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Journal of Bacteriology, March 2001, p. 1921-1927, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1921-1927.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The 
10 Region Is a Key Promoter Specificity Determinant for
the Bacillus subtilis Extracytoplasmic-Function 
Factors X and W
Jian
Qiu and
John
D.
Helmann*
Department of Microbiology, Cornell
University, Ithaca, New York 14853-8101
Received 22 September 2000/Accepted 20 December 2000
 |
ABSTRACT |
Transcriptional selectivity derives, in large part, from the
sequence-specific DNA-binding properties of the 
subunit of RNA
polymerase. There are 17 factors in Bacillus
subtilis which, in general, recognize distinct sets of
promoters. However, some factors have overlapping promoter
selectivity. We hypothesize that the overlap between the regulons
activated by the X and W factors can be
explained by overlapping specificity for the 
10 region:
X recognizes 10 elements with the sequence CGAC and
W recognizes CGTA, while both can potentially recognize
CGTC. To test this model, we mutated the X-specific
autoregulatory site (PX), containing the 10 element CGAC,
to either CGTC or GCTA. Conversely, the W autoregulatory
site (PW) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that
changes to the 10 element are sufficient to switch a promoter from
the X to the W regulon or, conversely,
from the W to the X regulon, but context
effects clearly play an important role in determining promoter
strength. It seems likely that these subtle differences in promoter
selectivity derive from amino acid differences in conserved region 2 of
, which contacts the 10 element. However, we were unable to alter
promoter selectivity by replacements of two candidate
recognition residues in W.
| |
INTRODUCTION |
While the sequencing of bacterial
genomes is proceeding at a rapid pace, functional annotation remains a
formidable challenge. Typically, half or more of all predicted open
reading frames encode proteins which have no functionally characterized
homologs or which are related only to large classes of proteins with a
wide range of functions (e.g., transporters or oxidoreductases).
Additional clues to gene function can often be gleaned from careful
analysis of operon organization (13, 23) or by identifying
groups of genes (stimulons) that are coordinately activated under
specific conditions (12, 19). Interpreting global
transcriptional profiles requires that genes be grouped into regulons
that share a common regulatory factor. Regulons activated by secondary
factors are often a significant component of the stimulons
activated in bacteria under specific stress conditions or in response
to changing environmental conditions (3-5).
Sequencing of the Bacillus subtilis genome revealed genes
for seven previously unidentified factors, all belonging to the extracytoplasmic-function (ECF) subfamily (15). Mutants
with alterations in these genes are viable and do not have obvious phenotypes, although the mutant strains are often somewhat more sensitive to selected stress conditions (7, 8). Therefore, to define the roles of the ECF factors in B. subtilis,
we have sought to identify target genes dependent on ECF factors
for their expression (10, 11).
ECF factors often positively regulate their own synthesis
(16, 21). The identification of the corresponding
autoregulatory promoters provides useful clues to promoter selectivity
which can then be used to search the genome for additional target
sites. To develop this strategy, we analyzed a large collection of
point mutations in the X-dependent
autoregulatory promoter (PX) to define bases
critical for activity (11). As expected for a
70 class holoenzyme, the critical bases are
clustered near 35 and 10 relative to the transcription start point
(tGtAACN17CGaC; bases with no allowable
substitutions are in uppercase). Using this information, we were able
to identify a number of promoters that are recognized by
X both in vivo and in vitro. However, some of
the target genes we identified were still transcribed, from the same
start point, even in a sigX null mutant (11).
This suggested that at least one other holoenzyme has an overlapping
specificity with X.
In parallel with these studies of the X
regulon, we also initiated an analysis of the
W regulon. Like sigX,
sigW is transcribed from an autoregulatory promoter element,
PW (TGAAACN16CGTA)
(9). Analysis of the genome revealed 15 additional operons
with candidate promoters identical to PW (in the
35 and 10 elements and spacer length), and all 15 of these sites
are W dependent both in vivo and in vitro
(10). Thus, promoter sequence comparisons have proven to
be a valuable approach to defining factor regulons.
Despite considerable sequence similarity between
PW and PX, these promoters
are exclusively recognized by the cognate in vivo and in vitro
(9-11). Sequence comparisons, in conjunction with the
mutational analysis of PX, suggested that this
selectivity might derive from the 10 region sequences.
PX contains the 10 element sequence CGAC, while
PW contains CGTA (9).
Characterization of the X and
W regulons also identified several promoters
with the 10 region sequence CGTC. In vitro, these promoters seem to
be recognized by both holoenzyme forms (9). In vivo, these
promoters seem to depend primarily upon X, but
often both X and W
contribute to expression.
It is difficult to accurately predict promoters based solely on 35
and 10 consensus sequences. Context effects may play a large role in
promoter selectivity, and important discriminatory elements may residue
outside the classically defined 35 and 10 elements. As a test of
our model for promoter recognition by X and
W, we have engineered mutations within the
10 element to convert PX into a
W-dependent promoter and, conversely, to
convert PW into a
X-dependent promoter. Our results indicate
that changes to the 10 region are sufficient for altering holoenzyme
selectivity both in vivo and in vitro. These observations support the
notion that the 10 region is a critical selectivity determinant for these two ECF factors.
| |
MATERIALS AND METHODS |
Bacterial strains, growth media, and antibiotics.
All
B. subtilis and Escherichia coli strains used in
this work are listed in Table 1. Strains
were grown at 37°C with vigorous shaking in Luria broth (LB) medium
unless otherwise indicated. In E. coli, ampicillin
resistance was selected by using 100 µg of ampicillin/ml. In
B. subtilis, antibiotics used for selection were
neomycin at 8 µg/ml, spectinomycin at 100 µg/ml, kanamycin at 20 µg/ml, and macrolides-lincomycin-streptogramin B at 25 µg/ml (lincomycin) and 1 µg/ml (erythromycin).
Mutagenesis of PX and PW.
To
introduce mutations into PX, 1 nmol of
oligonucleotide 313 (10 region, CGTAaa [10 consensus bases are in
uppercase]) or 314 (10 region, CGTAta) were mixed with 1 nmol of the
reverse oligonucleotide XH135 in 50 µl of TMED buffer (10 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 1 mM EDTA, 1 mM
dithiothreitol [DTT], 50 mM NaCl), heated at 95°C, and cooled
slowly to allow annealing. Deoxynucleoside triphosphates (10 µl) at
25 mM and 2 µl of Sequenase, version 2, were added, and the
oligonucleotides were extended at 37°C for 1 h. The duplex
product was purified using a Qiagen purification kit, digested with
HindIII and BamHI, and ligated into pJPM122
to construct pJQ1 and pJQ2, respectively. After transformation into
E. coli DH5
with selection for ampicillin resistance,
plasmids were recovered and the sequence of the promoter region was
verified by DNA sequencing. A plasmid containing a 10 element of
CGTCaa was obtained from the saturation mutagenesis studies by Huang and Helmann (11) and renamed pJQ3. The plasmids pJQ1,
pJQ2, and pJQ3 contain the 44 to +11 regions of
PX variants.
To introduce mutations into P
W,
P
W and its variants were amplified using the
forward primer XH180 (with a
HindIII site) and
one of
three reverse primers (416 to 418) (Table
2). The reverse
primers carry a
BamHI site and the indicated
10 element (Table
2). The
resulting PCR fragments were then cloned into pJPM122
(
26)
to construct pJQ8, pJQ9, and pJQ10, following the procedures
described
above. The plasmids pJQ8, pJQ9, and pJQ10 contain the
79 to +6
regions of P
W and its variants.
Construction and analysis of SP
reporter phage.
To
recombine the promoter-cat-lacZ fusions into the
SP prophage, each pJPM122 derivative was linearized by digestion
with ScaI and transformed into ZB307A [W168
SPc2
2::Tn917::pSK106 (MLSr)] (29) with selection for
neomycin resistance. To transduce each reporter fusion into various
genetic backgrounds, SP lysates were prepared by heat induction at
50°C for 10 min followed by continued incubation at 37°C for 90 min
(1). The resulting lysates were used to transduce
recipient strains using standard techniques (Table 1).
Expression levels for each reporter fusion were determined by
-galactosidase assays of cultures grown in LB media. Each strain
was
grown overnight in LB medium containing appropriate antibiotics
and
diluted 100-fold into LB medium without antibiotics. Samples
of cells
were taken after growth at 37°C for 3 h, harvested, and
frozen
at
80°C.
-Galactosidase activity was assayed as described
by
Miller (
20).
Overproduction and purification of W mutants.
Mutagenesis of sigW was achieved by two rounds of PCR with
Vent DNA polymerase (New England Biolabs). Primer 452 is located at the
5' end of sigW and contains a ribosome binding site and an
XbaI restriction site, and primer 453 is located at the 3' end of sigW and contains a XhoI restriction site.
Primers 454 and 455 introduce mutations in the codon of Arg75 on the
sense and antisense strands, respectively. Primer 456 and 457 introduce mutations in the codons for both Arg75 and Asn79. In the first round of
PCR, primer 452 and primer 455 or 457 were used to amplify the mutated
5' fragments of the sigW gene from B. subtilis
chromosomal DNA, and primer 453 and primer 454 or 456 were used to
amplify the mutated 3' fragments of sigW gene. In the second
round of PCR, the products from the first round were used as templates and oligonucleotides 452 and 453 were used as primers. The amplified fragments from the second round were digested with XbaI and
XhoI and cloned into pET17b to construct pJQ27 and pJQ28.
The sequences of both cloned mutant sigW genes were
confirmed by DNA sequencing.
For purification of
W mutants, pJQ27 and pJQ28
were transformed into
E. coli BL21/DE3 to generate
strains HE4524 and HE4525.
Both strains were grown to mid-logarithmic
phase at 37°C in 500
ml of LB medium with 100 µg of ampicillin/ml.
Isopropyl-
-
D-thiogalactopyranoside
(IPTG) was
added to 0.4 mM, and cells were harvested after further
incubation for
3 h. After centrifugation, the cell pellets were
suspended in 20 ml of disruption buffer (50 mM Tris-HCl [pH 8.0],
2 mM EDTA, 0.1 mM
DTT, 1 mM
-mercaptoethanol, 233 mM NaCl, 10%
glycerol) and lysed by
sonication, and the inclusion bodies were
recovered by centrifugation.
The inclusion bodies were washed
twice with 100 ml of TEDG buffer (50 mM Tris-HCl [pH 8.0], 0.1
mM EDTA, 1 mM DTT, 10% glycerol)
containing 0.5% (vol/vol) Triton
X-100 and 10 mM EDTA and then
dissolved in 10 ml of TEDG-6 M guanidine
hydrochloride. A portion (2.5 ml) was gradually diluted to 250
ml with TEDGX (TEDG containing 0.01%
Triton X-100) to allow renaturation
of
W
mutants and then loaded onto a 10-ml heparin-Sepharose CL-6B
column equilibrated with TEDGX. After being washed with 80 ml
TEDGX-0.2 M NaCl, the
W mutant proteins were
eluted with TEDGX-0.5 M NaCl. The peak fractions
were concentrated
with Centricon 10 from Amicon and stored at
80°C.
In vitro transcription assays.
Runoff transcription assays
were performed with DNA products from PCR as the templates.
PX and its variants were amplified from B. subtilis HB7022 [CU1065 SP7019
PX-cat-lacZ
(MLSr Neor)] chromosomal
DNA or plasmid pJQ1, pJQ2, or pJQ3 with primers 435 and 366. PW and its variants were amplified from plasmid
pJQ8, pJQ9, or pJQ10 with primers XH180 and 366. Primer 366 is
located within the cat gene, and the PCR-amplified
products contain the promoter regions and a 263-bp 5' fragment of
the cat gene.
B. subtilis core RNA polymerase (RNAP) (
14),
X (
8),
W
(
9), and
(
17) preparations have been
described previously.
Typical transcription reaction mixtures (20 µl)
contained 0.36
pmol of core RNAP, 4.5 pmol of
, 4.2 pmol of
, and
0.04 pmol
of template DNA in transcription buffer (20 mM Tris-HCl [pH
8.0],
10 mM MgCl
2, 50 mM KCl, 0.5 mM DTT, 0.1 mg
of bovine serum albumin/ml,
5% [vol/vol] glycerol, and the RNase
inhibitor RNasin from Promega
at 0.8 U/reaction), to which were added
nucleoside triphosphate
mixtures containing 10 nmol of ATP, GTP, and
CTP, 1 nmol of UTP,
and 0.6 pmol of
[
-
32P]UTP (3,000 Ci/mmol). Core RNAP,
,
and
were mixed on ice for
15 min to form RNAP holoenzyme before the
addition of template
DNA and incubation at 37°C for 10 min to allow
promoter binding.
Nucleoside triphosphates were added, and
transcription was allowed
to proceed for 7 min at 37°C prior to
addition of 6 µg of heparin/reaction
and an additional 11 min of
incubation. Reactions were terminated
by the addition of 80 µl of
stop solution (2.5 M NH
4 acetate,
10 mM EDTA, and
0.1 mg of glycogen/ml), extracted with phenol-chloroform,
and
precipitated with ethanol. The pellets were dissolved in 8
µl of
loading buffer (20 µg of xylene cyanol FF/ml, 20 µg of bromophenol
blue/ml, and 60 mg of urea/ml in 1× Tris-borate-EDTA buffer) and
subjected to 8 M urea-6% polyacrylamide gel electrophoresis. Reaction
products were visualized by using a Molecular Dynamics PhosphorImager
system and ImageQuant
software.
Experiments to test the selectivity of
W
mutant proteins were performed as described above or using a template
competition
assay containing both P
W and the CGTC
variant. To distinguish
the RNA products from the two fragments, the
PCR product carrying
the CGTC P
W variant was
digested with
DdeI. This reduces the size
of the PCR
fragment from 353 to 281 bp and leads to a concomitant
decrease in the
length of the runoff transcript. Transcripts were
quantified using a
Molecular Dynamics PhosphorImager system and
ImageQuant software, and
the molar ratios were calculated after
taking into account the
difference in UMP content of each runoff
RNA.
| |
RESULTS |
Activity of PX and PW variants in
vitro.
PX (the
X-dependent promoter preceding
sigX) and PW (the
W-dependent promoter preceding
sigW) have very similar sequences in the 10 and 35
elements, yet EX cannot recognize
PW nor can EW recognize
PX. Comparison of PX with
PW, in the vicinity of the 35 and 10
elements, reveals seven sequence differences that might account for the
mutually exclusive recognition of these two promoters (Fig.
1). Previous mutational analysis of
PX indicates that changing the sequences at five
of these positions does not eliminate promoter activity
(11), and alignment of known
X-dependent promoters supports the idea that
these are unlikely to be key selectivity determinants
(11). Nor is the difference in spacer length between
PX and PW
likely to be an important factor: both
X and W can recognize
promoters with either 16- or 17-base spacer regions (10,
11). These observations led us to focus our attention on the
10 element.
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FIG. 1.
Sequence comparison of PX and
PW. The sequence of the X-dependent
autoregulatory site, PX, is shown, with critical bases, as
judged by mutational analysis (11), in bold. Alignment of
PX with PW reveals a high degree of sequence
similarity with related 35 and 10 sequences, also in bold. The
numbers represent seven positions that might account for the mutually
exclusive recognition of these two promoters. Positions 1, 2, 4, and 7 are viewed as unlikely discriminatory features, since the corresponding
mutations in PX retain at least 25% of wild-type activity
in vivo (11). A change of A to T at position 5 also
retains activity in vivo and this activity is eliminated in a
sigX mutant. A variant of PX with a
single base deletion (), to generate a 16-bp spacer as in
PW, retains activity, although at a considerably
lower level than PX. This analysis, together with alignment
of promoters shown previously to depend on X,
W, or both, suggests a critical role for position 6 (located at 9 relative to the most common transcriptional start
point) in the discrimination of PX from PW
(9).
|
|
To determine whether the
10 element is the key determinant that
distinguishes between E
X and
E
W, we mutated the
10 elements so that
P
X acquired the
10 element
CGTC or CGTA and
P
W acquired the
10 element CGTC or CGAC (Table
3). Reconstituted RNAP holoenzyme was
then used to determine
promoter activity in runoff transcription assays
(Fig.
2). Under
our reaction conditions,
core RNAP alone (
'
2) could not
recognize
or transcribe from either P
X or
P
W, and enzyme reconstituted with
A recognized a
A-dependent promoter with high activity but
did not recognize either
P
X or
P
W (data not shown). Consistent with previous
reports (
9),
reconstituted E
X
directs transcription from P
X, but not
P
W, while E
W directs
transcription from P
W, but not
P
X.
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FIG. 2.
In vitro transcription of PX,
PW, and their variants by EX and
EW holoenzymes. The 10 elements of the promoters are
indicated. Downstream flanking bases are shown for the X
10 element, since one base was altered between the X2 and X3
variants. The dot indicates the expected runoff products. wt, wild
type.
|
|
Alteration of the P
X 10 element from CGAC to
CGTC, a single base change, allows in vitro recognition by the
E
W holoenzyme, but the level of transcription
is still less than
that achieved with E
X. One
additional base change, to generate a
10 region of sequence
CGTA
results in a promoter that can only be recognized by
E
W. Introduction of an adjacent T residue
(corresponding to position
7) (Fig.
1) results in a much stronger
promoter while retaining
the strong preference for the
E
W holoenzyme. Thus, two or three base changes
in the P
X 10 element
are sufficient to switch
this sequence from one exclusively recognized
by
X to one preferentially recognized by
W.
Similar results were found with the P
W variants.
When the
10 element of P
W was changed from CGTA
to CGTC, the resulting promoter
could be recognized by both
E
X and E
W with nearly
equal activity. One additional base change, to generate
a
10 sequence
CGAC, results in a P
W variant that is
preferentially
recognized by E
X. Thus, the
10 elements of P
X and P
W
determine whether the promoter
is
X and/or
W dependent in
vitro.
Activity of PX variants in vivo.
To test the
promoter activities of the PX and
PW variants in vivo, we cloned the promoters into
an SP-derived prophage to generate promoter lacZ
operon fusions. The resulting reporter fusions were transduced into
the wild-type strain (CU1065), mutant strains altered in
sigX (HB7007), rsiX (HB7013), sigW
(HB0020), or rsiW (HB0010), or the double sigX
sigW mutant (HB0030).
Consistent with previous work (
8),
P
X is active in CU1065 but not in the
sigX mutant (Fig.
3). As
expected for a
X-dependent promoter, activity
increases in the
rsiX anti-
factor
mutant. Unexpectedly,
activity of P
X is also reduced severalfold
in a
sigW mutant. The origin of this effect is unclear, since
previous analyses failed to reveal a comparable effect. Indeed,
activity of most
X-dependent promoters is
slightly elevated in
sigW mutant strains
(M. Cao and J. D. Helmann, unpublished data). The basis for this
discrepancy is not
clear.
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FIG. 3.
In vivo expression driven by PX,
PW, and their variants. -Galactosidase activities
(beta-gal) were measured for strains carrying the indicated promoter
variant. (A) Results for wild-type PX (CGACaa) and its 10
region variants. Note that the CGTAta variant used (X3) was
altered at three positions (underlined), since the X2 variant (Table 3)
did not have detectable activity in vivo. (B) Results for wild-type
PW (CGTA) and its 10 region variants. Error bars
represent the standard deviations from at least two assays.
|
|
The CGTC P
X variant has an in vivo profile
virtually indistinguishable from P
X: activity is
reduced to background levels in
the
sigX mutant strain (Fig.
3). This suggests that in vivo
W does not
contribute significantly to expression, despite the
fact that
E
W can recognize, albeit weakly, this promoter
in
vitro.
The CGTAaa P
X variant (bases flanking the four
10 consensus positions are in lowercase) did not show any measurable
activity
in any recipient strain. Notice that the CGTAaa
P
X variant has
a much weaker activity than the
CGTAta P
X variant in vitro, although
it is also
W dependent (Fig.
2). Consistent with the in
vitro transcription
results, the CGTAta P
X
variant is no longer dependent on
X for
expression, and instead, activity is reduced to background
levels (<1
Miller unit) in the
sigW mutant. As expected for a
W-dependent promoter, activity is elevated
slightly in an
rsiW mutant. In addition, activity is
elevated in the
sigX mutant and
decreased slightly in an
rsiX background. The latter results are
consistent with
previous observations that activities of
W and
X promoters are often mutually antagonistic:
increased activity
of one leads to decreased activity of the other
(
9,
10).
The basis for this effect is not yet understood.
Thus, despite
the fact that the in vivo activity of the
P
X CGTA variant is low
(~5 Miller units), it
has all the hallmarks of a
W-dependent
promoter
sequence.
Activity of PW variants in vivo.
As expected,
PW directs the synthesis of -galactosidase in
the wild type (CU1065), but activity is completely lost in the sigW mutant and actually increases slightly in the
sigX mutant strain. With the PW
variant with a CGTC sequence in the 10 region, mutation of
sigW has only a small effect on activity while a
sigX mutation leads to a significant decrease in activity.
In the double sigX sigW mutant, activity is reduced further
still, to near background. Thus, this single base change has converted
a W-dependent promoter to a one primarily
dependent on X in vivo (Fig. 3), consistent
with in vitro transcription results (Fig. 2). Similar results are seen
with the PW variant with a CGAC 10 region.
Taken together, the in vitro and in vivo expression studies demonstrate
that sequence changes localized to the
10 region
can convert a
X-dependent to a
W-dependent promoter and vice versa. However,
in each case the
total promoter activity is significantly reduced.
Thus, there
are likely to be other sequence features within the
promoter region
that, while not essential for recognition, nevertheless
help optimize
the promoter for activity with the cognate holoenzyme. In
addition,
the in vivo activity of these promoters may be affected by
regulatory
proteins not present in the purified in vitro
system.
Promoter specificity of W region 2 mutants.
Numerous previous studies indicate that region 2 of factors
interacts with the 10 element of promoter DNA (see references 3, 5, and 22 for reviews). Sequence comparisons of
X, W, and other factors identify two amino acids that are candidates for contacting the
10 region consensus element. Wilson modeled this region of ECF factors as an alpha helix, based on the three-dimensional structure of
this domain in E. coli 70
(28), and noted that there is a correlation between the
identity of surface-exposed amino acids and the 10 element sequences
recognized by the corresponding holoenzymes (Fig.
4) (28). Specifically, she
hypothesized that His64 and Ser60 of X
recognize the critical AC in the 10 element of
PX, while Asn79 and Arg75 of
W recognize the TA in the 10 element of
PW. To test this model, we mutated the
sigW gene to allow expression of W
variants having either one or both of these candidate selectivity determinants replaced with the corresponding residues from
X. The R75S and R75S N79H mutants were
expressed in E. coli and purified using procedures similar
to that used for wild-type W (9).
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FIG. 4.
Alignment of the 10 recognition domain (region 2) of
factors and a model for promoter recognition. (A) The portion of
conserved region 2 corresponding to the DNA 10 region recognition
helix (helix 14 in the crystal structure of a fragment of
70) (18) is shown. In E.
coli 70, residues Q437 and T440 have been
implicated in recognition of the T residue at the 12 position of the
10 region consensus (25, 27). Note that the 10 region
is written in inverted orientation (the transcriptional start site
would be to the left and the 35 region to the right). Genetic
experiments with B. subtilis H also
support a role for this region in 10 region recognition and
contribute to a model orienting the recognition helix with its
amino-terminal end at the downstream end of the 10 region
(2). The amino-terminal end of this helix contains
multiple aromatic amino acids (two Y and two W) that form a
single-stranded DNA-binding surface that makes contributions both to
promoter melting and to 10 region recognition (reviewed in reference
6). The corresponding region of W is shown
based on a multiple-sequence alignment of ECF family members
against other 70 family members (16). N79
of W is a candidate recognition residue that aligns with
70 Q437, while R75 aligns with the conserved W residues
in 70. (B) Modeling region 2 of W and
X as an helix reveals four amino acids that might
contribute to 10 region recognition as shown (28). In
each case, the conserved R and D residues would recognize the upstream
CG dinucleotide common to both X and W
promoters while the two amino-terminal residues would distinguish
between the downstream portion of the 10 element.
|
|
We used in vitro transcription assays to determine if the
W mutations affected promoter recognition.
Like
W, both the R75S and the R75S N79H
W mutant proteins recognize
P
W, but not P
X, in vitro.
Neither mutant
holoenzyme is able to recognize the CGAC
P
W variant. Thus, substitution
of either or both
of these amino acids failed to confer a
X-like
selectivity on the resulting holoenzyme. Both mutants, like
wild-type
W, can recognize the CGTC
P
W variant. Reasoning that perhaps
transcriptional
selectivity had been altered only marginally by these
amino acid
changes, we set up a mixed-template transcription system
containing
both the wild-type P
W and the CGTC
variant (see Materials and
Methods). The molar ratios of transcripts
from P
W to those from
the CGTC
P
W variant were 7.2, 9.9, and 9.8 for the
wild-type
W, the R75S mutant, and the R75S
N79H mutant, respectively. Thus,
these mutations did not allow
W to recognize the CGTC
10 region better
than the wild type, as
might have been expected if the mutant proteins
had a selectivity
more similar to that of
X.
| |
DISCUSSION |
We have used in vitro transcription and -galactosidase assays
to investigate the role of the PX and
PW 10 elements in recognition by
EX and EW. Altering
the 10 element of PX from CGAC to CGTA makes
the promoter W dependent, while changing the
10 element of PW from CGTA to either CGAC or
CGTC makes the promoter recognizable by EX.
This indicates that the 10 region is the major sequence element that
distinguishes a X-dependent from a
W-dependent promoter. In contrast, the 35
elements of promoters under ECF control often have similar sequence
features, including a conserved AAC trinucleotide motif
(21). Comparisons of known W- and
X-dependent promoters are consistent with this
model for the 10 region as a key selectivity determinant and have
failed to reveal any plausible discriminatory sequences in the 35 region.
Several lines of evidence suggest that other sequence elements also
function in promoter recognition and also play a major role in
determining promoter strength. For example, the B. subtilis genome contains 27 perfect matches to
TGAAACN16CGTA, including at least 16 active,
W-dependent promoters. However, the remaining
11 sites are not positioned upstream of genes and are thus unlikely to
be active promoters (10). Additional sequence elements are
postulated to help distinguish the 16 active
W-dependent promoters from the other 11 sites
with identical 35 and 10 elements. Previously, we suggested that
these additional sequence features might include upstream promoter
elements between 40 and 70, a pyrimidine-rich region just
downstream of the 35 element (Fig. 1), and additional sequence
determinants near the 10 element (10). The complexity of
promoter recognition is also apparent from the observation that
mutating the 10 element of PW from CGTA to CGAC
results in a X-dependent promoter, but
one much less active than PX (at least in vivo). Conversely, mutating the 10 element of
PX from CGAC to CGTA results in a weak
W-dependent promoter. Thus, while the 10
element plays a dominant role in promoter selectivity, overall activity
of the promoter is strongly influenced by context. The origins of these
context effects on promoter strength are not yet clear. It is possible that regulatory proteins present in vivo, but lacking in our in vitro
system, also play an important role in governing both promoter strength
and selectivity.
In previous studies, we found that promoters with a CGTC 10 element
could be transcribed by either the EX or
EW holoenzyme, but with variable efficiency
(9). In the present study, we found that in the context of
PX, the CGTC variant is much more active with
EX than with EW and
in vivo activity is completely eliminated in a sigX mutant. In contrast, when the CGTC 10 element occurs in the context of PW, the resulting promoter is recognized about
equally by the two holoenzymes in vitro and both appear to contribute
to in vivo expression. Thus, it is difficult to predict which
holoenzyme will play a dominant role in expression of candidate
promoters with a CGTC 10 element, although in most cases it appears
to be X. Two promoters of this class, both
dependent in vivo on EX, are found upstream of
the dltABCD and the pssA operons and control expression of genes involved in modification of teichoic acids and
phospholipid biosynthesis, respectively (M. Cao, J. Qiu, and J. D. Helmann, unpublished data). Other examples include promoters preceding
the abh, divIC, ywbN, and yrhH genes
(9) and a promoter identified by Petersohn et al. upstream
of the yjbC gene (10, 11, 24).
While our studies lend strong support to our model for DNA determinants
that distinguish X- from
W-dependent promoters, we were not successful
in identifying the corresponding amino acid determinants in factor.
A model, based on alignment of factor sequences (16)
and analysis of mutations known to affect 10 region recognition, was
developed in which two residues in an -helical portion of conserved
region 2 were proposed to contact the 10 element (28).
However, replacement of these residues in W
with the corresponding residues from X did not
alter promoter selectivity in vitro. This may indicate that these
residues play no role in promoter recognition. Alternatively, these
residues may recognize the conserved portions of the 10 element, such
as the initial CG dinucleotide, and other residues, not tested in this
study, may discriminate between the CGAC
(X-specific) and CGTA
(W-specific) 10 elements.
Understanding the promoter selectivity of factors is essential for
the use of consensus-directed approaches to defining factor
regulons, which in turn provides important insights into biological
function (10, 11, 24). One limitation of the consensus-directed methods is that they tend to identify those promoters, and only those promoters, that closely match a predefined consensus. This limitation can be circumvented, however, through the
use of DNA arrays to define factor regulons. Indeed, knowledge of
factor regulons will be key to the interpretation of genome-scale transcriptional profiling experiments in general, which often reveal
the activation and repression of multiple regulons in response to
stress conditions or environmental stimuli.
| |
ACKNOWLEDGMENTS |
We thank Megan Wilson and Iain Lamont for communication of
unpublished results.
This work was supported by grant GM47446 from the NIH.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu.
| |
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Journal of Bacteriology, March 2001, p. 1921-1927, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1921-1927.2001
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Top
Abstract
Introduction
