ABSTRACT
The specific interaction of the upstream element-containing promoter of the
Escherichia coli
acetate operon with either the RNA polymerase holoenzyme or its
[alpha]
subunit has been analyzed by the base removal method. Our results indicate
that: (i) direct and specific base contacts can be detected in the acetate
promoter-
[alpha]
subunit complex; (ii) base elimination in the upstream element of the acetate
promoter enhances the binding of RNA polymerase. A similar effect is observed
when studying the interactions between RNA polymerase and the
rrnB
ribosomal operon P1 promoter.
It has recently been shown that, in addition to its two major determinants (the -10 and -35 hexamers), the promoter strength of the
Escherichia coli
RNA polymerase can be greatly increased by a third
cis
-acting recognition element: the upstream (UP) module. Thus, in the case of
the
rrnB
P1 (promoter of the ribosomal RNA operon), Ross
et al
. (
1
) have demonstrated that interactions between the UP element,
which spans an (A+T)-rich region (residues -40 to -60), and the [alpha] subunit of the RNA polymerase induce a 30-fold stimulation, compared to the promoter
activity lacking the UP element. Studies performed on mutants of RNA polymerase
holoenzyme (RNAP) have shown that the C-terminal domain of the [alpha] subunit ([alpha]CTD) is directly involved in such stimulation (
1
-
3
). The three-dimensional structure of the C-terminal domain of the [alpha] subunit of RNAP, recently determined by NMR analysis (
4
), contains a spatial arrangement of four helices and two arms enclosing a
hydrophobic core. When incubated with a 25 bp DNA containing the
rrnB
P1 UP element, the region of the protein including in particular the amino acid
residues of helix 1, and the N-terminal end of helix 4 have been shown to participate in the binding
process. However, no detailed description of the structure of the [alpha]CTD-
rrnB
P1 UP complex has been provided so far, and the process of recognition between
the bases of the DNA and the amino acid residues of the [alpha] subunit remains to be determined (
5
).
Therefore, in this work, the binding of either purified [alpha] subunit alone, or total RNAP, to the promoter of the
ace
operon (the operon encoding the enzymes for acetate utilization) of
E.coli
has been studied by using the `missing contact' chemical approach described by
Brunelle and Schleif (
6
). We have shown that while the removal of certain bases between positions -32 to -50 of the
ace
P (promoter region of the
ace
operon)
interferes
with the binding of the [alpha] subunit, the elimination of single bases from either strand of the UP-like element results, in contrast, in an
enhanced
binding affinity of the RNAP. This finding suggests that disruption of the helix
in this particular region promotes local DNA flexibility that stabilizes the
RNAP-promoter complex. Similar results have been obtained when incubating RNAP
specifically with the well-characterized UP element of
rrnB
P1, which supports the concept that the interference footprinting method is an
efficient tool for studying the binding of RNAP to different UP module-containing promoters.
All chemicals were purchased from Fluka except hydrazine which was from Aldrich
Chemical Co. Restriction enzymes and DNA modification enzymes were obtained
from Promega Corp. The
E.coli
RNAP (100% [sigma]
70
-saturated) was from Epicentre Technologies. [[alpha]-
32
P]dATP (3000 Ci/mmol), [[alpha]-
32
P]dGTP (3000 Ci/mmol) and [[alpha]-
32
P]UTP (800 Ci/mmol) were obtained from Du Pont-New England Nuclear. Ni
2+
-nitrilotriacetic acid-agarose resin and pQE30 plasmid were from Quiagen
Inc. The Protein Pak Glass DEAE 5PW column was from Waters Co.
A
Bam
HI-
Dra
I DNA fragment containing the
rpoA
gene encoding the [alpha] subunit of RNA polymerase was amplified by polymerase chain reaction
(PCR) using
E.coli
K12 chromosomal DNA as template and two oligonucleotides, one carrying a
Bam
HI site within the following sequence, 5'-TAT
GGATCC
ATGCAGGGTTCTGTGACAGAGTTTC-3', and the other with a
Dra
I site in a sequence complementary to the 3'-end of the gene, 5'-TAT
TTTAAA
TGCCAGACGACGATTAGCAAC-3'. Following PCR, the fragment was restricted with
Bam
HI+
Dra
I and cloned into the compatible sites
Bam
HI+
Sma
I of the expression vector pQE-30 to create plasmid pJCD6. This plasmid encoded the [alpha] subunit under control of the
E.coli
phage T5 promoter. The
rpoA
gene contained a 6* His-tag at its 5'-end to facilitate the purification of the
corresponding protein by affinity chromatography.
Plasmid pJCD6 was transformed into
E.coli
strain JM105 (
endA
1,
thi
,
rpsL
,
sbcB
15,
hsdR
4, [Delta](
lac-proAB
), [F',
traD
36,
proAB
,
lacI
q
Z
[Delta]M15] (
7
). Cells were grown at 37oC in 2* YT broth (
8
) in the presence of ampicillin (50 [mu]g/ml) to an absorbance value of 0.7-0.8 at 600 nm, and treated with the inducer isopropyl-[beta]-thiogalactopyranoside (IPTG) at a final
concentration of 2 mM. After 2 h of induction, cells were harvested by
centrifugation, disrupted in a French pressure cell at 10 000 p.s.i. in buffer
A (10 mM Tris-HCl, pH 7.3, 10 mM [beta]-mercaptoethanol, 10% glycerol) containing 150 mM NaCl, 30 mM
imidazole and 1% Triton X-100. A high-speed supernatant (S30) was then prepared by centrifugation for 30
min at 30 000
g
and applied, in a first step, onto a Ni
2+
-nitrilotriacetic acid-agarose column equilibrated with buffer A. After
elution of the His
6
-tagged [alpha] subunit with buffer A containing 250 mM imidazole, column
fractions enriched with the protein were pooled and dialyzed against buffer A
containing 50 mM NaCl. In a second step, the corresponding fractions were
loaded onto a DEAE anion exchange column, and elution was achieved with a 0-500
mM NaCl linear gradient in buffer A. Pure [alpha] subunit protein was concentrated and dialyzed against buffer A
containing 50% glycerol by using a Vivaspin-50 (Vivasciences Co.) filtration device, and stored at -20oC. Protein concentration was estimated according to the method
of Bradford (
9
). Size exclusion chromatography showed that, after purification, [alpha] subunit was present as a homodimer in solution (
10
,
11
).
Two plasmids, pJCD7 and pJCD8, derived from pBluescript II KS vector (GenBank
accession no: X52327, Stratagene) were used to generate radioactively labelled
DNA fragments for gel retardation and base removal studies. Plasmid pJCD7
carried a 219 bp (base pairs)
Eco
RI-
Not
I fragment containing the
ace
promoter regulatory region (
12
), and plasmid pJCD8 had a 160 bp insert containing the
rrnB
P1 (
13
). In each case, the
Eco
RI+
Not
I fragment was end-labelled at the
Not
I site by [[alpha]-
32
P]- dGTP (top strand) or at the
Eco
RI site by [[alpha]-
32
P]dATP (bottom strand), using the Klenow fragment of DNA polymerase.
Premodifications of the promoter-containing DNA fragments were carried out by using the base removal
approach described in (
6
) and modified as follows: (i) promoter DNAs were chemically modified either by
formic acid (G+A reaction) or by hydrazine (C+T reaction) in conditions that
allowed an average of one modifiing event per DNA molecule (
14
) (Table
1
); (ii) after ethanol precipitation, modified DNAs were loaded on a non-denaturing 4% polyacrylamide gel and electrophoresed by using Tris-borate buffer; (iii) following autoradiography, the fastest-moving DNA molecules were electroeluted from gels and assessed
in subsequent binding experiments.
Retardation assays were performed essentially as already described (
15
). A typical assay mixture contained in 200 [mu]l: 12 mM HEPES-NaOH, pH 7.9, 4 mM Tris-HCl, pH 7.9, 95 mM KCl, 1 mM EDTA, 1 mM DTT, 9% (v/v) glycerol,
0.02% (v/v) Nonidet P-40, 2 [mu]g poly(dI-dC).poly(dI-dC) as bulk carrier DNA, 10 [mu]g bovine serum albumin, chemically-modified DNA probe (1.7 * 10
6
c.p.m.), and either [alpha] subunit (2.4 [mu]M) or RNAP (2 nM). After incubation for 10 min at 25oC, the mixtures were loaded on a 4% preparative polyacrylamide
gel at high-ionic-strength and electrophoresed for 1 h at 30 V/cm. Bands corresponding
to free and complexed DNA were visualized by autoradiography on the wet gel
after overnight exposure at 4oC. Labelled DNA was excised from the gel, eluted for subsequent piperidine
cleavage (
14
), and analyzed on a 6% sequencing gel to reveal the position of the interfering
modifications. Autoradiograms were analyzed by densitometry using a Personal
Densitometer SI from Molecular Dynamics.
Table 1
The plasmid pJCD9 used as a template in the
in vitro
transcription assay carried the
ace
P region followed by the transcription termination signal T1T2 of the
rrnB
operon (
16
), inserted between the
Eco
RI and
Afl
III sites of the pUC19 vector (GenBank accession no: X02514). Transcription
experiments were performed at 37oC in a buffer containing 50 mM Tris-acetate (pH 8), 100 mM KOAc, 8% glycerol, 0.1 mM EDTA, 8 mM MgOAc,
0.1 mM DTT and 500 U/ml RNasin. In a 20 [mu]l typical assay, 60 nM of supercoiled pJCD9 plasmid purified by
centrifugation in CsCl/ethidium bromide were incubated for 20 min, with 67 nM
of either wild-type RNAP or C-truncated [alpha]-256 RNAP to allow open complex formation. The mutant
RNA polymerase containing [alpha]-256 was prepared as described in (
17
). Transcription was initiated by adding a nucleotide mix of ATP, GTP and CTP
(200 [mu]M each), UTP (10 [mu]M), [[alpha]-
32
P]UTP (2.5 [mu]Ci at 800 Ci/mmol) and 0.1 mg/ml of heparin. After 5 min of incubation, the
reaction was stopped by adding 1 vol of formamide loading dye (98% deionized
formamide, 0.1% xylene cyanol, 0.1% bromophenol blue and 10 mM EDTA). Following
heat denaturation at 65oC, a 5 [mu]l aliquot of the assay was electrophoresed on a 6% sequencing gel and
visualized by autoradiography on a Kodak Biomax MR film at -70oC.
In a number of prokaryotic promoters, the presence of a third recognition
element interacting with RNAP, upstream from the -10 and -35 hexamers, remains to be demonstrated. For this purpose, the
promoter region of the
ace
operon (
12
) containing a putative UP element, centered around base -50, was studied in order to analyze in detail its interaction with the [alpha] subunit.
The 219 bp
Eco
RI/
Not
I insert obtained after restriction of plasmid pJCD7 (see Materials and Methods)
was labelled with [[alpha]-
32
P]dATP, and then used in a gel retardation assay. After incubation of this DNA
with a varying amount of [alpha] subunit followed by electrophoresis in a 4% native polyacrylamide gel, a
single DNA-protein complex was detected (Fig.
1
). This result indicates that [alpha] alone can form a
stable
and
specific
complex with the
ace
P DNA fragment, with a binding affinity in the range of that observed for the
rrnB
P1 (see fig. 3 in ref.
18
).
The precise location of the [alpha]-binding site within the
ace
P DNA was determined by the base removal method (
6
). In this approach, the 219 bp end-labelled DNA fragment containing
ace
P was depurinated or depyrimidated at a level of slightly less than one base
removed per DNA fragment. This partially modified DNA was incubated with the [alpha] subunit, then free and complexed DNA were separated by electrophoresis
on a non-denaturing gel. After cleavage at the positions of missing bases, DNA
molecules were separated on a sequencing gel to reveal the positions either
irrelevant or, instead, crucial to binding. Experiments were performed by
removing either G+A residues by treatment with formic acid, or C+T residues by
treatment with hydrazine (
6
,
14
). The corresponding electrophoretic patterns for both top and bottom DNA
strands are shown in Figure
2
A, and a summary of the effects of DNA modifications on [alpha] binding is presented in Figure
3
A. The comparison of band intensity in the lanes of
bound
and
free
DNA in Figure
2
A, indicates that missing bases at positions -41 to -50 in the
ace
P fragment significantly affect [alpha] binding to its DNA target. A weaker effect was observed for bases
spanning positions -31 to -37. All together these results show that (i) isolated [alpha] subunit of RNAP actually behaves as a DNA-binding module (
19
) capable of interacting specifically, by direct contacts, with a limited number
of bases of the UP element in the
ace
operon; (ii) recombinant [alpha] subunit which was purified as a homodimer, partly contacts a region of
the
ace
P that exhibits palindromy (Fig.
3
A). This specific mode of interaction with DNA might be related to the fact that
the [alpha] subunit does not contain the well-characterized helix-turn-helix motif detected in many other
bacterial DNA-binding proteins (
20
); (iii) most of the contacted bases detected by base removal experiments span a
region that overlaps the
ace
operator of the IclR repressor previously characterized by chemical and
enzymatic footprinting (
21
,
22
). Since these two sequences are, in this case, essentially coincident, one can
consider that the negative regulator IclR could partially inhibit transcription
by steric hindrance, i.e
.
, by preventing access of RNAP to the extended promoter of the
ace
operon.
The binding of RNAP to
ace
P was studied by using the same chemical interference approach as that described
above. Figure
2
B shows the autoradiograms of the depurination (G+A) and the depyrimidation
(C+T) footprints obtained for both the top strand and the bottom strand of
ace
P complexed with RNAP. Comparison of the band intensity of bound and free
fractions reveals two distinct regions where a missing base enhances the
affinity of RNAP for its promoter. The first region, between positions -5 and -14 (Fig.
3
B), including the
ace
P -10 hexamer, likely corresponds to the `melting domain' of the initiation
complex. This result is in agreement with the footprinting experiments previously performed with the promoter A1 of the
E.coli
RNAP and with the promoter sites of the RNA polymerases of bacteriophages T3,
T7, and SP6 (
23
,
24
). Of particular interest was the detection of an increased affinity of RNAP for
ace
P when the bases in the region -43 to -73 on either strand of DNA were removed (Figs
2
B and
3
B). The DNA between positions -43 and -62 in this region of the promoter shares common features with the
UP element of the ribosomal
rrnB
P1 (
1
).
These data provide evidence, on the one hand, that the mode of interaction of
RNAP with the UP element of the
ace
P is essentially dependent on the structure of DNA. On the other hand, base
removal interference experiments carried out with isolated [alpha] subunit have shown that only a few direct base contacts occur with a
region of DNA that partially overlaps the UP module. This quite different
behavior suggests that, when it is part of RNAP, the [alpha] subunit can recognize a higher order structure of the UP element rather
than its primary nucleotide sequence. This finding could be connected with the
previous observation that the purified [alpha] subunit of RNAP retains the ability to bind the UP element of the
ada
and
rrnB
P1 promoters (
1
,
25
).
Moreover, the DNA encompassing residues -66 to -73 partially overlaps the 3'-end of a proximal IHF-binding site recently characterized in the
ace
promoter/operator regulatory region (Fig.
2
B) (
26
). In several operons the IHF factor is known to facilitate the loop formation
responsible for the contacts between RNAP and DNA sequences located further
upstream from the
ihf
site. In our case, such a structural role for IHF could be mimicked by the
functional replacement of the IHF-binding site by a DNA of enhanced flexibility. The detection of both an
IHF-binding site and an UP element has been already reported in the case of
the
ilvGMEDA
operon of
E.coli
(
27
). Mutagenesis experiments recently performed on the promoter area have shown
that transcriptional activation of these two regions are functionally
independent (
28
).
Finally, it can be noted that, when they are missing, the four thymines (-92 to -95) in the top strand of the
ace
P
DNA also increase RNAP binding (Fig.
2
B). This long-distance stimulation of the promoter strength was previously detected in
several natural and hybrid promoters, and could be related to the presence of
A+T-rich tracts in the DNA (
29
-
32
).
Specific recognition between the [alpha] subunit of RNAP and the UP-element of
ace
P has been analyzed by
in vitro
transcription. In this assay, the supercoiled plasmid pJCD9 containing the
ace
P region upstream from the
rrnB
transcription termination signal T1T2, was incubated with either wild-type RNAP or mutant RNAP containing the C-truncated [alpha]-256 subunit (
17
). RNA transcripts generated in both reactions were then separated on a 6%
sequencing gel and revealed by autoradiography.
Comparison between tracks in Figure
4
shows that while, on the one hand, an equivalent amount of the control
transcript RNA-1 was synthetized in both cases, on the other hand, the
ace
RNA was only present at a level ~8-fold higher when transcription was initiated with wild-type RNAP. These results therefore demonstrate that the
specific recognition between RNAP and the UP-element is mediated by the C-terminus of the [alpha] subunit which stimulates the
ace
promoter expression.
In order to check the results presented above, base removal experiments were
performed on the
rrnB
P1-RNAP complex. Figure
2
C shows that, in this case again, two sites of increased affinity for RNAP can
be detected when the bases from positions -7 to -15, and from positions -40 to -53, were removed by chemical treatment of the bottom
DNA strand. These regions of functional importance could be related with the
`melting domain' and with the UP module of the
rrnB
P1 promoter, respectively. Indeed, this latter region coincides with the (A+T)-rich [alpha]-binding site which was characterized by DNase I and hydroxyl
radical protection assays (
1
,
2
).
Our results concerning two different UP module-containing promoters suggest that local DNA flexibility generated in the
UP element facilitates its recognition by RNAP. This observation is in
agreement with previous data that demonstrate the ability of intrinsically
curved DNA, upstream the -35 region, to increase the interaction of
E.coli
RNAP with its promoter (
33
,
34
). Moreover, by studying both
in vivo
and
in vitro
a series of mutations within the -10 to -35 region of the
proU
promoter of
Salmonella typhimurium
, it has been suggested that increased DNA flexibility is required for the
activation of these mutant promoters (
35
).
Furthermore, recent work carried out on the spacer DNA, a region that has been
proposed to contact specifically the [alpha] subunit, between the CRP-binding site and the
lac
promoter, has shown that it contains no specific sequence determinant for
lac
transcription activation (
36
).
Taken together, these results could explain the previous failure to define a
precise consensus [alpha]-binding site from all the UP elements detected so far.
This work was supported by grants from the C.N.R.S. (UPR 412), the Université de Lyon and the Institut Universitaire de France. We thank Emmanuelle
Duglas, Christian Van Herrewege and Alain Bosch for their help in preparing the
manuscript.
G+A reaction
C+T reaction
Mix at 0oC
5*106 c.p.m. DNA in 200 [mu]l H2O
5*106 c.p.m. DNA in 200 [mu]l H2O
20 [mu]l formic acid 4%
300 [mu]l hydrazine 98%
Incubate
30 min, 37oC
15 min, 25oC
Add and mix
200 [mu]l NaOAc 300 mM, pH 7.0 and 3 vol ethanol
Precipitate
5 min in liquid nitrogen
Centrifuge
10 min at 20 000 g
Dissolve in
200 [mu]l loading buffer [10% glycerol, 0.1% (w/v) bromophenol blue, and 0.1% (w/v) xylene cyanol]
Electrophoresis in
4% non-denaturing polyacrylamide gel (80:1) in 0.5* TBE buffer
REFERENCES
Return