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INTRODUCTION |
The tyrR gene of Haemophilus influenzae,
which was identified during the determination of the complete genomic
sequence of this organism (1), has been cloned and used as the basis of an expression system for the TyrR protein (2). The TyrR protein of
H. influenzae was shown to bind to operator DNA both
in vitro and in vivo. It is predicted that the
TyrR protein of H. influenzae controls a group of genes in
H. influenzae similar to those controlled by the TyrR
protein of Escherichia coli (2).
Limited trypsin digestion of the TyrR protein of E. coli
(513 amino acids) generated two stable subfragments, a 22-kDa
N-terminal domain and a 31-kDa central domain (3). The C-terminal
segment of E. coli TyrR was completely digested under
conditions of limited trypsin digestion. The central domain of the TyrR
protein of E. coli has a high degree of sequence similarity
to the analogous segments of a family of activators specific for the
54 form of RNA polymerase (4, 5). The central domain
also contains one or more segments that are important for the
self-association of TyrR monomers plus at least one ATP binding site
and a tyrosine binding site.
The ligand binding sites are important for regulating the interaction
of TyrR with its DNA targets. Tyrosine binds to the TyrR protein after
it associates with ATP. When the TyrR protein of E. coli
binds both ATP and tyrosine, there is an increase in operator affinity.
The binding of ATP alone has no effect on operator affinity (3, 6, 7).
When the TyrR protein of H. influenzae binds both ATP and
tyrosine, its operator binding ability is enhanced. However, one
observes a decrease in operator binding with this protein in the
presence of ATP alone (2).
The C terminus of the TyrR protein of E. coli contains a
helix-turn-helix motif, which is believed to be responsible for DNA binding (7). The TyrR protein of H. influenzae (318 amino
acids) has a high degree of sequence similarity to the central and
C-terminal domains of the TyrR protein of E. coli (2) but
lacks a counterpart to the N-terminal domain, a region that is critical
for positive regulation (8). Not surprisingly, the TyrR protein of
H. influenzae is unable to activate transcription from
promoters that are subject to stimulation by the TyrR protein of
E. coli (2).
Despite our current level of understanding of the TyrR protein of
E. coli and several related proteins in the NtrC
superfamily, little structural information is available for this group
of transcription factors. How these proteins are organized and how
their diverse regulatory functions are exerted remain unclear. Here we
report that the TyrR protein of H. influenzae can be
proteolytically cleaved at the boundary between two functional domains.
Conditions were established for obtaining each species in pure and
native form. The two separated domains reassociate to generate a
species whose properties were similar to full-length TyrR protein.
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EXPERIMENTAL PROCEDURES |
Strains, Bacteriophages, and Plasmids
The bacterial strains, bacteriophages, and plasmids used in this
study are described in Table I.
Materials
Phosphocellulose P-11, DEAE-Sepharose CL-6B, Sephacryl S-200,
and hydroxylapatite were purchased from Whatman, Sigma Chemical Co.,
Pharmacia Biotech, and Bio-Rad, respectively.
Buffer A contained 50 mM Tris (pH 7.5) with 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
Buffer B contained 50 mM
Na2HPO4/NaH2PO4 (pH
6.5) with 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
Buffer C contained 50 mM
Na2HPO4/NaH2PO4 (pH
7.5) with 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
Overexpression and Purification of TyrR Protein of H. influenzae
Plasmid pZZ, a derivative of pET-3a containing the coding
sequence of the TyrR protein of H. influenzae under the
control of the T7 promoter, was used to overexpress the TyrR protein
(2). The procedures for overexpression and purification were modified as described below.
Overexpression--
Plasmid pZZ was introduced into
E. coli BL21(DE3), selecting ampicillin resistance. A
single colony was picked and grown in 10 ml of L broth supplemented
with 50 µg/ml ampicillin overnight at 37 °C with shaking. The
saturated culture was then transferred into 1 liter of L broth
supplemented with 50 µg/ml ampicillin. In a 4-liter Erlenmeyer flask,
the culture was incubated at 37 °C on a rotary shaker. After the
A600 of the culture reached 1.0, isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM, and the culture was incubated
for another 4 h at 37 °C. Cells were harvested by
centrifugation at 5,000 rpm for 20 min (Sorvall RC2B).
Purification--
All steps were carried out at 4 °C. Cell
paste (40 g) was suspended in Buffer A (200 ml). The cells were broken
by three passes through a French pressure cell (Aminco) operated at
1,500 p.s.i. Cell debris was removed by centrifugation at 17,000 rpm
(J21C; Beckman) for 60 min. The supernatant was loaded directly onto a
DEAE-Sepharose CL-6B column (3 × 20 cm). The column was washed with Buffer A until the A280 was less than 0.1 and then developed using a linear gradient from 0 to 1.0 M
NaCl in 500 ml of Buffer A. Individual fractions were analyzed by
SDS-PAGE.1 The TyrR protein
eluted at a salt concentration of about 0.4 M. Fractions
containing the TyrR protein were pooled. At this stage, the TyrR
protein was about 70% pure (Fig. 1, lane 3). The pooled
protein fractions from previous chromatography were treated with
ammonium sulfate (50% saturation). The resulting protein pellet was
dissolved in 40 ml of Buffer B and dialyzed against Buffer B (1 liter,
three changes). The dialyzed protein was loaded onto a phosphocellulose
P-11 column (3 × 20 cm) that was pre-equilibrated with Buffer B. The column was washed with Buffer B until the
A280 fell below 0.1. Protein was eluted using a
0.1-1.0 M linear gradient of NaCl in 500 ml of Buffer B. TyrR was detected by SDS-PAGE, and the peak fractions were pooled. At
this stage, the purity of the TyrR protein was around 90% (Fig. 1,
lane 4). The pooled protein fractions were precipitated with
ammonium sulfate (50% saturation), and the resulting protein pellet
was dissolved in 30 ml of Buffer C. The dissolved protein was then
loaded directly onto a hydroxylapatite (Bio-Rad) column (3 × 15 cm) that was pre-equilibrated with Buffer C. The column was washed with
Buffer C and developed using a 0-1.0 M phosphate gradient.
The peak protein fractions, which were identified by SDS-PAGE, were
pooled. The final purity of the TyrR preparation was about 97% (Fig.
1, lane 5).
Purification of DNA Binding Domain
Sufficient urea was added to 15 mg of proteolytically nicked
TyrR (5 ml) to bring the concentration to 4 M (in Buffer C,
pH 7.5). The sample was then loaded onto a Sephacryl S-200 (high resolution; Pharmacia) gel filtration column (2.8 × 120 cm).
Buffer C containing 4 M urea was used as the mobile phase.
This procedure generated two well-separated peptide peaks of 28 and 8 kDa, respectively (Fig. 2B). The 28-kDa fragment was
discarded. The 8-kDa fragment, which contained the DNA binding domain,
was renatured by dialysis against Buffer C (five buffer changes).
Overexpression and Purification of the 28-kDa N-terminal
Fragment
Plasmid construction pZZ257, which is identical to pZZ, except
that it encodes only the first 257 amino acids of the TyrR protein of
H. influenzae, was constructed by introducing two
consecutive UAA (ochre) codons after amino acid 257 and deleting the
remainder of the TyrR gene. A polymerase chain reaction method was used to synthesize the desired DNA fragment. The fragment was inserted into
pET3a that had been cleaved with NdeI and BamHI
as described previously (2).
Overexpression--
pZZ257 was introduced into E. coli BL21(DE3), selecting for ampicillin resistance. To
overexpress the 28-kDa fragment, a single colony was inoculated into 1 liter of L broth supplemented with 50 µg/ml ampicillin. The culture
was harvested by centrifugation after the culture was grown at 37 °C
on a rotary shaker for 16 h.
Purification--
All steps were carried out at 4 °C. Cell
paste (15 g) was suspended in 50 ml of Buffer A. Cells were broken by
three passes through a French pressure cell (Aminco) at 1,500 p.s.i.
and then centrifuged at 15,000 rpm for 60 min. The pellet was
discarded, and streptomycin was added to the supernatant to a final
concentration of 1%. The resulting solution was clarified by
centrifugation at 12,000 rpm for 30 min. The pellet was discarded. The
supernatant from the previous step was loaded directly onto a
DEAE-Sepharose CL-6B column (3 × 20 cm) that had been
pre-equilibrated with Buffer A. The flowthrough material containing the
28-kDa fragment was pooled. About 95% of the desired protein was
recovered after this step. The 28-kDa fragment constituted about 80%
of the total protein recovered (Fig. 3, lane 4). Pooled
fractions from the previous step were treated with ammonium sulfate
(final concentration, 50% saturation at 25 °C). The resulting
precipitate was dissolved in 30 ml of Buffer B and dialyzed against 1 liter of Buffer B (three changes). The sample was loaded onto a
Phosphocellulose P-11 column (3 × 20 cm). The flowthrough
fractions were collected. About 85% of the total 28-kDa fragment was
recovered after this step, with a purity of about 90% (Fig. 3,
lane 5). The 28-kDa species was precipitated by 50%
saturated ammonium sulfate. The protein pellet was redissolved in 30 ml
of Buffer C and dialyzed against Buffer C (l liter, three changes). The
dialyzed protein was then loaded onto a hydroxylapatite (Bio-Rad)
column (3 × 10 cm). The column was washed with Buffer C until the
A280 fell below 0.1. The column was then
developed by a 0-1.0 M linear phosphate gradient.
Fractions containing the 28-kDa species were identified by SDS-PAGE.
The protein purity was about 95% after this step (Fig. 3, lane
6). About 30 mg of purified fragment were obtained from each liter
of cell culture.
Reconstitution of a Protein Preparation with DNA Binding
Properties Similar to Nicked TyrR
Equimolar amounts of purified, renatured DNA binding fragment
(61-mer) were mixed with the purified 28-kDa fragment. The mixture was
stored at 4 °C for 24 h in Buffer C.
Operator Protection Assay
pUC-aroF is a derivative of pUC19 that carries a 318-bp
BamHI-EcoRI fragment containing the
aroF promoter-operator region. The aroF promoter
has three operators, one of which contains a RsaI site. On
complete digestion by RsaI, three fragments of 467, 627, and
1899 bp are produced. If the TyrR protein binds to the aroF
promoter, the RsaI site within one of the TyrR operators will be protected. As a result, the digestion products will consist of
two DNA fragments of 627 and 2366 bp. Operator protection is reflected
by the presence of a DNA fragment of 2366 bp (2). Appropriate dilutions
of TyrR protein or protein fragments and pUC-aroF (5 pmol) were mixed
in Buffer C in the presence of 0.5 mg/ml bovine serum albumin in a
volume of 20 µl. After incubation at 37 °C for 30 min, 2 µl of
10× New England Biolabs buffer 1 (10 mM bis-tris
propane·HCl, 10 mM MgCl2, 1 mM
dithiothreitol, pH 7.0) and 1 µl of RsaI (10,000 units/ml)
were added. After incubation at 37 °C for 3 h, the digestion
products were analyzed by horizontal gel electrophoresis on 1.1% agarose.
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RESULTS |
Proteolytic Nicking of Purified TyrR Protein--
TyrR protein of
H. influenzae exists as a homodimer in solution. Each
monomer has a molecular mass of 36 kDa (2). When purified TyrR protein
was stored at 4 °C for 1 week, two additional species of about 28 and 8 kDa, respectively, became detectable (data not shown). After 3 weeks, more than 95% of the TyrR protein had undergone cleavage (Fig.
1, lane 6). Further incubation
at 4 °C for 2 more weeks did not lead to further breakdown (data not
shown). Sequential Edman degradation analysis showed that the
N-terminal amino acid sequence of the 28-kDa fragment was TISKFN, which
exactly matches the N terminus of the mature TyrR protein of H. influenzae (2). The N-terminal amino acid sequence of the 8-kDa
fragment was SAVISL, which matches that of residues 258-273 of the
TyrR protein of H. influenzae. Protease inhibitors such as
phenylmethylsulfonyl fluoride had no effect on the cleavage of TyrR.
Ligands of TyrR, namely
-S-ATP and L-tyrosine, altered the rate of TyrR cleavage. In the presence of 1 mM
-S-ATP, a nonhydrolyzable analog of ATP, the rate of cleavage of
TyrR was accelerated by 50%. L-tyrosine (0.3 mM) slowed the rate of cleavage by 30% (data not shown).
To investigate whether the TyrR protein of H. influenzae is
capable of self-cleavage, this protein was purified according to a
previously described protocol (2). The TyrR protein purified in the
alternative fashion also generated the same two fragments after
incubation at 4 °C.

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Fig. 1.
TyrR protein of H. influenzae:
purification and proteolytic cleavage. The TyrR protein and its
cleavage products were analyzed by 10% SDS-PAGE. The various species
were visualized by staining with Coomassie Blue. Arrows and
numbers refer to molecular masses. Lane 1, whole
cell lysate; lane 2, supernatant after French press and
centrifugation; lane 3, TyrR after passage through
DEAE-Sepharose CL-6B; lane 4, TyrR after passage over a
phosphocellulose P-11 column (sample missing); lane 5, TyrR
after hydroxylapatite column purification; lane 6, TyrR
incubated at 4 °C for 3 weeks.
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Isolation of 28- and 8-kDa Fragments--
Nicked TyrR was
indistinguishable from full-length TyrR in its ability to bind to the
aroF operator (Table I). To
study the operator binding ability of the 28- and 8-kDa fragments in
more detail, various approaches to the separation of these two
fragments were explored. Ion exchange chromatography on either
DEAE-Sepharose CL-6B or phosphocellulose P-11 matrices failed to
separate the two fragments. Gel filtration chromatography on Sephacryl
S-200 at neutral pH was also evaluated. When undenatured nicked TyrR was loaded, the two fragments always eluted together (Fig.
2A). A similar result was
obtained when gel filtration was carried out in 3 M NaCl
(data not shown). However, if nicked TyrR was pretreated in 4 M urea, and Buffer C containing 4 M urea was
used as the mobile phase for gel filtration chromatography, the two fragments could be separated (Fig. 2B). The 28-kDa fragment
eluted first, as expected; the 8-kDa fragment emerged later. In
attempts to renature the two fragments, fractions containing each
fragment were pooled and dialyzed against Buffer C. The 8-kDa fragment remained in solution after dialysis, whereas the 28-kDa fragment precipitated when the urea was removed.

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Fig. 2.
Separation of 8- and 28-kDa fragments.
Samples were taken from the column (Sephacryl S-200; 2 × 130 cm)
fractions (each fraction, 2.5 ml) and analyzed by 10% SDS-PAGE.
Peptides were visualized by staining with Coomassie Blue.
Numbers at the top refer to fraction numbers.
Arrows and numbers refer to the molecular masses.
A, separation under nondenaturing conditions. B,
separation in the presence of 4 M urea. C,
refractionation of reconstituted 8- and 28-kDa fragments in 50 mM phosphate buffer, pH 7.5.
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Overexpression and Purification of the 28-kDa Species--
To
obtain the 28-kDa fragment in native form, a truncated gene that
encoded amino acids 1-257 of TyrR was constructed. The resulting
construct led to high-level accumulation of the 28-kDa fragment in an
expression system based on the T7 promoter. Of special interest was the
fact that this species was produced in E. coli BL21(DE3) in
the absence of induction. The basis of this observation is not understood.
After the cells were broken, the 28-kDa fragment remained in soluble
form (Fig. 3, lane 1) and was
readily purified. Streptomycin precipitation of cellular nucleic acids
failed to co-precipitate the 28-kDa species (Fig. 3, lane
3), which is consistent with the absence of a DNA binding motif on
this fragment. Ion exchange matrices such as DEAE-Sepharose CL-6B and
phosphocellulose P-11 failed to bind the 28-kDa domain under conditions
that led to the retention of full-length TyrR (Fig. 3, lanes
4 and 5), which is consistent with a change in pI
caused by the removal of the DNA binding motif. However, the 28-kDa
species, like full-length TyrR, was retained effectively by
hydroxylapatite (Fig. 3, lane 6).

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Fig. 3.
Purification of the 28-kDa fragment.
After overexpression, the samples were analyzed by 10% SDS-PAGE.
Visualization was achieved by staining with Coomassie Blue. Lane
1, molecular mass standard; lane 2, supernatant after
French press and centrifugation; lane 3, supernatant after
treatment with streptomycin; lane 4, flowthrough material
from the DEAE-Sepharose CL-6B column; lane 5, flowthrough
material from the P-11 column; lane 6, the 28-kDa fragment
after hydroxylapatite column chromatography.
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Properties of the Purified 28-kDa Species--
To investigate
whether the 28-kDa form of TyrR had a secondary and tertiary structure
similar to that of the full-length TyrR protein, two experiments were
carried out. First, circular dichroism studies of the purified 28-kDa
fragment at different temperatures showed that this species had the
same heat stability as the full-length H. influenzae TyrR in
terms of
-helical content (Fig. 4). It was possible to estimate the
-helical content of each species at
different temperatures from the height of the negative peak at 220 nm.
Because the temperature at which the
-helical content was
half-maximal was almost identical for both the full-length protein and
the 28-kDa fragment, it is concluded that each species has the same
heat stability (Fig. 4). Second, the purified 28-kDa species was shown
to form heterodimers with full-length TyrR. This abolished the operator
binding ability of the full-length protein (Fig.
5).

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Fig. 4.
CD spectra of TyrR and the 28-kDa
fragment. CD spectra were determined on a JASCO spectropolarimeter
(J600). The buffer was 50 mM phosphate buffer (pH 7.5).
Numbers on the curve are the temperature ( °C) at which
the CD spectrum was taken. Wavelength range is indicated at the
bottom. A, full-length TyrR. B, the
28-kDa species.
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Fig. 5.
Heterodimer formation between full-length
TyrR and the 28-kDa fragment. Operator protection assays were
carried out as described under "Experimental Procedures."
Full-length TyrR (5 µM) afforded full protection of the
aroF operator (lane 2). This concentration of
TyrR was present in all additional reaction mixtures. Progressively
increasing concentrations of the purified 28-kDa fragment were added to
the reaction mixtures as follows: 2.5 µM, lane
3; 5 µM, lane 4; 10 µM,
lane 5; 15 µM, lane 6; 20 µM, lane 7. Lane 1 was the x174
HaeIII DNA standard, and the arrow with number
indicates the length (bp) of each band. The appearance of a DNA band of
2366 base pairs is indicative of full operator protection (2).
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In Vitro Reconstitution of the 8- and 28-kDa Species--
To
confirm that the 28-kDa species can physically associate with the 8-kDa
fragment in vitro, the purified 28-kDa species was mixed
with an equimolar amount of the purified 8-kDa species. After
incubation at 37 °C for 30 min, the mixture was subjected to native
Sephacryl S-200 gel filtration chromatography (3 × 120 cm; pH
7.5). It was found that the elution of the 8-kDa species coincided with
that of the 28-kDa species (Fig. 2C). This result supports
the idea that these two fragments can associate in
vitro.
Operator Binding Properties of the 8-kDa Domain, the 28-kDa Domain,
Nicked TyrR, and Reconstituted Mixtures--
The full-length TyrR
protein of H. influenzae efficiently binds to a target
within the aroF operator. DNA binding ability is inhibited
by
-S-ATP and L-tyrosine (2). Proteolytically nicked
TyrR was identical to uncleaved TyrR in operator binding ability and
response to cofactors (Fig.
6A; Table I). The purified 28-kDa fragment failed to bind to the aroF operator (Fig.
6B). The renatured 8-kDa fragment, which contains the DNA
binding motif of the TyrR protein, engaged the aroF operator
with the same apparent affinity as full-length TyrR. The binding of the
8-kDa fragment to operator was unaffected by
-S-ATP and
L-tyrosine (Fig. 6C; Table
II). When the 28-kDa fragment was added
to a mixture containing the 8-kDa fragment, the operator binding
properties were unaffected, but this mixture now responded to
-S-ATP
and L-tyrosine by binding to the operator in a less
effective manner (Fig. 6D).

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Fig. 6.
Operator protection assays. For each
assay, TyrR or fragments (5 µM) and the amount of
operator (5 µg) were kept constant. -S-ATP was added to each
reaction as follows: 0 mM, lane 1; 0.25 mM, lane 2; 0.50 mM, lane
3; 0.75 mM, lane 4; 1.0 mM,
lane 5; 1.25 mM, lane 6. Lane
0 was the x174 HaeIII DNA standard, and
arrows with numbers refer to the corresponding band DNA size
(bp). A, nicked TyrR. B, the purified and
renatured 8-kDa fragment. C, the purified 28-kDa species.
D, the reconstituted 8- and 28-kDa species. For a more
complete description of this assay, see "Experimental
Procedures."
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Table II
Operator binding constants for TyrR of E. coli, TyrR of H. influenzae
and its fragments
The association constants (Ka,
µM3) were estimated from operator protection
assays (Fig. 6). Half-maximal protection was estimated from the
condition when the 2366- and 1899-bp bands were of equal intensity.
Calculation of Ka was as described elsewhere (S. Zhao and R. L. Somerville, manuscript in preparation). -S-ATP was
present at 1 mM, and L-tyrosine was present at
0.3 mM.
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Association In Vivo between the 28- and 8-kDa Species--
The
ability of the separated fragments of TyrR to reassociate in
vitro raised the question of whether a similar form of
reconstitution might occur in vivo. To address this
possibility, we used a sensitive aroF reporter system based
on the
(tyrR) strain SP1313(
JC1) (11). One or more
plasmids that encode the TyrR protein segments of interest were
introduced into this strain. In the absence of TyrR, reporter enzyme
levels were strongly elevated (1,500 Miller units; Table
III). When either the E. coli
or H. influenzae TyrR proteins were synthesized, there was a
drop of about 30-fold in reporter enzyme levels to 50-60 Miller units.
When either the 8-kDa species or the 28-kDa species was expressed,
essentially no repression was observed. However, when both the 28- and
8-kDa species were expressed within the same cell through the use of compatible plasmid constructs, there was clear-cut evidence for repression. The reporter enzyme levels were reduced by a factor of 3.5 from 1,500 to 420 Miller units. To explore the structural specificity
of in vivo reassociation, plasmid constructs encoding the
8-kDa species with (His)6 extensions at either the
N-terminal or C-terminal ends were evaluated. Significant reductions in
the level of reporter enzyme were not observed in either case, even when the levels of the histidine-tagged species were raised via isopropyl-1-thio-
-D-galactopyranoside induction.
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Table III
-Galactosidase levels in E. coli strains carrying different TyrR
species
Cells were grown in minimal ampicillin (50 µg/ml)-tyrosine (0.5 mM) medium. The values are reported in Miller's units (9).
Each value shown above is the result of at least three independent
assays.
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It is concluded that reconstitution of operator-binding forms of TyrR
can occur in vivo. However, either this process is
inefficient or the reconstituted species are subject to degradation or
turnover, thereby preventing reductions in promoter activity to control (TyrR+) levels.
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DISCUSSION |
The results presented here suggest that the TyrR protein of
H. influenzae is organized in the form of two domains of 28 and 8 kDa. The two species remain stable in solution and readily
reassociate, indicating a distinct domain structure for the TyrR
protein of H. influenzae and a strong interaction between
the two domains. The fact that the TyrR protein of H. influenzae can be resolved into two separate functionally active
domains provides new insight into the structure-function relationships
of this protein. Because the TyrR protein of H. influenzae
bears significant sequence similarity to the TyrR protein of E. coli and the other members of the NtrC superfamily of
transcription factors, studies of the TyrR protein of H. influenzae have the potential to contribute to our understanding of structure-function relationships of other members of the NtrC superfamily of transcription factors.
Proteolytic cleavage occurred without adding any additional reagents to
the purified preparations of the TyrR protein. The basis of this
cleavage is not understood. Two possibilities can be proposed. First,
the cleavage could be catalyzed by a protease of E. coli.
This hypothetical protease, which is present in trace amounts, may have
been co-purified with the TyrR protein of H. influenzae by
each of the two methods that were used. If this were the case, the
enzyme that cleaves the TyrR protein must be a currently unknown
protease of E. coli, because the cleavage site in TyrR,
which is between a glutamine-serine peptide bond, matches no currently
known protease target site in E. coli. Second, the cleavage
may be a self-digestion process. If this were the case, the cleavage of
the TyrR protein of H. influenzae could have important
regulatory implications. For example, it could be either a way to
regulate the amount of TyrR protein itself (via turnover) or a way to
generate a separate functionally active form of TyrR that has a
different role than undamaged TyrR in vivo. The fact that
none of the common protease inhibitors affected the cleavage process
but known cofactors of the TyrR protein did alter the rate of cleavage
is consistent with a self-cleavage mechanism. However, none of the
available facts conclusively prove a self-cleavage mechanism. For
example, the effects of cofactors on proteolytic cleavage could be
explained by assuming that the cofactors alter the conformation of TyrR
so as to either facilitate or hinder proteolytic cleavage. Similarly,
the ineffectiveness of common protease inhibitors could indicate that
the enzyme that cleaved TyrR is not a serine protease. Additional
detailed studies need to be carried out to elucidate the mechanism of
the cleavage process.
Most prokaryotic repressors must form dimers to bind to operator DNA
targets. For the TyrR protein of E. coli, the central domain
is responsible for dimerization and ligand binding, whereas the C
terminus of TyrR containing the helix-turn-helix motif is mainly
responsible for DNA recognition (6). Given the high degree of sequence
similarity between the TyrR proteins of E. coli and H. influenzae, it is reasonable to suppose that the TyrR protein of
H. influenzae is also composed of a dimerization/ligand binding domain (the 28-kDa domain) and a DNA binding domain (8-kDa domain). The finding that the 8-kDa domain alone can bind to an operator target with an affinity similar to that of the full-length TyrR protein of H. influenzae was unexpected. Two possible
explanations can be considered. First, the dimerization of the TyrR
protein of H. influenzae may not be solely determined by the
28-kDa domain. The 8-kDa domain containing the helix-turn-helix DNA
binding motif may be able to dimerize on its own. However, the
structural elements responsible for the dimerization within the 8-kDa
domain need to be defined. Second, it is possible that the 8-kDa
species can bind to target DNA as a monomer. This is less likely,
because the binding of full-length TyrR to operator was inhibited by
the 28-kDa fragment, both in vitro and in vivo,
presumably as a consequence of the formation of inactive heterodimers
(Fig. 5; Table III). This observation strongly supports the notion that
dimer formation precedes the binding of the TyrR protein to its DNA target.
The binding of cofactors has long been known to alter the DNA binding
ability of TyrR protein (3). How the signal generated by the binding of
cofactors in the central domain of TyrR is transmitted to the DNA
binding domain to regulate transcription remains unclear. The ability
of the 28- and 8-kDa domains to reassociate to yield a species of
TyrR that can mimic the cofactor response of full-length TyrR promises
to provide a useful tool for addressing this question.