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Journal of Bacteriology, December 1999, p. 7558-7565, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Regulation of Sigma 54-Dependent Transcription by
Core Promoter Sequences: Role of 
12 Region Nucleotides
Lei
Wang,
Yuli
Guo, and
Jay D.
Gralla*
Department of Chemistry and Biochemistry and
Molecular Biology Institute, University of California, Los Angeles,
California 90095
Received 29 July 1999/Accepted 29 September 1999
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ABSTRACT |
The tetranucleotide core recognition sequence (TTGC) of the sigma
54 promoter 
12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo
and in vitro. Deregulated promoters were identified, implying that this
core element can mediate the response to enhancer-binding proteins.
These promoters had in common a substitution at position 12
(consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were
identified. Their analysis indicated that positions 13 (consensus G)
and 15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the 13 and 15 positions contribute to closed-complex formation, whereas the 12 position has a
stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that
the 12 region core contains specific subsequences that direct the
diverse RNA polymerase interactions required both to produce RNA and to
restrict this RNA synthesis in the absence of activation.
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INTRODUCTION |
Core sequences of promoters
typically contain specific critical elements within 40 bp upstream from
the transcription start site. There are often pairs of such sequences,
which may have overlapping functions. Such pairs include the well known
10 and 35 sequences for the major bacterial promoters of
Escherichia coli and related bacteria and TATA and initiator
elements for major mammalian promoters (7, 21). Such
elements may have multiple purposes. These include specifying the type
of transcription components that will bind, contributing to the amount
of mRNA that will be produced, and possibly contributing to the
response to regulators. Although consensus sequences are simple to
derive from analysis of databases, individual promoters rarely, if
ever, match the consensus sequence. This suggests that core promoters sequences have evolved not just to give specific, abundant transcripts but to give physiologically appropriate amounts of mRNA. There have
been relatively few studies on how these core sequences contribute to
the regulation of RNA amounts.
Recently, we began to address this question in the case of promoters
recognized by the sigma 54 form of bacterial RNA polymerase. Sigma 54 holoenzyme mediates enhancer-dependent transcription in bacteria
(7, 13, 14, 19). The polymerase recognizes a pair of
promoter elements termed the 12 and 24 elements (17, 18). The 24 element is always contacted when holoenzyme is bound, and it appears to be dominant in specifying promoter occupancy (11, 22, 30). However, the 12 element, with the central consensus sequence TTTGCA (29), also contributes
to binding affinity (2, 26). The latter element may play a
more complex role in RNA synthesis, beyond simply assisting in promoter
recognition (2, 4, 15, 20, 24, 27, 29). Changes in the
highly conserved GC doublet have long been known to have the potential to reduce binding affinity (1, 2) and transcription in vivo (4, 15, 16, 24, 29). However, we showed previously that the
consensus 12 element did not specify the largest amount of RNA in
vivo. Instead, changes in the upstream TTT consensus half of the
element could increase transcription in conjunction with the wild-type
downstream GCA half of the element (29).
Prior studies have suggested that the 12 region sequences can
contribute to determining the level of basal transcription. One in
vitro study showed that a nonconsensus sequence had a higher level of
unregulated transcription than a consensus sequence (27). An
in vivo study showed that a 12 region double mutation (D3) gave
detectable RNA under conditions where the consensus promoter is fully
repressed (29). Thus, the D3 mutation caused a defect in
regulation, leading to leaky basal transcription. However, D3 was not a
stronger promoter than the wild type under conditions of strong
activation. These results imply that the 12 region can contribute not
only to the specificity of transcription but also to its regulatory response.
As these prior studies used a limited series of site-directed mutants,
it was not possible to explore the effects of the full range of DNA
sequence changes on the function of the 12 region sequences. In the
present study we used random mutagenesis within the core 12 sequences
to make libraries of potential 12 region sequences. Two screens were
used to attempt to identify mutants with different properties that
might be specified by the 12 region. These screens were designed to
find nucleotides that are most important for specifying minimal
transcription and also those critical for proper regulation. The
results identify nucleotides within this element that contribute to
separate controls for regulatory response and production of RNA.
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MATERIALS AND METHODS |
Mutagenesis and screens.
The low-copy-number plasmid pRS415
(23), derived from pBR322, carries the lacZ gene.
The new plasmid pRS415-M12 has lacZ expression driven by the
consensus M12-glnHP2 promoter. This promoter contains the
natural glnHP2 NtrC and integration host factor (IHF) binding sites and promoter elements except for the substitution of T to
G (14 in reference 6 [see reference
29]; renumbered 13 in this paper). Random
mutagenesis of the 12 region and screening used pRS415-M12.
Two oligonucleotide mixtures were used for mutagenesis. The 33-mers
contained glnH promoter sequences except for the TTGC from
15 to 12. Those four positions contained equal amounts of each of
the four nucleotides. The two mixtures were used together to generate
random substitutions within the TTGC sequence of the M12 promoter of
pRS415-M12 by using a standard site-directed mutagenesis protocol
(Stratagene). The resulting library was transformed into Epicurian Coli
XL1-Blue supercompetent cells and was screened initially on X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyryanoside)-Luria-Bertani medium (LB)-0.2% (NH4)2SO4-2%
glucose plates. On these plates the parent consensus promoter M12 is
white, whereas the bypass promoter D3 is blue, after overnight
incubation at 37°C. Blue colonies were taken to be bypass mutants.
Colonies that turned blue more slowly were not deemed to have passed
the screen. White colonies were transferred to X-Gal-G-gln plates (500 ml of G-gln medium contains 5.25 g of
K2HPO4, 2.25 g of
KH2PO4, 7.5 g of agar, 10 ml of 20%
glucose, 1.0 ml of thiamine [10.0 mg/ml], 0.215 ml of 1 M
MgSO4, 1 g of L-glutamine, and 100 µg of
ampicillin per ml). Colonies that remained white after overnight
incubation at 37°C were taken to be nonfunctional mutants. Colonies
that turned blue slowly were not deemed to have passed the screen.
Promoter DNA from both types of colonies was sequenced by using
standard dideoxy methods.
In vitro transcription.
Standard one-round in vitro
transcription was used. The activated transcription reaction mixture
contained 75 nM NtrC, 100 nM sigma 54, 25 nM IHF (a gift of Steven
Goodman), 5 nM supercoiled DNA template pBR-M12 or M12 derivatives, 10 mM carbamyl phosphate, 0.25 U of E. coli core RNA polymerase
(Epicentre Technology), 0.5 mM GTP, 0.5 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.
The reaction mixture was preincubated without GTP, UTP, and CTP for 20 min at 37°C, and then the missing nucleotides and heparin
(final
concentration, 100 µg/ml) were added. After 10 min the
reactions were
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.
In vitro bypass transcription was at 47°C with the same components,
except without NtrC. All components were preincubated
for 5 min to form
closed complexes in the absence of nucleotides
and heparin. The
nucleoside triphosphates, including radioactive
UTP, were then added
(without heparin). After 20 min, the reaction
mixtures were processed
as described
above.
Band shift analysis.
The band shift analysis was as
described previously (10). Briefly, the DNA probes were
prepared by annealing two complementary DNA strands. The bottom strand
always contained the sequence from 29 to +1. The complementary top
strands were different lengths, being truncated at either +1, 9, or
12. 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.
Band shift assay mixture contained 10 nM DNA and 15 nM RNA polymerase.
The assays were initiated by mixing sigma 54 and core
polymerase on ice
in a molar ratio as 2.5:1 for 30 min. The 10-µl
reaction mixtures
also contained 6.0 ng of dI-dC per µl in 1×
buffer (50 mM HEPES-HCl
[pH 7.9], 100 mM KCl, 10 mM MgCl
2, 0.1
mM EDTA, 1 mM
dithiothreitol, 0.05 µg of bovine serum albumin
per ml, 2.8%
polyethylene glycol 8000). The mixtures were incubated
at 37°C for 10 min and subjected to 5% polyacrylamide gel electrophoresis,
which was
run at 350 V, while bathed in ice. After electrophoresis,
the
radioactive bands were visualized and analyzed with a
phosphorimager.
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RESULTS |
We created libraries by targeting the 12 region for random
substitutions (Fig. 1). Oligonucleotides
were prepared with a mixture of all four nucleotides present in
critical positions. The remaining positions correspond to the wild-type
glnH promoter sequence. These oligonucleotides were used as
mutagenic primers in standard site-directed mutagenesis protocols (see
Materials and Methods). Thus, the collection of mutated glnH
promoters would be expected to contain randomly selected nucleotides in
the mutagenized positions.
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FIG. 1.
Scheme for isolation of 12 region mutations that allow
deregulated expression (bypass) or lead to loss of expression
(nonfunctional). The 12 region positions indicated by Xs were
replaced randomly to create a library of promoters. Bypass mutants are
blue on nonactivating LB. Nonfunctional mutants are white on activating
G-gln medium. The plasmid contains the consensus M12 sequence in the
context of the glnHP2 promoter with upstream IHF and NtrC
sites.
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Two libraries were created, one randomizing four nucleotides within the
most highly conserved core of the 12 region (underlined in
ATTTGCAT [29]) and one
randomizing all eight nucleotides. The eight-nucleotide library gave an
insufficient number of colonies, but the four-nucleotide library gave
more than 4,000 colonies and therefore was subjected to screening.
These four nucleotides are moderately well conserved; in a prior
compilation the TTGC nucleotides are present in 11, 11, 15, and 16 of
the 16 promoter sequences surveyed (29).
Screens (Fig. 1) were developed for loss of regulation and for loss of
function. Two plate tests were used to assess whether sigma
54-dependent expression occurs under either activating or nonactivating
conditions. The reference reporter in both screens is a plasmid
containing the M12 promoter upstream from the beta-galactosidase gene;
M12 is a glnHP2 derivative with a consensus 12 core
sequence and is responsive to nitrogen availability through activator
NtrC (6, 29).
The behavior of the M12 reporter plasmid in plate tests is consistent
with properly regulated expression of the promoter. On G-gln plates,
which are known to have low nitrogen availability, the transformed
colonies were blue, indicative of promoter-directed beta-galactosidase
expression. This is expected, as promoter expression is activated under
these conditions. Below we use the loss of blue color on G-gln plates
as an indicator of mutant promoters that have lost function. On
nitrogen-rich LB plates, transformed colonies were white, as expected
from the repression that accompanies the availability of excess
nitrogen. Below we use the appearance of blue colonies on LB plates to
indicate mutated promoters that have lost proper regulation.
Screen for promoters exhibiting unregulated basal
transcription.
This screen for loss of proper regulation was
tested for appropriateness by using the D3 mutant promoter
(29). The D3 mutation is a double substitution within the
tetranucleotide core sequence that will be subject to mutation in the
screen. Although D3 has not previously been tested on plates, it was
shown previously to yield detectable levels of mRNA in liquid LB
(29). We found that D3 induced blue color under conditions
in which the M12 wild-type parent remained white. We infer that the
screen for blue colonies on LB-X-Gal plates can detect low levels of
unregulated expression. We have previously termed mutants with this
property bypass mutants (28, 29), and Fig. 1 outlines the
protocol used to detect bypass promoter mutants.
A total of 184 of the 4,340 colonies obtained from mutagenesis showed
obvious blue color after overnight incubation on X-Gal-LB
plates. The
184 colonies were transferred to fresh LB-X-Gal plates
and were found
to maintain the blue color. As this number was
sufficient for further
analysis, colonies that turned blue more
slowly were not analyzed
further. Slightly more than half of the
blue colonies (97 colonies)
were sequenced, yielding 43 unique
promoter sequences. None of these
were wild type or contained
just a single substitution; all 43 contained multiple substitutions
restricted to the tetranucleotide that
was targeted. Two promoters
had double changes, 20 had triple changes,
and 21 had quadruple
changes. The D3 promoter sequence was one of the
two double changes
that passed the bypass screen. This D3 promoter is
known to lose
its bypass expression in a strain lacking sigma 54 (
29). Thus,
bypass expression should be sigma 54 dependent,
a property consistent
with the analysis of sequences and of in vitro
transcription (see
below).
The frequency of mutation at each position was calculated and is
presented in Fig.
2. A total of 148 nucleotide substitutions
are represented in this collection. The
numbering system used
here (
15 to
12 for the sequence TTGC) differs
from one used
previously (
29) to conform with systems used
for other promoters
(
10). The most stunning result is that
the C at position
12
(
12C) was not present in any of the 43 colonies. This 0% appearance
of the consensus
12 nucleotide is far
less than the 25% expected.
The other consensus nucleotides were
present at frequencies much
closer to the 25% expected for random
substitution, with frequencies
of 16, 16, and 23% for
15T,
14T,
and
13G, respectively. We
infer that it is necessary to mutate the
12C in order to obtain
bypass mutants.
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FIG. 2.
Distribution of mutations obtained from the bypass
library. The frequency of appearance of each nucleotide in each of the
four promoter positions is shown. The consensus nucleotides are
underlined.
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The blue-white screen did not identify any promoters with single
substitutions, even for
12C. This is consistent with prior
study of
site-directed
12C substitutions, which did not lead
to detectable
transcription in liquid LB (
29). There were two
double-substitution mutants that showed a bypass phenotype, and
both
contained a
12C substitution. One of these double substitutions
had
the same sequence as the D3 sequence from site-directed mutagenesis
studies, which was shown previously to produce mRNA in a deregulated
manner (
29). The data indicate that in addition to the
change
of the
12C, at least one other substitution is required to
obtain
detectable unregulated
transcription.
In order to see if changing
12C is strictly required, we made and
screened another library. In this case only the trinucleotide
TTG of
the core tetranucleotide was subject to random substitution;
the
12
position was held fixed as the wild-type C. If
12C substitution
is
required for unregulated transcription, no colonies in this
library
should pass the bypass screen. A total of 2,400 colonies
were obtained,
and 2 of these were blue. This 0.1% frequency is
far less than the 4%
obtained when the entire tetranucleotide
core, including
12C, was
targeted for mutagenesis. When the DNAs
from these two colonies were
sequenced, both turned out to have
the
12C changed as well as to have
other changes. This indicates
that mutations within the TTG alone are
unlikely to be sufficient
for bypass transcription and supports the
necessity for changes
that must include substitution for the C at
12.
The data in Fig.
2 show that there is a bias towards changing this C to
T in the
bypass library but that the substitution to thymine is not
required.
The key point is that the presence of the
12C is required
to
hold unregulated expression in check and that changing it is
required
to obtain
12 region-dependent bypass
expression.
Screen for promoters of lowest function.
The original library,
with the core TTGC tetranucleotide targeted, was also screened for
promoters that directed the lowest expression. Colonies that were blue
on LB-X-Gal plates were excluded from the screen. The remaining
approximately 4,000 non-bypass colonies were transferred to
G-gln-X-Gal plates, where activation causes the consensus M12 promoter
to induce blue color formation. Seventy-two colonies were definitively
white on these plates, and these were thus identified as being
nonfunctional. Light blue colonies were not further analyzed.
Sequence analysis of plasmids from these 72 colonies revealed 37 unique
promoter sequences. No single mutations were identified.
This need for
multiple mutations is consistent with prior experiments
using single
substitutions created by site-directed mutagenesis
(
29). In
that case all of the single substitutions retained
at least 30% of the
M12 mRNA production level in liquid media
under activating conditions.
Four nonfunctional promoters contained
double mutations, 22 contained
triple mutations, and 11 contained
quadruple mutations. Thus, a total
of 118 nucleotide changes are
represented in this collection. None of
the 37 promoters had the
same sequence as any of the 43 promoters that
passed the screen
for bypass
expression.
The frequency with which each base appears in the nonfunctional library
is displayed in Fig.
3. As there are 37 promoters,
one would expect each substitution to be present in 9, based
on
random inclusion of the four nucleotides. The data indicate that
there is a very strong bias against the
12C being mutated to
a T;
only 1 of the 37 promoters had this change. By contrast,
all other
possible substitutions in the four positions each occurred
between 5 and 15 times, much closer to the expected 9 for random
changes. Recall
that the
12T, largely absent in this library,
was strongly preferred
among promoters that passed the bypass
screen. Taken together, the data
suggest that promoters with a
12C-to-T change have a high probability
of being deregulated
and thus giving some basal expression. This may be
sufficient
to yield an expression level that exceeds the level for
indicating
lack of function in this screen; that is, such colonies are
likely
to be blue.
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FIG. 3.
Distribution of mutations obtained from the
nonfunctional library. The frequency of appearance of each nucleotide
in each of the four promoter positions is shown. The consensus
nucleotides are underlined.
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There are only minor differences in the frequencies with which each of
the four positions are mutated in this nonfunctional
library. The
retention of the wild-type nucleotide was 14, 22,
16, and 32% for the
15T,
14T,
13G, and
12C, respectively.
This indicates that the
nonfunctional promoters have a stronger
tendency to retain the
12C
nucleotide and a greater tendency
to substitute for the
15 and
13
nucleotides. The retention of
the
12C has been discussed and probably
relates to minimizing
unregulated expression. The more frequent
substitution at
15
and
13 may indicate that these positions are
slightly more important
in retention of minimal
function.
Further indications of which nucleotides are more important come from
analysis of the individual nonfunctional promoters with
the fewest
sequence changes. Only four nonfunctional promoters
had the minimum of
two nucleotide changes (Fig.
4). All four
of
these promoters had substitutions in common positions,
15 and
13. This commonality supports the importance of these nucleotides,
as
weakly suggested by the analysis of the total spectrum of nonfunctional
substitutions presented above.
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FIG. 4.
12 tetranucleotide sequences of the
double-substitution mutants and two selected triple mutants.
Substitutions within the consensus TTGC are underlined.
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Overall, the analysis suggests that the
12C is required to prevent
unregulated expression and that the
15T and the
13G
are especially
important in maintaining expression. Analysis of
individual promoters
lends further support to this view. Two of
the nonfunctional promoters
with double mutations (N1 and N2 in
Fig.
4) are closely related to
promoters from the bypass library
(B2 and B3 in Fig.
4). Each
nonfunctional mutant has a bypass
partner with the same inactivating
changes at both
15 and
13.
However, each bypass partner has an
additional change substituting
for the
12C. This comparison suggests
that the latter change
could conceivably restore low-level unregulated
expression even
to a nonfunctional
promoter.
In vitro transcription.
We tested several of the promoters
from the library for transcription in vitro. The promoters were
transferred into a vector with a downstream terminator to allow direct
visualization of any RNA produced. They were transcribed in vitro by
using activated NtrC and IHF in the same glnHP2 promoter
context used for the in vivo selection. Six promoters were selected
from the nonfunctional library. Four of these were the double mutants
N1, N2, N3, and N4 (Fig. 4). These were chosen because they have the
fewest substitutions and are thus more likely to show residual function
in vitro; prior study of protein mutants showed that proteins testing
as nonfunctional on plate tests could nonetheless show residual
function in vitro (25). Two triple mutants (N5 and N6) were
also chosen, as random examples of more highly mutated promoters.
Figure
5A shows the results of
transcription of promoters from the nonfunctional library. The two
triple mutants, N5 and N6,
gave amounts of RNA that were at the limit
of detection, less
than 1% of the consensus M12 amount. Three of the
four double
mutants (N1, N3, and N4) gave RNA amounts that averaged
10% of
the M12 level. One mutant (N2) gave substantial amounts of RNA,
slightly more than half of the wild-type amount. Thus, five of
the six
nonfunctional mutants gave small amounts of mRNA in vitro,
consistent
with the lack of expression in vivo; double mutant
N2 was the
exception. Based on the very low level associated with
the triple
mutants, we expect that the large majority of promoters
in this
library, all triple or quadruple mutants, would show very
low function
in this test.
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FIG. 5.
In vitro transcription. (A) Transcription of bypass (B
and D3) and nonfunctional (N) mutants in the presence of NtrC under
standard conditions is shown at the top. Amounts of RNA compared to
those for the M12 consensus promoter are shown at the bottom. (B)
Transcription at 47°C in the absence of NtrC. Lanes M12, markers for
fully activated transcription from the consensus promoter under
standard conditions. The arrows denote the correct transcript.
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Mutants screened from the bypass library were also tested by in vitro
transcription. In this case, the reaction mixtures lacked
activator
NtrC to attempt to mimic bypass expression in vivo.
Preliminary
experiments under standard conditions did not show
detectable
transcription without the activator (not shown). It
is known that
bypass transcription in vitro can be weak and is
enhanced by altering
solution conditions, particularly by lowering
the ionic strength and
raising the temperature (
27). We explored
such alterations
in conditions to see if the bypass mutants could
be distinguished from
the M12 parent and also from the nonfunctional
mutants. One condition,
the use of 47°C, allowed the bypass mutants
to be distinguished from
the M12 parent and from the nonfunctional
mutants. The best signal
under these suboptimal conditions (Fig.
5B, left panel) corresponds to
5% or less of the activated signal
from the consensus promoter.
However, the weak bypass signal was
detectable at the four promoters
selected from the bypass library
and was greater than that of the M12
control and greater than
the signal from mutants screened from the
nonfunctional library
(Fig.
5B, right panel). We infer that bypass
promoters can be
distinguished from the M12 and nonfunctional promoters
in vitro
but that their transcription is very
weak.
The bypass signal for the D3 promoter is at least twofold weaker than
that seen previously in vivo for the same promoter in
liquid LB
(
29). Collectively, signals for these bypass promoters
are
much weaker than signals from in vitro transcription with
bypass
mutants of the sigma 54 protein (data not shown and reference
27). Bypass promoters are also not transcribed very
well in
the presence of activator in vitro (Fig.
5A). They have not
been
studied in vivo, except for the strong D3 promoter, which is not
severely defective in activated transcription. Their in vitro
defects
are discussed
below.
DNA binding.
Recently, the pathway for promoter recognition
and melting has been analyzed by using band shift assays with a variety
of promoter probes (9, 10, 12). It was proposed
(10) that open-complex formation includes the following
three sequential steps: (i) formation of a closed complex, (ii)
formation of a junction complex that includes a structure with 12
intact and an adjacent single-stranded segment, and (iii) spreading of
melting to include binding to downstream single-stranded DNA. Optimal regulated transcription is presumed to require the use of all three of
these interactions sequentially. These studies suggested that low-level
bypass transcription could be triggered by inactivation of the
protein-DNA junction complex. That is, proper formation of a 12/11
junction complex is presumed to be required to keep regulation intact.
If it is not formed due to a defect in either the protein or the DNA
partner, then melting might spread downstream inappropriately and give
unregulated bypass transcription.
We tested these ideas with the promoter mutants isolated from the two
screens. Three band shift probes are relevant (Fig.
6A). Probe T1 corresponds to
double-stranded promoter DNA, and
binding to it assesses closed-complex
formation. Probe T12 corresponds
to a tight binding fork junction, and
binding to it helps assess
the ability to recognize the appropriate
12/
11 fork junction.
Probe T9 has its junction in a downstream
location, and binding
to it helps assess downstream single-strand
binding (
10). We
used the promoter mutants identified here,
in the forms of these
three probes, to assess the consequences of
mutation.
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FIG. 6.
Band shift analysis of mutant promoters in the form of
various probes. (A) The three types of probes: T1 is double-stranded
DNA, T9 has a double-strand-single-strand junction at position
9/8, and T12 has a double-strand-single-strand junction at
position 12/11. The asterisks indicate the positions of radioactive
labeling. Each group of three mutant probes uses a unique labeled
bottom strand. Thus, the extent of binding between mutants may not be
compared directly and was calculated by comparison to free probe run in
parallel (not shown). (B) Top, holoenzyme binding to the M12 consensus
sequence and the indicated mutant promoters in the forms of the three
probes. Bottom, Row T1 shows the fraction of each probe shifted,
normalized to M12. Rows T12/T1 and T9/T1 show the relevant binding
ratios for each promoter.
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Figure
6B shows the use of each promoter, in the form of each of the
three probes, in a band shift protocol with sigma 54
holoenzyme. We
compare the results for the bypass mutants to those
for the
nonfunctional mutants N1, N3, and N4. Results for nonfunctional
mutant
N2 are also presented, but recall that this mutant is unique
among the
nonfunctional mutants in exhibiting substantial function
in
vitro.
The binding to each probe for each promoter was measured several times,
and the average data are compiled at the bottom of
Fig.
6B.
Double-strand binding for each promoter is normalized
to that for the
M12 consensus parent (line T1).
12 junction binding
was assessed by
determining the ratio of binding of the T12 probe
to that of the T1
probe; the normalization to T1 ensures that
any loss in signal of T12
binding is not a consequence of interactions
specific to closed-complex
formation. The data for T9 binding
are similarly normalized to assess
single-strand binding independent
of double-strand
binding.
First, we consider the results for the bypass mutants. The data (Fig.
6B) show that they have the strongest defects in recognition
of the
12 fork junction. That is, the ratio of binding to the
T12 fork probe
compared to binding to the T1 double-strand probe
is approximately
fivefold less than that for M12 parent for this
group. The mutants have
relatively minor defects in the other
interactions; the extent of T1
binding and the T9/T1 ratio for
this group are 40 to 100% of the value
for the M12 consensus.
In this regard they resemble the previously
obtained bypass mutants
of sigma 54 protein, which also are defective
in recognition of
this critical fork junction. The results are
consistent with qualitative
data obtained recently for the D3 promoter
(
10). We infer that
this group of mutants is most defective
in recognition of the
12/
11 fork
junction.
This loss of junction recognition is also typical of protein bypass
mutants (
10). However, in one regard these bypass promoter
mutants do not resemble the bypass protein mutants. The bypass
protein
mutant
N works in part by unmasking both double-strand
and
single-strand binding determinants (
5,
10). However,
these
promoter bypass mutants do not show increased T1 or T9 binding
compared
to the M12 parent (Fig.
5B). Thus, the bypass promoters
show only one
of the two altered properties associated with bypass
proteins, probably
accounting for the weaker bypass transcription
from the promoters
compared to the
proteins.
Next, we consider the results for the nonfunctional mutants. The three
mutants, N1, N3, and N4, that transcribe poorly in
vitro have common
behavior in the band shift assays. By far the
strongest deviation from
the M12 parent is in T1 binding. This
binding is three- to fivefold
less than from that of M12. By contrast,
the T12/T1 ratio for these
three mutants is reduced by less than
a factor of 2. Thus, this group
of nonfunctional mutants has band
shift properties distinguishable from
those of the group of bypass
mutants. Bypass mutants have their
strongest defect in junction
recognition, whereas nonfunctional mutants
have their strongest
defect in closed-complex formation. The only
exception among the
eight mutants studied is N2, which functioned well
in vitro but
not in vivo and has a defect in junction
recognition.
All of the eight mutants retain a recognition component that includes
the single-stranded DNA downstream from the
12 region
consensus
sequence. This is indicated by the stronger binding
to the T9 probe
than to the T1 probe for all eight mutants (Fig.
6B, bottom row).
However, as discussed above, none of the promoter
mutants show the even
greater preference for the T9 probe that
was observed with strong
bypass mutants of the sigma 54 protein
(
10). Thus, the data
indicate that single-strand binding is
preserved in this collection of
promoters that have various mutations
in the
12 region consensus
sequence. We infer that the
12 consensus
sequence does not have a
direct effect on the ability to recognize
downstream single-stranded
DNA, although indirect effects are
discussed
below.
| |
DISCUSSION |
These experiments have demonstrated multiple roles for the
nucleotides within the core of the 12 element of sigma 54-dependent promoters. Two clear roles have been identified, and the contributions of individual nucleotides to these roles have been assessed. The most
unexpected result is that the presence of a single consensus nucleotide
is necessary for ensuring that unregulated transcription does not
occur. Such an observation has not been made for the large sigma 70 family of promoters. In those cases, deregulation has been observed
only when nonconsensus core elements are made consensus by mutation;
this can make a promoter such as lacUV5 so strong as to
eliminate the requirement for activator. Thus, the use of consensus
sequences to restrict basal transcription appears to be unique for the
enhancer-dependent sigma 54 promoters. The data also show the more
expected role for the 12 element of determining induced RNA levels.
In the discussion that follows, we elaborate on how the 12 region
sequence appears to control both the level and the regulatory response
of promoter-dependent RNA synthesis.
Regulatory response.
Each of the 43 bypass promoters
identified in the genetic screen had a substitution for the 12
consensus C. Another experiment showed that if this C is held fixed,
one cannot isolate bypass mutants by changing other core 12 region
positions. Thus, the retention of C at 12 is critical for maintaining
proper regulation. In support of this, we note that the 12C is the
most conserved nucleotide in natural promoters (17, 29).
Nonetheless, substitution at 12 alone is not sufficient to give
detectable levels of unregulated transcription in vivo (29).
At least one additional substitution is required. Although the
deregulated transcription can be detected in vitro, it appears to be
stronger in vivo.
A common property of the deregulated promoters is a defect in RNA
polymerase recognition when binding is tested with a probe
that mimics
a physiologically relevant fork junction. Band shift
experiments showed
a fivefold-lowered binding to such probes (probes
T12) when they
contained substitutions that deregulated promoter
expression in vivo.
By contrast, the deregulated promoters were
recognized fairly well when
in the form of a double-stranded probe,
which mimics closed complexes
(probes T1). Thus, the common defect
is specifically related to fork
junction recognition. The deregulated
promoters have as a common
feature the loss of C at
12, and so
this nucleotide would appear to
be critical for fork junction
binding. This is reasonable, as the
12C
is within the physiologically
relevant fork junction itself; it is the
terminal base pair at
the upstream fork of the open complexes formed at
sigma 54
promoters.
Recent work has independently led to the speculation that recognition
of this junction is critical for regulation (
10).
The idea
is that sigma 54 needs to be directed to this junction
in order to
prevent inappropriate downstream DNA recognition and
melting in the
absence of activator. The present data support
that speculation by
demonstrating that promoters with deregulated
expression have in common
a mutation within the terminal base
pair of the junction that likely
inhibits its recognition. However,
these promoter mutations do not
enhance binding to the downstream
single-stranded regions, as occurred
with bypass mutations in
the sigma 54 protein (
5,
10). This
correlates with the weaker
transcription from the promoter mutants.
Overall, the data suggest
that proper regulation is enhanced by
12
region-directed recognition
of the appropriate fork junction and by
proper masking of the
full single-strand binding region of the protein
component.
Minimal promoter function and a perspective on the function of the
12 region core.
No single nucleotide was dominant in the results
of the genetic screen for loss of promoter function. The data showed a
slight bias towards mutating the 13 and 15 positions. These same
two positions were jointly mutated in the four double-substitution mutants that passed the screen for nonfunctional mutants. The appearance of these double mutants may be understood in the context of
a prior study of site-directed single-substitution mutants (29) and the deregulated mutants just discussed. In that
study no single substitution was strongly defective, consistent with the requirement for double substitution indicated in the present study.
The four double mutants represent the minimal changes that lead to loss
of expression as judged by the loss of blue color
in
lacZ
promoter fusions. These four promoters had the sequence
from
15 to
12 of NTNC, as only the T and C are in common. The
retention of the
consensus
14T and
12C in this set of loss-of-function
mutants is
explicable in terms of other data. First, as just discussed,
substitution for the
12C can lead to unregulated expression,
likely
allowing some inappropriate use of DNA downstream from
the
12 region.
Had this substitution occurred, the ensuing unregulated
expression may
have been sufficient to lead to blue color. Therefore,
one expects the
12C to be retained in the double mutants, as
was observed. Second,
the
14T that is retained was shown to be
particularly repressive in
the prior study of single-substitution
mutants. Therefore, its presence
would be expected to contribute
to maintaining low levels of
expression. Thus, the NTNC sequence
would be expected to give low
levels of regulated
expression.
These interpretations may be viewed in context to give an overview of
the role of the
12 region. Although the
24 region
is dominant for
recruitment of sigma 54 polymerase (
11,
30),
the
12 region
contributes to enhance closed-complex formation
(
2).
Lowering the level of closed complexes can lead to lowering
of
expression levels (see above) (
8). Thus, the sequence of
the
12 region likely contributes to expression, in part, by specifying
the extent of promoter
occupancy.
The two halves of the consensus central
12 element appear to have
separate but overlapping roles. Each half responds differently
when
mutated; single substitutions at TT can raise transcription,
and single
substitutions at GC can lower it (
29). With regard
to the
GC, as shown above, retention of the
12C is critical for
proper
regulation, in the sense of preventing leaky transcription.
The data
also are consistent with the
13G being important for
allowing an
efficient positive response to the regulator. That
is, when
transcription for the 11 promoters (Fig.
5B) is normalized
to promoter
binding (T1) (Fig.
6, bottom) to calculate the response
per occupied
promoter, the two highest ratios are for D3 and M12;
these are the only
2 of the 11 promoters to retain the
13G. Thus,
the downstream half
element is required for providing regulation,
with a
12C contributing
to negative regulation and a
13G probably
contributing to positive
regulation. This view is consistent with
the appearance of this GC
doublet in 15 of the 16 promoters recently
surveyed (
29).
The role of the
15 and
14 consensus TT appears to be different.
Single changes here can increase RNA levels (
29) but when
coupled with changes in the
13G can decrease expression. Neither
thymine is particularly important for regulation, so a role in
determining maximal transcription is suggested. Seven of the 16
promoters surveyed do not retain Ts at both positions (
29),
so obviously the TT is dispensable to obtain transcription. Taken
together, the data suggest that the TT most likely plays a role
in
modulating expression levels, making them appropriate to the
need for
the specific operon that is controlled by the
promoter.
Overall, the central TTGC of the
12 region of the collected promoters
may be considered as being constructed of two interrelated
halves. Both
halves can contribute to the affinity of closed-complex
formation
(
3). The C of the highly conserved GC contributes
to
directing the polymerase to the precise position at which the
melted
fork junction will be created during activation; in its
absence the
restriction is lost and deregulation can occur. The
TT is highly
variable among promoters, and this variation creates
sequences with the
potential to direct promoter-specific differences
in RNA levels. Thus,
the two halves together ensure that appropriate
amounts of properly
regulated RNA are produced. Consensus elements
for promoters for other
holoenzymes have not been analyzed at
this level of detail. In general,
roles in specifying amounts
of RNA are generally discussed more
prominently than roles in
regulation. It would be interesting to learn
if the features found
in this study are particular to sigma 54 promoters or are generally
applicable.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant GM35754.
We thank S. Goodman (USC) for IHF and F. Govantes and R. Gunsalus
(UCLA) for plasmids.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095. Phone: (310) 825-1620. Fax: (310) 267-2302. E-mail: gralla{at}mbi.ucla.edu.
| |
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Journal of Bacteriology, December 1999, p. 7558-7565, Vol. 181, No. 24
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Top
Abstract
Introduction
