-rich sequences between their palindromic arms (18), and
it is of interest that, in the case of the strong box of
tyrP, removal of any of the four bottom-strand adenine
residues in this location has a significant effect on binding (Fig. 1).
No effect or interaction is seen with equivalent positions in the weak
box. A strong TyrR box can be bound either by a TyrR dimer or by a TyrR
hexamer, but a weak TyrR box can be bound by the hexamer only when a
strong box is nearby and on the same face of the helix. Ethylation
interference studies have already shown that the central region of the
strong box appears to be more intimately associated with the protein
than the corresponding region of the weak box (11). The
depurination studies confirm this important difference, with
depurination of the central region of the strong box, but not the weak
box, significantly affecting binding. Subtle differences can also be
seen in the relative effects at positions +4 and +5 of the strong and
weak boxes. In addition, there is a strong effect at 3 in the top
strand of the weak box which is not present in the strong box. Taken
together, these may indicate that although, in general, similar bases
in both boxes are involved in critical interactions with the TyrR
protein, subtle but important differences exist in the relationship of
the protein to each box.
Removal of bases from the +9 position of the strong box and the 9
position of the weak box had a major impact on TyrR binding (Fig. 1).
These two positions are located in the interior region of the operator,
and their removal may have an effect on the structure of the operator.
Previous studies involving ethylation interference (11) and fluorescent probes (5) have indicated
that base pairs at positions ±9 are in close proximity to the TyrR
protein, but mutational studies have suggested that these base
pairs play a less significant role in TyrR binding (11).
Deletion of bases immediately outside the palindromic arms of the
two boxes appears to facilitate binding of the TyrR protein to the
operator (Fig. 1A and B). Similar effects were also seen when the
internal bases +3T of the top strand of the weak box and 1G of the
bottom strand of the weak box were removed (Fig. 1A and B). In all
these cases we assume that the deletion alters DNA structure in a way
which enhances the binding affinity of TyrR, perhaps by increasing flexibility.
Specific interactions between threonine-494 of TyrR and base pairs
at positions ±5 of the TyrR boxes.
From the results of missing
contact probing with the mutant TyrR protein TA494, it appears that the
threonine at position 495 in TyrR makes specific contacts with adenine
and thymine residues at the ±5 positions in the TyrR boxes. The weak
box contains symmetrical base pairs (A · T)(T · A) at
positions ±5, whereas the strong box contains asymmetrical base pairs
(A · T)(G · C) at the equivalent positions.
Previous genetic studies have shown that changing the threonine residue
at position 494 into a serine causes a slight increase in the
DNA-binding activity of the TyrR protein (27), whereas an
alanine replacement at this position results in a significant reduction
in the binding activity (11). This indicates that it is the
hydroxyl group but not the methyl group of T495 that plays an important
role in DNA binding. Direct contacts can occur through hydrogen bonding
between the hydroxyl group of T494 and the N6 of the adenine or the O4
of thymine.
Youderian and coworkers have carried out extensive genetic studies to
investigate the importance of the threonine residue at position 81 of
the Trp repressor in DNA binding (6, 17). Their results have
suggested a direct contact between T81 of the Trp repressor and the
symmetrical base pairs (G · C)(C · G)3 and (A · T)(T · A)4 of the trp operator. In this case, it has
been proposed that the hydroxyl group of T81 forms a hydrogen bond with
the O4 group of the thymine residues at the ±4 positions and that the
methyl group makes a hydrophobic contact with the C-5 atom of the
cytosine residues at the ±3 positions (6).
A direct contact between a threonine residue in the DNA-binding domain
of a regulatory protein and A · T base pairs in an operator has
also been found in the PurR-DNA crystal structure (20). In
this complex, the O
of T15, which is the first residue in the
recognition helix, hydrogen bonds to the O4 of thymine at position 7'
via a water interface, and the O of T16 hydrogen bonds
simultaneously to the N6 of adenine and the O4 of thymine at position 6.
A model of specific interactions has been proposed for the invariant
(G · C)(G · C)8 base pairs, where the side chains of H494
and R484 of TyrR donate hydrogen bonds to the O6 and N7 of the guanine,
respectively (11). This proposal, together with the
assumption of the specific contact of T495 as described above, allows
the prediction of the orientation of the recognition helix of the TyrR
protein on the 22-bp palindromic sequence of the TyrR boxes. This
prediction positions the recognition helices of a TyrR dimer on the
operator in same orientation as those for the 
repressor
(12), the Cro repressor (3, 7), CAP
(19), and the 434 repressor (1) and in the
opposite orientation to those for the Lac repressor (15),
the PurR repressor (20), and the Tet repressor
(23).
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Australian Research
Council. J. S. Hwang is the recipient of an Overseas Postgraduate Research Scholarship from the Australian Government and a Malaysian Alumni Melbourne University Postgraduate Scholarship.
We thank Jing Hong An, Yan Jiang, and Thu Betteridge for technical
assistance and Barrie Davidson and coworkers for wild-type TyrR protein.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Melbourne, Parkville,
Victoria 3052, Australia. Phone: 61 3 9344 5679. Fax: 61 3 9347 1540. E-mail: aj.pittard{at}microbiology.unimelb.edu.au.
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Journal of Bacteriology, April 1999, p. 2338-2345, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
