Journal of Bacteriology, November 2000, p. 6503-6508, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transcription Initiation-Defective Forms of
54 That Differ in Ability To Function with a
Heteroduplex DNA Template
Mary T.
Kelly,
John A.
Ferguson III, and
Timothy R.
Hoover*
Department of Microbiology, University of
Georgia, Athens, Georgia 30602
Received 14 August 2000/Accepted 6 September 2000
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ABSTRACT |
Transcription by
54-RNA polymerase holoenzyme
requires an activator that catalyzes isomerization of the closed
promoter complex to an open complex. We examined mutant forms of
Salmonella enterica serovar Typhimurium
54
that were defective in transcription initiation but retained core RNA
polymerase- and promoter-binding activities. Four of the mutant
proteins allowed activator-independent transcription from a
heteroduplex DNA template. One of these mutant proteins, L124P V148A,
had substitutions in a sequence that had not been shown previously to
participate in the prevention of activator-independent transcription.
The remaining mutants did not allow efficient activator-independent transcription from the heteroduplex DNA template and had substitutions within a conserved 20-amino-acid segment (Leu-179 to Leu-199), suggesting a role for this sequence in transcription initiation.
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TEXT |
Bacterial RNA polymerase holoenzyme
consists of a core enzyme (
2
') and a dissociable
subunit. Bacteria often contain multiple
factors with
individual sequence specificities that direct holoenzyme to different
classes of promoters (22).
54 is a
distinctive bacterial sigma factor that does not share significant sequence homology with other sigma factors. Transcription initiation by
54-RNA polymerase holoenzyme
(
54-holoenzyme) occurs through a distinct
mechanism that involves an activator or enhancer-binding protein
(3).
54-Holoenzyme binds to promoters
to form stable closed complexes. Isomerization of these closed
complexes to transcriptionally active open complexes requires an
enhancer-binding protein, which generally binds to sites upstream of
the promoter and contacts
54-holoenzyme through DNA
looping. This isomerization requires nucleoside triphosphate hydrolysis
by the enhancer-binding protein.
Deletion analysis of
54 has revealed that the protein
consists of at least three functional regions (7, 16, 26, 32, 33). The 50 amino-terminal residues of
54
constitute region I, which is rich in glutamine and leucine residues and plays a direct role in transcriptional activation (16,
17). Region II of
54 from enteric bacteria, which
extends from residue 50 to residue 120 and is highly acidic, appears to
influence the rate of open complex formation, as well as suppress
nonspecific DNA binding by holoenzyme (4, 32). Region III
consists of 360 carboxy-terminal residues and contains determinants for
binding of both core RNA polymerase and promoter DNA (7, 27, 28,
33). Additional functions for region III are likely, as some
substitutions in this region disrupt one or more steps in transcription
initiation following initial promoter recognition (11, 18).
When region I of
54 is deleted, the resulting holoenzyme
assumes a conformation believed to reflect polymerase isomerization (6). This isomerization is inhibited by a peptide containing the region I sequence, indicating that region I can elicit its effects
on holoenzyme in trans (6, 14). Holoenzyme
containing
54 with region I deleted fails to initiate
transcription under normal conditions but can initiate transcription
independently of enhancer-binding protein from a transiently melted or
premelted DNA template (6, 29). These studies suggest that
region I inhibits isomerization of the holoenzyme to a form that can
stably associate with single-stranded DNA (4). Productive
interaction with the enhancer-binding protein apparently relieves this
inhibition and permits transcription initiation in a reaction that
requires nucleotide hydrolysis by the enhancer-binding protein
(14).
We previously isolated several mutant forms of Salmonella
enterica serovar Typhimurium
54 that were defective
in transcription initiation but retained core- and DNA-binding
activities (18). Further analysis of these mutant
54 proteins, the results of which are presented here,
revealed that certain amino acid substitutions in regions I and III
allowed the holoenzyme to initiate transcription from a heteroduplex
DNA template in the absence of enhancer-binding protein, suggesting that sequences within these regions influence isomerization of the
holoenzyme. The remaining
54 mutant proteins had amino
acid substitutions within region III and represented a second class of
mutants that did not allow efficient transcription from the
heteroduplex template in the absence of enhancer-binding protein. The
strains and plasmids used in this study are described in Table
1.
L179P retains core- and promoter-binding activities.
Several
mutant forms of
54 were isolated previously that failed
to function normally at the
54-dependent
glnA promoter (glnAp2) but retained
promoter-binding activities (20). We included here a new
54 mutant protein, L179P, that was identified using the
same screening procedure following random mutagenesis of a
plasmid-borne copy of ntrA (encodes
54) with
the Epicurian Coli XL1-Red strain (Stratagene, La Jolla, Calif.). We
previously used a gel mobility shift assay with labeled oligonucleotides that corresponded to the
9 to
29 region of the
Sinorhizobium meliloti nifH promoter to examine interactions between the mutant forms of
54 and core RNA polymerase,
as well as the binding of the mutant proteins to promoter sequences
(20). One oligonucleotide was double stranded over its
entire length (double-stranded probe), while in the second
oligonucleotide residues
11 to
9 of the template strand were single
stranded (fork junction probe). Guo and Gralla first demonstrated fork
junction DNA-binding activity of
54 using these probes
(15), and we subsequently found that the
54-holoenzyme bound the fork junction probe
significantly better than it did the double-stranded probe
(18).
We examined the binding of the L179P-holoenzyme to the fork junction
and double-stranded probes. As observed with the wild-type holoenzyme,
the L179P-holoenzyme shifted the fork junction probe more efficiently
than the double-stranded probe (Fig. 1).
L179P produced less of the holoenzyme-shifted species than wild-type
54, suggesting that this mutant protein had reduced
affinity for either core RNA polymerase or fork junction DNA. These gel
mobility shift data, however, demonstrate that like the other mutant
forms of
54 described previously (20), L179P
still binds the core and directs the holoenzyme to promoter sequences.

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FIG. 1.
Gel mobility shift assay with double-stranded and fork
junction probes. Binding of mutant forms of
54-holoenzyme to the S. meliloti nifH
promoter was analyzed by a gel mobility shift assay as previously
described (18). Two different DNA probes were used for the
gel mobility shift assays. One probe contained 21 bp of double-stranded
DNA that included residues 9 through 29 of the nifH
promoter. The second probe had 18 bp of double-stranded DNA, which
included residues 12 through 29 of the nifH promoter
plus a three-base 5' overhang of the template strand that corresponded
to residues 9 through 11. Binding reaction mixtures contained 5 nM
DNA probe and 6 µg of sonicated calf thymus DNA/ml along with 300 nM
core RNA polymerase (lanes 1 and 2), 600 nM hexahistidine-tagged
54 (lanes 3 and 4), 300 nM core plus 600 nM
hexahistidine-tagged 54 (lanes 5 and 6), and 300 nM core
plus 600 nM hexahistidine-tagged L179P (lanes 7 and 8). Core RNA
polymerase and histidine-tagged 54 proteins were
purified as previously described (20, 23). The
double-stranded probe was used in odd-numbered lanes, while the fork
junction probe was used in even-numbered lanes. The holoenzyme-shifted
(h) and 54-shifted ( ) species are indicated. Unbound
probes are not shown.
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Mutant forms of
54 can be cross-linked to DctD.
S. enterica serovar Typhimurium
54 can be
chemically cross-linked to the enhancer-binding protein S. meliloti C4-dicarboxylic acid transport protein D
(DctD), suggesting that
54 is a primary target for the
enhancer-binding protein (20). Consistent with this
hypothesis, mutant forms of DctD that failed to activate transcription
and cross-linked poorly to
54 have been isolated
(31). Further support of this hypothesis comes from the
observation that
54 bound to heteroduplex DNA in the
absence of core RNA polymerase undergoes a conformational change in a
reaction that requires enhancer-binding protein and nucleotide
hydrolysis (8).
We wished to determine if any of the
54 mutant proteins
were deficient in cross-linking to DctD and therefore altered in the ability to make productive contact with the enhancer-binding protein. Since the
54 mutant proteins were isolated originally
based on their failure to function with nitrogen regulatory protein C
(NtrC) at glnAp2 in vivo (18), we first verified
that these mutant proteins were also defective in functioning with DctD
in vivo. For these assays, we used DctD(
1-142), which
is a truncated, constitutively active form of the protein that lacks
the first 142 amino-terminal residues (21). We examined the
ability of the mutant forms of
54 to function with
DctD(
1-142) in activating transcription from a
plasmid-borne dctA'-'lacZ reporter gene in Escherichia coli strain YMC11, which otherwise lacks
54.
DctD-mediated transcriptional activation with the mutant forms of
54 ranged from 1 to 19% of the activity achieved with
wild-type
54 (Table 2).
These values were consistent with the activities observed for the
mutant
54 proteins with NtrC at glnAp2 in
vivo (18).
Each of the mutant forms of
54 was purified and examined
for the ability to cross-link to DctD(
1-142) using
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
as the cross-linking reagent as described previously (20).
All of the mutant
54 proteins cross-linked efficiently
to DctD(
1-142) (data not shown), indicating that none
of them were defective in gross interactions with this enhancer-binding
protein. Since the cross-linking assay provides only a qualitative
assessment of interactions between
54 and DctD, we
cannot rule out the possibility that some of the mutant proteins had
reduced affinities for DctD or that some of the substitutions in these
mutant proteins interfered with specific contacts between
54 and DctD required for transcriptional activation.
Certain mutant forms of
54 allow
activator-independent transcription.
We initially used an in vitro
transcription assay to examine the abilities of the
54
mutant proteins to support transcription from a supercoiled template that carried the S. enterica serovar Typhimurium
glnA promoter region. In this transcription assay, heparin
was added immediately before the nucleotides to prevent reinitiation
but allow transcription from stable open complexes. None of the
54 mutant proteins supported transcription under these
conditions, except the L46P mutant protein, which had given the highest
activity in vivo (Table 2). The level of transcript generated with the L46P mutant protein was ~5% of that generated with wild-type
54 (Fig. 2). Addition of
heparin to the transcription assay reaction after addition of the
nucleotides did not alter the results of the assays with the mutant
proteins (data not shown). These data confirmed our earlier in vivo
findings that these mutant
54 proteins were defective in
their function at glnAp2 (18).

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FIG. 2.
In vitro transcription with mutant forms of
54 from the glnA promoter regulatory region
on a supercoiled DNA template. Transcription assays were carried out as
described previously (24, 31). Assay reaction mixtures
contained 400 nM maltose-binding protein-NtrC, 20 mM carbamoyl
phosphate, 100 nM core RNA polymerase, and a 300 nM concentration of
either hexahistidine-tagged 54 (lane 1), L37P (lane 2),
L46P (lane 3), E32K G189V (lane 4), L124P V148A (lane 5), L179P (lane
6), L199P D231G (lane 7), or L333P (lane 8). The arrow indicates the
transcript from the glnA promoter.
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Previous studies demonstrated that when region I of
54
is deleted, the resulting holoenzyme can initiate transcription in the absence of enhancer-binding protein from a premelted, heteroduplex DNA
template (6). We used this linear, heteroduplex template to
determine if any of the mutant
54 proteins would allow
activator-independent transcription. The template spanned nucleotides
60 to +28 of the S. meliloti nifH promoter, and the DNA
strands from
10 to
1 were noncomplementary (6).
The wild-type
54-holoenzyme was only able to initiate
transcription from the heteroduplex template weakly (Fig. 3, lane
1). Likewise, holoenzymes formed with the
L179P mutant protein, the L199P D231G double-mutant protein, or the
E32K G189V double-mutant protein initiated transcription from the
heteroduplex template poorly. In contrast, holoenzymes containing L37P,
L46P, L333P, or the double mutation L124P V148A initiated transcription
efficiently from the heteroduplex template (Fig. 3, lanes 2 to 4 and
8). Amino acid substitutions in
54 that result in
activator-independent transcription have been described previously
(10, 11, 26, 29), with the substitutions in these mutant
proteins occurring primarily within a leucine-rich patch of region I
and a very limited number of sites in region III. In general, these
previous activator-independent mutant proteins retained significant
activity in vivo. In contrast, holoenzymes with L37P, L333P, and the
double mutation L124P V148A were severely impaired in the ability to
function in vivo and in that regard are more like region I deletion
mutant enzymes which do not support transcription initiation from
homoduplex DNA templates (8). This result is not surprising,
however, given that the mutant proteins in this study were chosen based
on their failure to function at glnAp2 in vivo
(18).

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FIG. 3.
In vitro transcription assays using a heteroduplex DNA
template. Transcription assays were carried out using a heteroduplex
DNA template that spanned residues 60 to +28 of the S. meliloti
nifH promoter and was noncomplementary between residues 10 and
1. The heteroduplex template was generated using two oligonucleotides
as previously described (6). Transcription assays were
conducted as previously described (6), except that 7.5 µCi
of [ -32P]CTP (3,000 Ci/mmol) was used to label the
transcripts. Reaction mixtures contained 100 nM core RNA polymerase,
300 nM hexahistidine-tagged 54 proteins, and 30 nM
heteroduplex DNA template. The 54 proteins assayed were
the wild type (lane 1) and the L37P (lane 2), L46P (lane 3), L333P
(lane 4), E32K G189A (lane 5), L179P (lane 6), L199P D231G (lane 7),
and L124P V148A (lane 8) mutant proteins. The arrow indicates the
28-base transcript.
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Alanine scanning mutagenesis of the region around Leu-333.
The
similarities between the L37P and L333P mutant proteins in the ability
to support activator-independent transcription (Fig. 3) and the reduced
affinity for fork junction DNA (18) prompted us to examine
conserved residues in the vicinity of Leu-333. Leu-333 lies within a
well-conserved region of
54 that was shown previously to
be cross-linked to DNA upon UV irradiation of
54-promoter complexes (5). It is also close
to a putative helix-turn-helix motif that has been implicated in
recognition of the
12 region of the promoter (12, 23).
When we compared the sequences of this portion of
54
proteins from 25 different bacteria, we noted that it was similar to a
motif from single-stranded DNA-binding proteins (Fig.
4A). This motif from single-stranded
DNA-binding proteins,
(R/K)-N4-5-(K/R)-N4-6-(Y/F)-N5-8-(Y/F/N)-N6-9-(Y/C)-N9-(E/D)-N7-9-(Y/W)-N4-8-(Y/F/E)-N11-12-(R/K), is thought to stabilize the binding of single-stranded DNA by stacking
interactions between aromatic residues and bases and electrostatic
interactions between basic residues and phosphate groups of
single-stranded DNA (25).

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FIG. 4.
Alanine scanning mutagenesis of conserved residues near
Leu-333. (A) Amino acid sequence of the 54 protein from
S. enterica serovar Typhimurium near Leu-333. The PILEUP
program was used to compare sequences around Leu-333 from
54 proteins from 25 bacteria. The sequence shown spans
residues 328 to 404. Leu-333 is marked with a dot over the sequence.
Boldface letters indicate residues that are identical in at least 22 of
the 25 54 protein sequences, and italic letters indicate
residues that are similar in at least 22 of the 54
protein sequences. Arrows indicate the residues where alanine
substitutions were introduced. The stars indicate residues that align
with the single-stranded DNA-binding motif, which is indicated on the
bottom line. The sequence that is cross-linked to promoter DNA upon UV
irradiation (5) is indicated, as is a putative
helix-turn-helix motif that appears to be involved in DNA binding
(23). (B) Glutamine synthetase activities observed with
54 mutant proteins. Cells were grown in a modified E
medium that lacked sodium ammonium phosphate and was supplemented with
acid-hydrolyzed Casamino Acids at 1 mg/liter as described previously
(18). Glutamine synthetase activities of strains that
express the alanine substitution 54 mutant proteins were
carried out using the -glutamyl transferase assay as described
previously (18). Glutamine synthetase activities were
expressed as micromoles of -glutamyl hydroxymate per minute per
milligram of protein, and all assays were done at least twice. Error
bars show one standard deviation for each data set. The wild-type and
mutant ntrA alleles, which were carried on plasmids, were
under the control of the E. coli lac promoter-operator and
were overexpressed by including 100 µM
isopropyl- -thiogalactopyranoside in the growth medium. The
no-protein designation indicates the glutamine synthetase activity from
S. enterica serovar Typhimurium strain TRH134 without a
plasmid-borne ntrA allele. (C) Repression of
ant'-'lacZ reporter gene in S. enterica serovar
Typhimurium strain TRH107 by 54 proteins.
-Galactosidase assays were done in triplicate with strain TRH107,
carrying plasmids that encode the 54 proteins indicated,
as described previously (1, 18). Error bars show one
standard deviation for each data set. The no-protein control shows the
activity for strain TRH107 with no plasmid-borne ntrA
allele.
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We introduced alanine substitutions at five conserved positions near
Leu-333 by site-directed mutagenesis as described previously (18). Substitutions were introduced at Trp-328, Lys-331,
Arg-336, Arg-342, and Gln-351. Two of these residues, Arg-336 and
Arg-342, are located within the region of the protein that can be
cross-linked to promoter DNA and are also part of the sequence that
resembles the single-stranded DNA-binding motif. Gln-351 occurred in
all 25 of the
54 protein sequences that we compared,
while Trp-328 occurred in 20 of these sequences and lysine or arginine
occurred at position 331 in 20 of these sequences. During the course of
our experiments, Chaney and Buck (11) introduced alanine
substitutions at Trp-328, Arg-336, and Arg-342 in Klebsiella
pneumoniae
54 and reported that the R336A mutation
allowed activator-independent transcription.
The alanine substitution mutant proteins that we generated were
overexpressed at comparable levels, and each conferred glutamine prototrophy on an S. enterica serovar Typhimurium strain
that lacked
54 (strain TRH134), indicating that these
mutant forms of
54 could function with NtrC at
glnAp2 (data not shown). Glutamine prototrophy is not a
quantitative indicator of the functionality of
54 mutant
proteins, so we examined the abilities of these mutant proteins to
initiate transcription from glnAp2 by measuring glutamine synthetase activities. Strains expressing the alanine substitution mutant proteins had glutamine synthetase activities that were comparable to that of a strain that expresses wild-type
54 (Fig. 4B).
We also assessed the DNA-binding activities of the alanine substitution
mutant proteins by examining their abilities to repress the
transcription of an ant'-'lacZ reporter gene in vivo. For these assays, we used an S. enterica serovar Typhimurium
strain (TRH107) that contained a partially deleted P22 prophage bearing an ant'-'lacZ reporter gene in which the S. meliloti
nifH promoter overlapped the ant promoter
(1). Binding of the
54-holoenzyme to the
nifH promoter in the prophage represses transcription from
the ant'-'lacZ reporter gene (1, 18).
Overexpression of wild-type
54 repressed transcription
from the ant'-'lacZ reporter gene about sevenfold (Fig. 4C).
With the exception of R336A, the mutant
54 proteins
repressed transcription from the ant'-'lacZ reporter gene as
well as or better than wild-type
54. Interestingly, the
R336A mutant protein, which functioned as well as wild-type
54 from glnAp2, only repressed transcription
from the ant'-'lacZ reporter by ~25%, suggesting that the
R336A-holoenzyme had reduced affinity for promoter sequences. Chaney
and Buck (11) similarly observed that the K. pneumoniae
54 R336A mutant protein had reduced
affinity for the nifH promoter but supported ~70% of the
level of expression from a glnAp2-lacZ reporter
gene that was achieved with wild-type
54. Arg-336 is in
a region of the protein that can be cross-linked to DNA following UV
irradiation of
54-promoter complexes (5), and
it has been proposed that Arg-336 participates in a
54-DNA interaction that helps keep the closed complex
from undergoing isomerization in the absence of enhancer-binding
protein (11).
Conclusions.
Region I of
54 functions as an
intramolecular inhibitor that prevents isomerization of the closed
complex to the open complex, but it also has a role in DNA melting in
response to the enhancer-binding protein (6, 13, 29). Two of
the mutants that were analyzed here, L37P and L46P, had single amino
acid substitutions in region I and allowed activator-independent
transcription from the heteroduplex template, indicating that these
amino acid substitutions disrupt the inhibitory effect of region I on
the holoenzyme. Leu-37 is within a small leucine-rich patch, residues
Leu-25 through Leu-37, that was shown previously to be important for
mediation of the inhibitory effect of region I on the holoenzyme
(10, 26, 30). Substitution of arginine for Leu-37 in
E. coli
54 was shown previously to allow
activator-independent transcription. The L37R mutant protein, however,
remained responsive to enhancer-binding protein in vitro, allowing
NtrC-mediated activation to about 50% of the level observed with
wild-type
54 (26). The L37P mutant protein
described in this study appeared to be more severely impaired in the
ability to respond to enhancer-binding protein, which likely reflects
disruption of secondary structure in the protein by the proline
substitution. Casaz and coworkers identified two other areas within
region I, residues 15 to 17 and 42 to 47, that are important for
inhibition of the conformational change in the holoenzyme and
responsiveness to the activator (10). Consistent with these
previous results, the L46P mutant enzyme was capable of
activator-independent transcription from the heteroduplex template and
also appeared to be impaired in the ability to respond to
enhancer-binding protein, but not as severely as the L37P mutant protein.
Not all of the mutant
54 proteins with amino acid
substitutions in region I that we used in our study were capable of
activator-independent transcription, as the E32K G189V double-mutant
protein failed to activate transcription from the heteroduplex
template. Since both substitutions in this double-mutant protein are
needed for loss of function (18), we cannot rule out the
possibility that the substitution of valine for Gly-189 interfered with
the ability of the protein to initiate transcription from the
heteroduplex template.
Holoenzymes formed with L37P or L333P were capable of efficient
activator-independent transcription from the heteroduplex template
(Fig. 3) and also had diminished fork junction DNA-binding activities
(18). These observations suggest that these two
54 mutant proteins stabilize the same conformation of
holoenzyme, indicating that region I may function together with a
sequence around Leu-333 in region III to prevent isomerization of the
closed complex in the absence of enhancer-binding protein. We
identified here a second sequence within region III that also appears
to be important in keeping holoenzyme locked in the closed complex conformation prior to activation. Like the L333P mutant protein, the
L124P V148A mutant protein allowed activator-independent transcription from the heteroduplex DNA template (Fig. 3). Unlike the L333P mutant
protein, the L124P V148A mutant protein retained fork junction DNA-binding activity (18), suggesting that the L124P V148
mutant protein stabilizes a conformation of the holoenzyme that differs from that of the L333P-holoenzyme. Leu-124 and Val-148 lie within the
minimal core-binding domain of
54, which spans residues
120 to 215, and Leu-124 is also located in a portion of the protein
that is protected from hydroxyl radical cleavage by core RNA polymerase
(9). It is possible that the sequence around Leu-124
interacts directly with core RNA polymerase to prevent isomerization of
the closed complex prior to activation.
The remaining mutant forms of
54 examined here, L179P,
E32K G189V, and L199P D231G, did not allow efficient
activator-independent transcription from the heteroduplex DNA template.
Gel mobility shift assays showed that these mutant forms of
54 retained core RNA polymerase and fork junction
DNA-binding activities (18; Fig. 1). Taken together,
these findings indicate that these mutant forms of
54
represent a different class of mutant proteins. While both amino acid
substitutions were required for loss of function in the two double-mutant proteins (18), all of these mutant proteins
had an amino acid substitution within a well-conserved 20-amino-acid segment (Leu-179 to Leu-199) in the core-binding domain of
54. It is possible that this segment of
54 participates in some function in transcription
initiation following formation of the closed complex, such as mediation
of signal transduction from the enhancer-binding protein.
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ACKNOWLEDGMENTS |
We thank Frank Gherardini and John Olson for helpful comments on
the manuscript and Sydney Kustu for providing the NtrC and NtrB
proteins. We also thank Paula Buice for her assistance in isolating the
L179P mutant protein.
This work was supported by award MCB-9974558 to T.R.H. from the
National Science Foundation.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 527 Biological Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2675. Fax: (706) 542-2674. E-mail:
trhoover{at}arches.uga.edu.
Present address: Department of Biochemistry, Conway Institute of
Biomolecular and Biomedical Sciences, University College Dublin,
Belfield, Dublin 4, Ireland.
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Journal of Bacteriology, November 2000, p. 6503-6508, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.