From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1569
Received for publication, October 19, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Twenty-one conserved positively charged
and aromatic amino acids between residues 331 and 462 of sigma 54 were
changed to alanine, and the mutant proteins were studied by
transcription, band shift analysis, and footprinting in
vitro. A small segment corresponding to the rpoN box was found to
be most important for binding duplex DNA. Two amino acids, 52 residues
apart, were found to be critical for maintaining transcriptional
silencing in the absence of activator. These two activator
bypass mutants and several other mutants failed to bind the type of
fork junction DNA thought to be required to maintain silencing. The two
bypass mutants showed a binding pattern to DNA probes that was unique,
both in comparison to other C-terminal mutants and to previously known
N-terminal bypass mutants. On this basis, a model is proposed
for the role of the C terminus and the N terminus of sigma 54 in
enhancer-dependent transcription.
Sigma 54 is unique among bacterial sigma factors with regard to
both amino acid sequence and transcription mechanism. It is not a
member of the sigma 70 family of proteins and is uniquely required to
transcribe from enhancer-dependent promoters. As with most
sigmas, promoter recognition involves two DNA sequence elements separated by a defined number of base pairs. Initially the holoenzyme binds the two sigma 54-specific promoter elements, termed In addition to binding duplex DNA, sigma factors bind single-stranded
DNA and structures with single strand and duplex DNA juxtaposed (fork
junction structures, Ref. 8). In the case of sigma 54, these latter
interactions are central to control by the holoenzyme (6, 9-11).
Within the inactive closed complex containing sigma 54 holoenzyme, a
single base pair adjacent to the The motifs on sigma 54 that direct these various DNA interactions are
not well established but appear to involve primarily N-terminal and
C-terminal sequences. A short N-terminal region is required for
regulation; numerous deregulated bypass forms of sigma have been
identified with changes in the first 50 amino acids (19-22).
Holoenzymes containing these mutant forms of sigma can transcribe
in vitro in the absence of activator. These holoenzymes have
also lost the capacity to bind the fork junction structure associated
with the silent state of the closed complex. They have also gained an
ability to interact with downstream single-stranded DNA (9).
By contrast, mutations in the C-terminal region can destroy general DNA
binding and this property is not shared by N-terminal mutants (16, 18).
Because the Subregions of potential importance within the C terminus have been
identified. Among these are the following: a block of 10 almost
completely conserved amino acids termed the rpoN box (25), a segment
originally suggested to have the potential to form a helix-turn-helix
(HTH, Ref. 26), and a segment that can be cross-linked to DNA (27).
Collectively, these segments and others that have proposed functions
(28) cover a region of ~150 amino acids. This extensive region may
contain activities that contribute to recognition of duplex DNA, fork
junction DNA, and single strand DNA.
In studies of sigma 54 and sigma 70, the most prominent residues
involved in DNA recognition are positively charged and aromatic amino
acids (24, 28-32). The C terminus of sigma 54 contains many such
residues, and these have a tendency to cluster within the motifs
suggested to be important. To learn the role of these residues and to
identify the array of functions within the C terminus we have mutated
each of these residues individually, purified the mutant proteins, and
characterized them using biochemical assays. The collection displays a
rich array of properties, which allows the role of the C terminus in
both regulation and DNA binding to be understood to a much greater extent.
Strains, Plasmids, and Site-directed Mutagenesis--
The
plasmid pAS54, derived from expression plasmid pJF5401, carries the
Escherichia coli sigma 54 gene (33). This plasmid was
subjected to site-directed mutagenesis at the desired positions with
QuikChange site-directed mutagenesis kit (Stratagene). The presence of
the correct mutation was confirmed by sequencing. The strain E. coli YMC109 lacking a wild-type chromosomal copy of the sigma 54 gene was used as the host.
Protein Purification--
Sigma 54 and its derivatives were
partially purified by modified methods based on those described (13).
In brief, 10 ml of Luria-Bertani medium with 100 µg/ml ampicillin was
inoculated with YMC109 cells transformed with expression vector that
carries the appropriate sigma 54 mutant. The cell culture was grown to 1 OD at 30 °C with vigorous aeration. The culture was shifted to
43 °C and grown for another 3-4 h to induce expression. Cells were
collected and suspended in 300 µl of buffer S (10 mM
Tris-HCl, pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol) and disrupted by
sonication. After centrifugation of the cell lysate, the pellet
containing the inclusion body was dissolved in 150 µl of buffer S
plus 4 M guanidine-HCl and 0.1% Nonidet P-40 (nonionic
detergent). Sonication was used to help dissolve the pellet, which was
then dialyzed, first against buffer S with 1 M
guanidium-HCl, then against buffer S, and finally against buffer S with
40% glycerol. After each dialysis, the undissolved material was
discarded. The protein concentration was estimated on an
SDS-polyacrylamide gel against known protein markers.
In Vitro Transcription--
Standard one-round in
vitro transcription was used as described previously (13).
The activated transcription reaction mixture contained 100 nM NtrC,1 45 nM sigma 54 holoenzyme (core RNA polymerase is from
Epicentre Technology), 5 nM supercoiled DNA template pTH8,
10 mM carbamyl phosphate, 0.05 mM GTP, 0.05 mM CTP, 4 µCi of [32P]UTP, 50 µM unlabeled UTP, and 3 mM ATP in
transcription buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 50 ng of bovine
serum albumin, and 3.5% polyethylene glycol), in a total reaction
volume of 10 µl. When NtrC is present, the reaction mixture was
preincubated without GTP, UTP, and CTP for 20 min at 37 °C. When
NtrC is absent, the reaction mixture is preincubated with GTP and CTP
for 20 min at 37 °C. ATP was always present at the concentration of
3 mM. After preincubation, the missing nucleotides and
heparin (final concentration, 100 µg/ml) were added to the reaction
mixture and incubated for 10 min. The reaction was stopped by the
addition of urea-saturated formamide dye, and the mixtures were loaded
on 6% denaturing polyacrylamide gels for electrophoresis. The data
were analyzed with a phosphorimager.
Band Shift Analysis--
The band shift analysis was done as
described (9). Briefly, the probes were prepared by annealing two
complementary DNA strands. The lengths of bottom strand and top strand
are specifically described in the legends. The annealing mixture
contained 4 pmol of labeled bottom-strand DNA and 6 pmol of top strand
in 10 mM HEPES, pH 7.9, 80 mM NaCl. The mixture
was boiled for 2 min and gradually cooled to room temperature.
Annealing was monitored by 10% polyacrylamide gel electrophoresis.
10 µl of band shift assay mixture contained 1 nM DNA, 15 nM RNA polymerase, and 6.0 ng of poly(dI-dC) per µl in
1× HEPES buffer (50 mM HEPES-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.05 µg/ml
bovine serum albumin, 2.8% polyethylene glycol 8000). To detect the
induced interaction by NtrC, 625 nM NtrC was added into the
mixture to act from solution. The mixtures were incubated at 37 or
15 °C for 10 min and subjected to 5% polyacrylamide gel
electrophoresis, which was run at 350 V in ice. After electrophoresis, the radioactive bands were visualized and analyzed with a phosphorimager.
DNase I Footprinting--
The nifH promoter
fragment from We identified 21 highly conserved positively charged and aromatic
amino acids within the 150 amino acid region from residue 319 to 469. Each of these was conserved in at least 18 of the 21 sigma 54 sequences
available at the start of this study. They were changed individually to
alanine by site-directed mutagenesis. The plasmids carrying mutated
sigma 54 genes were transferred individually to a strain lacking
endogenous sigma 54. The 21 mutants and the wild-type sigma protein
were partially purified in 2 batches of 11 proteins each. In general
the proteins were judged to be 50-80% pure. The single exception was
R383A, which was poorly induced, possibly because of a folding defect,
and thus was much less pure. As a comparison for this form of sigma a
mock purification was done from a strain lacking plasmid and thus
containing only contaminating proteins. The 22 forms of sigma were used
in equal amounts for biochemical experiments, as judged from staining
of the protein gels. The holoenzymes were formed using a 4:1 ratio of
sigma to core.
Activator-independent Bypass Transcription in Vitro--
Although
the N terminus is the primary locus of in vitro deregulated
bypass mutants, a recent in vitro study found this property to be associated with a C-terminal change (23). This mutant, R336A, is
within the collection just described. To determine whether R336 is
unique or belongs to a family that determines the requirement for
activator, all altered sigma 54 holoenzymes were tested with a protocol
for activator-independent transcription in vitro.
The activator-independent bypass transcription assay was done using
plasmid pTH8 that carries the glnAP2 promoter as a template (19).
Holoenzyme, template DNA and ATP, GTP, and CTP were preincubated to
allow the potential for initiation without activator. Then, heparin was
added to destroy any residual closed complex. [32P]UTP
was added to initiate a round of transcription. No activator was
included, and in control experiments one round of activated transcription with wild-type holoenzyme was used for comparison. The
template contains a downstream terminator, permitting a discrete RNA to
be assayed.
In this assay, wild-type and 19 of the 21 mutant proteins showed very
low levels of activator-independent transcription, less than 5% of the
amount produced by activated wild-type holoenzyme (Fig.
1). Two of the mutant sigmas give high
transcription levels. R336A gives a 90% signal in this assay,
consistent with expectations from the prior report (23). In addition
K388A gives a high signal, at approximately the 50% level. We note
that these residues are not close to each other. One is within the
region that can cross-link to DNA, and the other is within or directly
adjacent to the putative HTH motif. We also note that residues that are
very close to each of these bypass mutants, such as Lys-331 and Arg-342
that surround Arg-336, and Arg-383, Tyr-389, and Arg-394 that surround
Lys-388, do not show this property when mutated. These
properties and others to be assayed below are collected in Table
I. We infer that silencing in the absence
of activator is conferred by widely separated amino acids within the C
terminus. In contrast to bypass mutants in the N terminus, the
silencing determinants do not involve extensive adjacent stretches of
amino acids.
In Vitro Transcription Activated by NtrC--
Next, the 22 proteins were assayed for transcription using a protocol that strictly
depends on activator. In this protocol heparin is added after activator
but prior to addition of initiating nucleotides. The experiment is done
under optimal conditions for in vitro transcription,
including 37 °C and high concentrations of all proteins. Under these
conditions transcription depends strictly on activator with very little
bypass transcription. This stringency was shown in prior experiments
using strong N-terminal bypass mutants (19) and is also true for the
C-terminal bypass mutants just described (not shown). Fig.
2 shows the results of this activated
transcription assay using the mutant holoenzymes. The data from several
experiments is compiled in Fig. 2B, where the transcription
level is normalized to that of the wild-type holoenzyme.
Under these conditions, 15 of the 21 mutant holoenzymes behave
normally, giving a level of activated transcription not significantly different from wild-type. Of the six defective mutants, four are in the
rpoN box region, K455A, R456A, Y461A, and R462A. A fifth mutant is
R383A, which because of its low purity cannot be firmly assigned as
being defective in activated transcription. The remaining mutant, F355A
is the least defective of the group (see also Ref. 30). We infer that
residues within the rpoN box region are particularly important for
obtaining a signal in the activated transcription experiment. Other
segments of the C terminus seem to be less important in this assay. We
note that the two mutants that gave bypass transcription in Fig. 1 are
not in the rpoN box, suggesting that the determinants for silencing and
those required to lead to activated transcription are at least
partially separable.
In the next experiment, the efficiency of the activated transcription
assay was lowered to see if secondary defects would turn up in residues
outside the rpoN box region. This simply involved lowering the
temperature from 37 to 15 °C. Under these conditions, several other
mutants showed partial defects (Fig. 3).
Now only 8 of the 21 mutants retained a transcription level of greater than 80% of the wild-type protein (Fig. 3B). The four
mutants in the rpoN box that showed defects under optimal conditions
were now even more defective. R383A and F355A were not much affected by
the change in temperature. Seven mutants showed defects only in this
assay, and they were not clustered in any region, being scattered
between amino acids 336 and 460. It appears that residues throughout
the C-terminal region play some role in activated transcription. These
include Arg-336 and Lys-388, which play a primary role in silencing and
now are seen to have their activated transcription levels reduced to
half of the wild-type level under these suboptimal conditions. We note
that the data makes no clear distinction between positively charged and
aromatic residues; both types are among the six residues that are most
defective and among the eight residues that show no defect in either
assay.
DNA Binding--
These data indicate that the rpoN box residues
play a primary role in transcription. A secondary role is played by
residues scattered throughout the C-terminal 150 amino acids. As closed complex formation is a prerequisite for transcription we next assayed
duplex DNA binding using the 21 mutant holoenzymes. The stability of
closed complexes is generally less than that of open complexes and so
two different assays were used, DNase footprinting and a band shift
analysis. A fragment of the nifH promoter, which contains
the central consensus sequence of the
Closed complex footprints are known to cover these two elements but
yield incomplete protection; the interaction does not extend very far
beyond the elements, in particular leaving the transcription start site
unprotected (34, 35). Under the conditions of our experiment, the
overall promoter occupancy with wild-type holoenzyme in closed
complexes was 50-70% for the best protected positions in optimal
experiments. As seen in Fig. 4, the
footprints gave partial protection over the
The most dramatic result is that each of four mutations in the rpoN box
lead to by far the lowest extent of protection (R455A, R456A, Y461A,
and R462A in Fig. 4B). R455A and R456A have been shown
elsewhere to bind core polymerase and form holoenzymes, so this is not
the source of the defect in DNA binding (31). These four mutants are
the same ones that showed the greatest defect in transcription so the
cause of this is very likely to be an inability to bind duplex DNA to
form a closed complex. The fifth change in the rpoN box, K460A, is
partially down in DNA binding, mimicking its partial loss of
transcription under suboptimal conditions (compare Figs. 4B
and 3B).
Other mutations, scattered about the C-terminal region, show partial
reductions in closed complex formation using this assay. These
partially defective mutants range over a nearly 150-amino acid region.
This is roughly the same collection of mutants that showed partial
defects in transcription under suboptimal conditions. Moreover,
approximately one-third of the mutants show little evidence of defects,
being at or near the levels of wild-type holoenzyme in both this assay
and the suboptimal transcription assay. It appears that both binding
and transcription have similar amino acid requirements.
Because protection within closed complexes is fairly weak, we sought to
confirm these results using a band shift assay. At very high
concentrations of holoenzyme most mutants bound DNA well (not shown),
and so the concentration was lowered to 15 nM to reveal
significant differences (Fig. 5). Two
shifted bands could be seen, a lower one that was not very sensitive to
protein concentration or to mutation and an upper one with an intensity that varied with the protein type and its concentration (Fig. 5A). The upper band was used in the quantitative analysis
(see Fig. 5B).
For the most part, the result of the band shift analysis was consistent
with that of the footprint analysis. There were only three exceptions;
F355A, F402A, and R383A, which bound better in the band shift analysis
than in the footprint analysis. These three mutants were partially
defective in both assays, and the higher extent of binding seen in band
shifts was better correlated with the extent of transcription under
suboptimal conditions. To learn if the level of binding by R383A was
specific, we compared it to a mock-purified protein preparation
obtained from nontransformed cells (Fig. 5A,
host). No upper band was present in the mock control, indicating that the R383A signal likely comes from the cloned sigma 54. However, because of the low purity of this protein and the uncertainty
that misfolding is the cause of its low induction, we cannot be certain
of the cause of the lowering of its binding to DNA.
These experiments were repeated at lower temperature (15 °C, not
shown), but the results were not different. Four of five rpoN box
mutants showed very little binding in both footprint and band shift
assays. Several other mutants (K388A, K400A, F403A, R421A, and K460A),
showed reduced binding in both assays. It appears that the rpoN box
region is the most critical for DNA binding, but other residues
throughout the C terminus make an important quantitative contribution
to affinity. In many cases, the lowering of occupancy within closed
complexes likely accounts for the lowering of activated transcription
under suboptimal conditions.
Binding to Probes Containing Fork Junctions and Single-stranded
DNA--
After initial recognition to form a closed complex, sigma 54 holoenzyme is bound in a silent state. The closed complex has base pair
The results of binding to the T12 fork probe are shown in Fig.
6B, lower panel. The data show that there is a
very wide variation in the extent to which this probe is bound by the
different mutants. 10 of 21 mutants showed nearly undetectable levels
of binding. Eight of these are in a segment from amino acids 400 to 462 that contains the rpoN box but extends well beyond it. It is not
surprising that rpoN box mutants would fail to bind probe T12 as they
are defective in general DNA recognition. The extension of the fork junction binding defect into the region adjacent to the rpoN box is
interesting. However, because these two mutants F403A and R421A have
partial defects in DNA binding, we cannot assess how much of their
defect in T12 recognition is specifically related to fork junction recognition.
The two other mutants that do not recognize the fork junction probe T12
are R336A and K388A. These are the two sigma mutants identified as
bypass mutants in the activator-independent transcription assays
described above. The failure of these C-terminal bypass mutants to bind
this probe mimics the behavior of the N-terminal bypass mutants assayed
previously (9, 36). The results indicate that failure to bind this
probe is a common property of bypass mutants.
Sigma needs to retain the ability to recognize templates with
single-stranded DNA near the start site to transcribe efficiently. We
assessed this property by assaying binding to probe T9 (see Fig.
6A, Ref. 9). This probe contains the full
However, the two striking exceptions are mutants R336A and K388A. The
contrast is remarkable in that if one compares the upper and
lower panels of Fig. 6B, these two mutants
uniquely display strong T9 binding and a complete absence of T12
binding. Recall that these are the only two mutants that show the
in vitro transcription bypass phenotype. That is, the bypass
phenotype in this group of 21 mutants strictly correlates with a lost
ability to bind the optimal fork junction while retaining the ability
to recognize downstream single-stranded DNA.
Additionally, the mutant Y401A appears to have unique properties. It
shows significantly reduced T9 binding but binds reasonably well to T12
and very well to duplex DNA. T9 binding should rely critically on
recognition of downstream single-stranded DNA. This suggests that the
tyrosine might be a determinant that assists in recognition of the
single-stranded DNA in the open complex.
These two C-terminal mutants share one property that is quite different
from all the N-terminal bypass mutants that have been subjected to the
same assay. N-terminal bypass mutants bind probe T9 much better than
does wild-type protein (Ref. 9 and unpublished data). Fig.
6B shows that the C-terminal bypass mutants R336A and K388A
do not share this property as their ability to bind T9 is essentially
unchanged. Thus their defect is associated much more selectively with
the loss of the ability to bind the T12 fork junction, as will be
discussed below.
Effect of Mutation on Response to Activator in a Band Shift
Assay--
Recently, we developed a band shift assay in which sigma 54 holoenzyme-DNA complexes change their mobility in response to added
activator (10). The altered complex appears not to contain activator
but rather to represent an activator-induced conformational change (6,
10, 11). Because the change does not require ATP hydrolysis, it appears
to represent an early step in response to the interaction with
activator. Regardless of the details, this assay measures the capacity
of the sigma 54 holoenzyme to make a response to activator. We used a
high concentration of NtrC to allow it to act from solution (37) as the
probe shown to be responsive lacks the activator NtrC binding site. The
probe used, based on prior results, is shown in Fig.
7A. It contains two
determinants of interest. One is the optimal fork junction, which
terminates at base pair
As shown in Fig. 7C, the wild-type holoenzyme binds this
probe, and a new weak upper band is formed in response to activator (compare the two leftmost lanes; band indicated by the
arrow). In the absence of activator, this upper band is
absent for the wild-type and all mutant holoenzymes. Each mutant
holoenzyme binds to this probe similar to the prior experiment using
the closely related probe T12 containing only the optimal fork
junction. In the presence of activator, most of the mutant holoenzymes
give an upper band (Fig. 7B, upper panel). The
exceptions are simply the rpoN box mutants; this is not surprising as
they were found not to bind DNA in the prior assays. The other mutants
that show partial defects in forming this band are also partially
defective in general DNA binding, so the data do not show specific
defects in response to activator. Indeed the intensity of this new band correlates well with activated transcription levels under suboptimal conditions (compare Figs. 7B and 3). The amount of this band
is always fairly small, probably because the activation system
(activation from solution and probe mimics), is weak.
One aspect of the results is new and revealing. Several of these
mutants (K460A, R421A, F403A, K400A, K388A, and R336A) bind the optimal
fork junction very poorly (see the absence of a strong lower band in
both B and C in Fig. 7). Nonetheless, each of
these gives a normal amount of upper band in response to activator. This indicates that there is an uncoupling of events associated with
two important processes, binding to the optimal fork junction and the
response to activator, implying that the two have different determinants, which was not known previously. Below we put these results in context and attempt to ascertain the functions directed by
the C-terminal domain of sigma 54.
A variety of biochemical phenotypes were observed when conserved
positively charged and aromatic amino acids in the C terminus of sigma
54 were mutated. 16 lysines and arginines and 5 phenylalanines and
tyrosines were changed individually to alanines. The strongest defects
were associated with seven of these; three arginines, three lysines,
and one tyrosine. Five failed to transcribe and four of these were
located within the highly conserved rpoN box between amino acids 455 and 462. Footprinting and band shift assays indicated that the defect
was at the level of DNA binding. With regard to regulation, no mutants
were identified that bound DNA normally but failed to transcribe.
However, two widely separated mutants, R336A and K388A, were found to
be deregulated in that they lost the ability to keep the holoenzyme
silent in the absence of activator. Band shift assays identified unique
properties associated with these mutants that can account for their
in vitro bypass transcription phenotype. Mutations in other
residues within this 150 amino acid region showed detectable but less
severe defects. Below we discuss the implications of these results for
the mechanism of action of the sigma 54.
Primary DNA-binding Determinants--
Data from both footprint and
band shift assays indicate that mutations in the rpoN box are far more
damaging to DNA binding than mutations elsewhere. Prior systematic
studies of mutants defective in DNA binding have either excluded the
rpoN box (28) or focused on it in isolation (31). The current data
provide a side by side comparison showing strong effects of rpoN box
mutations and lesser effects at a variety of other residues. The rpoN
box has the conserved sequence
ARRTVTKYRE, with the bold
residues changed to alanine and the underlined residues showing the
strongest defects. The very hydrophilic nature of the sequence and its
high density of positive charges are consistent with a role in DNA binding.
Existing data, although indirect, suggests that the rpoN box is most
likely to have a direct role in recognition of the promoter Transcription Regulation Mutants--
Two clear cut regulatory
mutants were found within the collection, R336A (found previously in
Ref. 23) and K388A. These mutants transcribed at the 50-90% level in
the absence of activator compared with fully activated transcription.
Both showed minor defects in DNA binding and in transcription under
suboptimal conditions. None of the other mutants showed any detectable
tendency toward this activator bypass transcription. These 19 nonbypass
proteins included those with a similar type of amino acid changed at
locations near to bypass mutants. For example, R336A was a bypass
mutant, whereas R342A and R331A were not, and K388A was a bypass
mutant, whereas R394A and R383A were not. The data indicate that there is remarkable selectivity for these two well separated individual residues with regard to the ability to silence transcription in the
absence of activator. This selectivity is in marked contrast to the
bulk of bypass mutants, which reside in the N terminus; numerous
changes throughout the N-terminal 50 amino acids can lead to the bypass
phenotype (19, 20, 22, 33). The contrast indicates that the N terminus
is a discrete regulatory motif, whereas the C terminus contains
scattered residues that contribute to regulation.
No positive control or pure isomerization mutants were found that bound
DNA normally but failed to melt it and transcribe. This supports the
view that transcriptional regulation and aspects of DNA recognition may
be inextricably linked. The linkage was proposed to occur through the
Implications for the Mechanism of Silencing and
Activation--
The data revealed unique binding properties of the two
bypass mutants, and this has implications for the mechanism by which transcription is silenced prior to activation. Approximately one-half of the mutants showed very significant impairment in binding a probe
(T12) that contained a double-strand/single-strand junction at position
Overall, the data support the proposal that silencing is maintained by
sigma binding to the fork junction created when nucleotide at position
The mutants that lose binding to the T12 fork probe are scattered over
a wide region of the C terminus, and many mutants of this type exist in
the N terminus of sigma 54 (9, 22, 36). The number and dispersion of
these amino acids make it clear that most mutants that lose T12 binding
are not in residues that contact the fork directly. Instead, there is
likely to be a regulatory structure that supports T12 fork binding and
includes these residues. The structure would promote both the
maintenance of the silenced state and the changes that occur at the
fork with activation. The changes that accompany activation include an
unmasking of the determinants that strengthen binding to downstream
single-stranded DNA (6, 9, 11, 38). All N-terminal bypass mutations studied thus far (9, 38)2
display this enhanced downstream binding, indicating that their effects
extend beyond simply interfering with binding the silencing fork
structure. The two C-terminal mutants are shown here to behave in a
simpler manner; they retain downstream binding, but they do not
strengthen it.
Thus these two mutants are unique in that they simply fail to bind the
T12 fork probe. This raises the possibility that Arg-336 and Lys-388
directly recognize the silencing fork junction. The numerous other
mutated residues, largely N-terminal, that fail to bind the T12 probe
may be part of the large network of interactions that indirectly
support this binding and allow it to change as activation occurs.
The changes that occur upon activation have been proposed to involve a
switching of sigma binding preference from the bottom strand of the
fork to the top strand (6). It has not been possible to cleanly
separate the determinants on sigma that are required for activation
from those required for silencing (33). The current data show that
several mutants that have lost binding to the bottom strand fork
structure still retain the ability to initiate a response to activator.
This involves binding using the top strand of the fork. Thus the
determinants for silencing and activation are not the same within the C
terminus, and indeed it is still not clear if the C terminus contains
simple determinants of the response to activator. Perhaps these
determinants are primarily within the N terminus (19, 22, 33, 36), and
changes within this regulatory module are transmitted to the C
terminus, which appears to be in close physical proximity (40). This
transmission would alter a complex structural network and switch the
interaction with the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
24 and
12
in reference to locations that include conserved nucleotides (1). The
holoenzyme typically remains tightly bound until signal transduction
leads to activation by a protein bound to a remote enhancer element
(2-5). The holoenzyme is initially inactive, because it cannot open
the DNA and engage the transcription start site; the enhancer protein
overcomes this block, thus allowing the DNA to open and transcription
to begin (reviewed in Ref. 6). The use of enhancers and the common
regulation at the DNA melting step differentiates this class of
holoenzymes from all others in bacteria (reviewed in Ref. 7). All sigma
factors use the common core enzyme so these differences are solely
attributed to the nature of the sigma factor.
12 recognition element is
transiently melted (12). This provides a transient double-strand/
single-strand fork junction structure. Interaction at this fork is
repressive in the sense that the conformation of sigma bound to it
helps keep the holoenzyme silent by blocking its ability to melt DNA.
Activators can overcome this silencing by triggering conformational
changes in both sigma and holoenzyme (6, 10, 11). Both the silent state
and the active state rely on a complex network of interactions that
centrally involve the promoter
12 element (9, 13-15). Interactions
with the
24 elements are simpler and are the dominant factor in
directing general DNA binding (16-18).
24 interaction is dominant for DNA binding, it is
presumed that the C terminus recognizes this element. The C-terminal
region also contains determinants that are needed for activation of the
holoenzyme (21, 23) and both ends of the protein have been proposed to
participate in recognition of the
12 promoter element (17, 24).
Recently a deregulated, bypass point mutation has been identified
within the C-terminal region (23). It is obvious that the C-terminal region of sigma 54 has a particularly complex array of functions.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
59 to +29 was synthesized by Operon technology. The
bottom strand DNA was labeled with [
-32P]ATP and
annealed with the complementary top strand. The 10-µl reaction
mixture contained 0.8 nM DNA, 45 nM holoenzyme,
and 6.0 ng of dI-dC per µl in 1× HEPES buffer (as described above)
and incubated at 37 °C for 10 min. Then 0.8 µl of DNase I solution (1 mg/ml in 10 mM MgCl2 and 5 mM
CaCl2) was added into the mixture and incubated for 1 min.
The reaction was stopped with 10 µl of stop solution (20 mM EDTA, pH 8, 1% (w/v) SDS, 0.2 M NaCl). The DNA was precipitated and dissolved in 5 µl of urea-saturated
formamide dye and loaded on 10% denaturing polyacrylamide gels for
electrophoresis. The data were analyzed with a phosphorimager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bypass transcription at the E. coli glnAP2 promoter in vitro.
A, autoradiograph of RNA made from a panel of mutants
designated by the numbered amino acids. B, transcription
levels from the average of two experiments normalized to that of
activated transcription by the wild-type protein.
Mutant properties
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Fig. 2.
Activated glnAP2 transcription at
37 °C. A, autoradiograph of RNA. B,
transcription levels from three experiments normalized to
wild-type levels.
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Fig. 3.
Activated glnAP2 transcription at
15 °C. A, autoradiograph of RNA. B,
transcription levels from three experiments normalized to wild-type
levels.
24 and
12 elements, was used
in both assays.
12 and
24 elements
(compare the two leftmost lanes in Fig. 4A) and no
protection further downstream. We used three of the strongest bands to
analyze the quantitative extent of protection using phosphorimager
technology. Fig. 4A shows that position
1 is of equal
intensity in the two leftmost lanes, without and with
wild-type holoenzyme. This band is used to normalize the signal to
ensure that binding by the 21 mutants is not improperly analyzed
because of altered loading or extent of digestion. The degree of
protection was judged by comparing the signals of mutants with wild
type at the
21 and
10 band positions. That is, after normalizing
the signal from each lane to equalize the
1 band intensity, the
protection using bands at
21 or
10 was taken as the extent of
binding in the closed complex. The data were not significantly
different using the two bands, and the results using
10 are
quantified in Fig. 4B. In this display, wild type is taken
as 1.0 so the extent of protection by each mutant can be compared
directly to wild type.
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Fig. 4.
DNase I footprinting with holoenzymes on a
59 to +29 nifH promoter fragment. A,
autoradiographs. Numbers at the left indicate promoter
positions as determined from parallel sequencing experiments. Mutant
sigmas used are defined at the top with the left-most
lane lacking sigma (
). B, average levels from three
experiments for protection at
10 relative to wild-type
holoenzyme.
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Fig. 5.
DNA band shift assay with duplex
nifH DNA. A, autoradiograph. The
arrow points to the band shifted by holoenzyme.
B, the average binding (three experiments) normalized to
wild-type.
11 transiently melted (12). The enzyme has a high affinity for DNA
containing such double-strand/single-strand fork junction structures
(8, 9). When artificial fork probes are used that mimic parts of this
structure, the affinity is highest when the bottom strand unpaired
nucleotide is present (Fig. 6A, probe T12). We tested the binding of the C-terminal mutants to this probe to learn if they retained the ability to recognize such
junctions.
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Fig. 6.
DNA band shift assay using two
nifH fork junction probes. A, the
bottom strand DNA from 29 to +1 was radioactively labeled (as
indicated by asterisks) and annealed with different lengths
of top strand. T9 has a double-strand/single-strand junction at
9/
8. T12 has a double-strand/single-strand junction at
12/
11.
B, autoradiographs of binding of holoenzymes to the T9 and
T12 probes. The binding was quantified, and the average data from
duplicates is shown at the bottom.
12 and
24 duplex elements but does not contain the tight binding fork junction. Most important, it contains single-stranded DNA from position
8 to
+1. Fig. 6B, upper panel shows that many mutants
have lowered levels of T9 binding compared with wild type (Fig. 6,
left). The worst binders here are primarily the same mutants
that failed to bind probe T12, probably because they are largely
defective in recognition of the duplex part of the probe (discussed above).
12 and contains an unpaired bottom strand
nucleotide (as discussed above). The other is the nontemplate (top) single strand, which is required for response to
activator. Thus the probe could potentially be bound in at least two
types of complexes, one involving the optimal fork junction and one resulting from a response to activator. We will assay each of the
mutant holoenzymes to determine whether they have retained the ability
to sense the presence of activator.
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Fig. 7.
Band shifts of holoenzyme in the presence of
NtrC. A, the probe used in the band shift assay begins
at 59 and ends at the indicated positions. B, the
top autoradiograph is in the presence of NtrC. At the
bottom, the levels are quantified. C,
controls in the absence of NtrC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
24
element. The
24 region interactions are known to be dominant for
DNA-binding. That is, there are no known protein mutations that destroy
24 binding but still allow the holoenzyme to bind to the DNA. This is
in contrast to the existence of many mutations that interfere with
12-region binding but still allow the holoenzyme to become promoter
bound (17, 18). Many mutations that alter
12 recognition also have
regulatory defects (17, 19), which are not seen in any of the rpoN
mutants studied here. The strong and selective defect in DNA binding by
the rpoN box mutants contrasts with the much weaker effects on binding
of many mutations scattered elsewhere in the C-terminal region. These
other mutants also include several with regulatory defects. The
contrast suggests that the rpoN box may contact the
24 region with
the rest of the C terminus contributing to the complex network of
interactions that use the
12 region for both binding and regulation.
12 recognition element because certain promoter mutations there lead
to bypass transcription (14, 15). Mutants with greater effects on
isomerization than on binding have been reported (9, 19, 21, 38, 39), and some of the mutants identified here may fall in that category. Some
of these are within the N terminus where there is at least one cluster
of residues with a role in mediating activation (33, 36). Overall, it
appears that both silencing and activation likely rely on a complex
network of interactions involving the N-terminal regulatory module,
residues scattered about the C terminus, and DNA sequences overlapping
the
12 promoter element.
12 and the bottom single-stranded fork. We have suggested previously
that binding to structures of this type is required to fully maintain
the silenced state (6, 9). However, the data make it apparent that loss
of binding to this structure is not sufficient to impose the bypass
phenotype. Most of these mutants also show reduced ability to bind a
probe (T9) that presents single-stranded bottom strand DNA but has the
fork junction placed in a physiologically inappropriate location. There
are only two exceptions, R336A and K388A, that fully retain T9 binding;
these are the same two mutants that show the bypass phenotype. We note
that other mutants such as those at positions 400, 403, 421, and 460, bind duplex DNA no differently from K388A but do not show bypass
transcription. Thus it appears that selective loss of fork junction T12
binding by K338A and R336A is a prerequisite for bypass transcription. But in addition, a bypass mutant needs to retain the ability to bind
downstream single-stranded DNA.
11 is unpaired. Prior data indicate that silenced binding is
strongest using the unpaired bottom strand nucleotide (Ref. 9, see also
Ref. 11). The current data go on to demonstrate that many mutants that
lose this binding still cannot transcribe without activator, because
they have also lost the ability to bind the single-stranded DNA in the
melted bubble. Only the two mutants that retain this ability can
transcribe without activator.
12 junction from the bottom to the top strand of
the fork, which may be required to initiate activation.
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ACKNOWLEDGEMENTS |
---|
We thank Yuli Guo and other members of the group for advice.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM35754.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.
To whom correspondence should be addressed. Tel.: 310-825-1620;
Fax: 310-267-2302; E-mail: gralla@chem.ucla.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M009587200
2 Y. Guo and J. Gralla, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NtrC, nitrogen regulator protein C; nifH, nitrogen fixation H.
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