From the School of Biochemistry, The University of
Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom,
§ Department of Chemistry, University of California, Davis,
California 95616, and ¶ Department of Molecular Genetics, National
Institute of Genetics, Mishima, Shizuoka 411, Japan
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ABSTRACT |
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A cysteine-tethered DNA cleavage agent has been
used to locate the position of region 2.5 of Promoter recognition requires sequence-specific contacts by the
transcriptional apparatus. At most promoters these contacts are made
upstream from the transcription start point. Once the transcriptional
apparatus has bound the promoter to form a closed complex, an
isomerization event occurs to generate the open complex, forming the
single-stranded template required for transcription. The bacterium
Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription
elongation with subunit composition The aim of the study presented in this paper is to complement the
genetic study with biophysical data to support the suggested role of
region 2.5. We wanted to show that, in open, transcriptionally competent complexes at E. coli promoters, region 2.5 of
Strains and Materials--
E.coli strain DH5 Plasmids Encoding Promoter Fragments--
The KAB-TTcon (23) and KAB-TG (24)
promoters were cloned on EcoRI-HindIII fragments
in the galK fusion vector pAA121. The promoter
galP1(19T8A9A) (25) was cloned as an
EcoRI-HindIII fragment in pBR322. The sequence of
the three promoters is shown in Fig. 1.
For FeBABE cleavage reactions and transcription run-off assays,
850-base pair PstI-HindIII fragments were
prepared from plasmid DNA purified on caesium chloride gradients. In
addition to the promoter under study, these fragments also contained
two additional promoters, pX and pbla. For the
FeBABE cleavage analysis, the template strand was labeled at the
HindIII end with [ Proteins--
Single cysteine mutants of Conjugation of Reconstitution of RNA Polymerase Holoenzyme--
A 10-fold molar
excess of DNA Cleavage by FeBABE--
RNAP holoenzyme (300 nM)
was mixed with 32P-end-labeled promoter fragment (0.4 nM) in a reaction volume of 35 µl (20 mM
HEPES, pH 8.0, 5 mM MgCl2, 50 mM
potassium glutamate, 50 µg/ml bovine serum albumin, 5 µg/ml
poly(dI·dC)) and incubated at 37 °C for 20 min. Complexes were
challenged with heparin (200 µM at 37 °C for 5 min).
DNA cleavage was initiated by the addition of sodium ascorbate (2 mM) followed by incubation at 37 °C for 20 min. Modified DNA was extracted with phenol/chloroform and precipitated with ethanol
before analysis on a 6% polyacrylamide gel containing 6 M
urea. Gels were calibrated with Maxam-Gilbert G+A sequence ladders and
were processed and scanned using a PhosphorImager (Molecular Dynamics).
In Vitro Transcription--
The activity of RNAP reconstituted
with wild-type, un-conjugated, and conjugated Modeling of RNA Polymerase-Promoter Interactions--
Modeling
of region 2.5 interactions with promoter DNA is based on secondary
structure prediction and genetic evidence. Secondary structure
prediction suggests that Glu-458 is part of a Construction and Conjugation of
In this study we analyzed the interaction of E. coli RNA
polymerase-carrying FeBABE with different promoters. We chose promoters that were sufficiently strong such that, in our conditions, open complex formation would not be disrupted by the introduction of the
bulky FeBABE probe into region 2.5 of Specificity of FeBABE Cleavage--
In our first experiment, we
examined the cleavage of a labeled PstI-HindIII
fragment purified from pAA121 carrying the KAB-TG promoter (Fig. 1),
using RNAP containing
In our second set of experiments, both strands of the consensus
extended Nontemplate Strand Cleavage--
Fig.
5a shows the patterns of
cleavage on the nontemplate strand of the galP1(19T8A9A),
KAB-TG, and KAB-TTcon promoters in open complexes with RNAP holoenzymes
carrying Template Strand Cleavage--
Fig. 5b shows cleavage on
the template strand of the different promoters by FeBABE, located at
different positions in This study and previous work (37) has shown that the cysteine
residues present in the wild-type The major conclusion from this study is that, in open complexes, region
2.5 of 70 in
transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have
engineered
70 to introduce a unique cysteine residue at
a number of positions in region 2.5. Mutant proteins were purified, and
in each case, the single cysteine residue used as the target for
covalent coupling of the DNA cleavage agent
p-bromoacetamidobenzyl-EDTA·Fe (FeBABE). RNA polymerase
core reconstituted with tagged
derivatives was shown to be
transcriptionally active. Hydroxyl radical-based DNA cleavage mediated
by tethered FeBABE was observed for each derivative of RNA polymerase
in the open complex. Our results show that region 2.5 is in close
proximity to promoter DNA just upstream of the
10 hexamer. This
positioning is independent of promoter sequence. A model for the
interaction of this region of
with promoter DNA is discussed.
INTRODUCTION
Top
Abstract
Introduction
References
2
'. Promoter
specificity is principally afforded by a separate subunit,
, which
associates with the core enzyme to give holoenzyme
(RNAP)1 but dissociates once
sequence-specific promoter DNA contacts are no longer required (1). The
70 subunit, encoded by rpoD, is one of
several
subunits utilized by E. coli and is responsible
for directing the transcription of most genes during vegetative growth.
RNAP is capable of sequence-specific transcription initiation in the
absence of other transcription factors. Factor-independent
transcription is reliant on the ability of
70 to make
stable contacts with the promoter DNA (1, 2). E. coli
promoters contain two very conserved motifs, the
10 and
35 hexamers
(3), and several less-conserved sequences including the UP element (4)
and the extended
10 motif (5). The extended
10 motif
(5'-TGXTATAAT-3') can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within
the
35 region (6, 7). Therefore this TGX motif is able to
compensate for a poor
35 hexamer. The TG motif has been shown to be
important for promoter activity in several other bacterial species
(8-11). Work from many laboratories has defined the regions within RNA
polymerase that are responsible for sequence-specific contacts within
promoter DNA. Regions 2.4 and 4.2 of
70 contact the
10
and
35 hexamers, respectively (1, 2), whereas the C-terminal domain
of the
subunit (
CTD) contacts the UP element (4). Recent work
from this laboratory has indicated that a newly defined region of
70, region 2.5, is responsible for making
sequence-specific contacts with the extended
10 motif (12). This
region was identified by screening for altered or relaxed specificity
mutants of
70 capable of compensating for down-mutations
within the extended
10 motif. One relaxed specificity mutant,
70 E458G, was isolated (12). The E458G substitution
partially suppressed the effect of changing the G·C base pair of the
TG motif, suggesting a role for the side chain at position 458 in contacting the extended
10 motif.
70 is located near to promoter DNA, just upstream of the
10 hexamer. To do this, we exploited a novel method that relies on
tethering a DNA cleavage agent to a single specific amino acid side
chain of a protein (13). The reagent
p-bromoacetamidobenzyl-EDTA·Fe (FeBABE) has one reactive
group facilitating covalent attachment to cysteine side chains, whereas
a second group holds a single metal atom in a tight coordination
complex (14). Under appropriate conditions, the divalent cation can
participate in the generation of hydroxyl radicals, which attack
deoxyribose units, resulting in DNA strand scission (15). Recently,
this chemistry has been applied to the study of the interaction of
CTD of E. coli RNAP with promoter DNA. Hydroxyl radical
DNA cleavage mediated by FeBABE showed that the two
CTD subunits are
arranged asymmetrically, contacting different halves of the UP element
and that activator contact patches are available on both subunits (16,
17). To identify sites on promoter DNA that are near to region 2.5 of
70 in open complexes, amino acids in this region were
replaced with cysteine for conjugation with the DNA cleavage agent FeBABE.
EXPERIMENTAL PROCEDURES
(supE44
lacU169(
80 lacZ
M15)
hsdR17 recA1 endA1 gyrA96
thi-1 relA1) was used for all cloning (18).
Unless indicated otherwise, chemicals were purchased from Sigma,
radiochemicals were from NEN Life Science Products, synthetic
oligonucleotides were from Alta Bioscience at the University of
Birmingham, restriction and DNA-modifying enzymes were from New England
Biolabs and Taq DNA polymerase from Boehringer Mannheim.
70--
Mutants of
rpoD encoding a single cysteine were generated by megaprimer
polymerase chain reaction (19) using plasmid pGEMD(S), which contains a
cysteine-free rpoD gene as a template (20, 21). Mutagenic
oligonucleotides complementary to the template strand of
rpoD used were CYS454
(5'-CCATCCGTATTCCGTGTCACATGATTGAGACCATC-3'), CYS459
(5'-GTGCATATGATTGAGTGTATCAACAAGCTCAAC-3'), and CYS461
(5'-TGATTGAGACCATCTGTAAGCTCAACCGTATT-3'), encoding mutations
corresponding to cysteine substitutions at positions 454, 459, and 461 of
70. In the first round of polymerase chain reaction,
a mutagenic oligonucleotide and oligonucleotide D13346
(5'-GGTCGCAGAATCCAGCGGC-3'), annealing to the coding strand of
rpoD were used to amplify a "megaprimer" fragment of
rpoD. In the second round of polymerase chain reaction, the
megaprimer and oligonucleotide D12444 (5'-GCAGATTAATGATATCAACCGTCGT-3') annealing to the template strand of rpoD were used to
amplify a 540-base pair fragment of rpoD. Plasmid cloning
vector Pinpoint-Xa1 (Promega) was used for direct cloning of polymerase
chain reaction products according to the manufacturer's instructions,
and the recombinants were used for sequencing. A cysteine substitution at position 458 had previously been constructed (22). Restriction enzymes XhoI and BamHI were used to subclone
internal fragments of rpoD containing the desired mutations
into pGEMD(S) (21). Construction of the other single cysteine mutants
used (422C and 581C) are described elsewhere (21).
-32P]ATP and T4
polynucleotide kinase or the nontemplate strand was labeled at the
HindIII end using [
-32P]dATP and the
E. coli DNA polymerase Klenow fragment.
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Fig. 1.
The nucleotide sequence of three promoters
used in this work; KAB-TG, KAB-TTcon, and
galP1(19T8A9A). The promoter consensus recognized by
the E. coli 70 holoenzyme is also shown. The
35 and extended
10 sequence elements are
underlined.
70 were
purified by the method described previously for the wild-type protein
(20, 26), with the important modification of the exclusion of
thiol-containing reducing agents (dithiothreitol or
-mercaptoethanol) from all steps in purification. Purified mutant
subunits were dialyzed against storage buffer (10 mM
Tris-HCl, pH 7.6, at 4 °C, 10 mM MgCl2, 0.1 mM EDTA, 100 mM KCl, and 50% glycerol) and
stored at
20 °C.
70 with FeBABE--
FeBABE was
synthesized and characterized as described previously (27). Single
cysteine derivatives of
70 were used for conjugation
with FeBABE based on the method of Murakami et al. (16).
Conjugation was initiated by mixing 1.2 ml of 6.67 µM
protein solution in conjugation buffer (10 mM Hepes, 200 mM KCl, and 2 mM EDTA, pH of 8.0) with 9 µl
of 18 mM FeBABE in Me2SO. After incubation at
37 °C for 4 h, excess unreacted FeBABE was removed by dialysis
against storage buffer. The efficiency of conjugation was determined by
estimating free side chains of both conjugated and un-conjugated
proteins with the fluorescent reagent CPM
(7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin) (Molecular
Probes) (27).
70 was mixed with core RNAP and incubated at
20 °C for 30 min (26).
70
derivatives was measured by in vitro transcription assays
(28). Fragments used for cleavage analysis were also used as templates for in vitro transcription. The derivatives of the
gal promoter were expected to generate run-off products of
51 nucleotides. DNA template (5 nM) and RNAP holoenzyme
(100 nM) were preincubated in 12 µl of transcription
buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 2.5 mM MgCl2, 50 µM EDTA, 0.5 mM dithiothreitol, 25 µg/ml bovine serum albumin, 2.5%
glycerol) for 5 min at 37 °C. Transcription was initiated by the
addition of unlabeled nucleotide triphosphates ATP, CTP, GTP (200 µM), and UTP (10 µM), 0.5 µCi of
[
-32P]UTP (800 Ci/mmol), 100 µg/ml heparin.
Reactions were stopped by the addition of an equal volume of run-off
stop mix (20 mM EDTA, 80% deionized formamide, 0.1%
bromphenol blue, 0.1% xylene cyanol). Transcripts were analyzed on a
6% polyacrylamide gel containing 6 M urea and scanned
using a PhosphorImager.
-helix starting at
Val-454. An octapeptide from Val-454 to Asn-461 was constructed using
the molecular modeling package Quanta by Molecular Simulations, Inc.
and energy minimized to place amino acid side chains in sterically
favorable positions. This peptide was then manually docked into the
major groove of a model of B-form DNA based on the sequence of KAB-TG
from position +1 to
29 with the carboxyl group of Glu-458 making
base-pair edge hydrogen bond interactions with N6 of adenine at
position
15 and N4 of cytosine at position
14 (i.e. the
template strand of the TG motif). In this position, no steric clashes
were observed. The model was extended to include the helix of region
2.4 (helix 14) derived from the crystal structure of a tryptic fragment
of
70 (29). The atomic coordinates for the fragment
(1SIG) were obtained from the Protein Data Bank (Brookhaven National
Laboratory, Upton, Long
Island2). Genetic studies
show that Gln-437 and Thr-440 are involved in interactions with
position
12 (30, 31). The coordinates obtained were used to dock
helix 14 with residues 437 and 440 in hydrogen-bonding contact with the
base pair edge at
12 (see Fig. 7). The minor groove at the center on
the
10 element must be placed on the inside of a curvature for
efficient promoter recognition by E. coli RNAP (32). In
addition, many studies show that recognition of
10 and
35 elements
is accompanied by promoter bending and suggest that the major groove of
the
10 hexamer widens to accommodate
(33). Such a promoter
structure would allow favorable interactions between basic residues
Arg-441 and Arg-446 and the phosphate backbone (T-A base steps of the
10 promoter consensus element distort double-strand DNA in solution) (34). The model in Fig. 7a places the carboxyl group of
amino acid 448 1.0 nm from the amino group of residue 454. This would allow for a flexible unstructured 7-amino acid loop connecting the two
helices shown. Methods of probing for single-stranded DNA assign
position
12 as the upstream limit of the open complex (35, 36). The
TG motif at position
14/15 would thus remain in a region of
double-stranded DNA, whereas helix 14 is shown within the transcription
bubble (Fig. 7b).
RESULTS
70 Mutants with
FeBABE--
In previous work, we used suppression genetics to identify
a region of the
70 subunit of RNA polymerase, region
2.5, that interacts with the extended
10 motif of bacterial promoters
(12). In this work, we have exploited a tethered DNA-cleaving agent to
show that region 2.5 of
70 is in close proximity to the
extended
10 motif. The reagent used was FeBABE, which is covalently
attached to
70 by conjugation with a cysteine residue.
Starting with an rpoD gene that had been mutated to remove
all three native cysteine codons, single cysteine codons were
introduced at amino acid positions 422, 454, 458, 459, 461, and 581. Residue 422 lies within region 2.3, adjacent to region 2.4, which is
known to contact the
10 hexamer. Residues 454, 458, 459, and 461 are
within region 2.5, postulated to contact the extended
10 motif, and
residue 581 lies in region 4.2, which is known to contact the
35
hexamer element. The FeBABE reagent was conjugated to the single
cysteine proteins, and the efficiency of conjugation was determined.
The FeBABE conjugation yields were: 422C, 46%; 454C, 82%; 458C, 59%, 459C, 60%; 461C, 56%; 581C, 71%.
70. Thus, Figs.
2 and 3
show the transcription activity of RNAP holoenzyme preparations
containing single-cysteine derivatives of
70 before and
after conjugation with FeBABE. With the exception of 422C, all
derivatives retained at least 80% activity compared with wild-type
before conjugation and at least 70% activity after conjugation with
FeBABE.
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Fig. 2.
Transcriptional activity of RNAP carrying
70 derivatives. The figure shows run-off
transcripts from promoters (i) KAB-TG and (ii) KAB-TTcon. The single
cysteine derivative is indicated. Transcripts generated by RNAP
containing
70 tagged with FeBABE are indicated by a
+.
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Fig. 3.
Quantification of the transcription activity
of RNAP carrying 70 derivatives. The activity of
the
70 derivatives relative to wild-type
70 (100%) at KAB-TTcon (a) and KAB-TG
(b). The black bars represent activity of
derivatives before conjugation, and the gray bars show
activity after FeBABE conjugation and correction for the presence of
unconjugated
70.
70 tagged with FeBABE positioned
at 461. This DNA fragment contains the KAB-TG promoter as well as the
pbla and pX (these promoters are located upstream
from the EcoRI site in the pAA121 vector). The results in
Fig. 4 show that cleavage is observed
with holoenzyme reconstituted from FeBABE-conjugated 461C mutant
.
In contrast, no cleavage was observed with holoenzyme containing either
wild-type
70 or unconjugated 461C. The results (Fig. 4,
lane 4) clearly show that cleavage is restricted to the
three promoters. Further analysis revealed that similar patterns of
FeBABE-mediated cleavage are observed at the KAB-TG, pbla,
and pX promoters (data not shown).
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Fig. 4.
DNA cleavage pattern of a promoter fragment
by RNAP carrying FeBABE attached at position 461 of
70. The fragment used contained the KAB-TG,
pbla, and pX promoters. Lane 1, no
protein; lane 2, sequence calibration; lane 3,
core RNAP; lane 4, RNAP containing the 461C derivative of
70; lane 5, RNAP carrying FeBABE at Cys-461.
The three promoters KAB-TG, pbla, and pX are
indicated.
10 promoter galP1(19T8A9A) and the semi-synthetic promoters KAB-TG and KAB-TTcon (Fig. 1) were analyzed for cleavage by
holoenzymes carrying
70 protein tagged with FeBABE at
different positions. The first promoter, galP1(19T8A9A), is
a derivative of galP1, which has been changed to introduce a
consensus
10 element. It is an extended
10 promoter containing a UP
element but has a
35 hexamer with no homology with the consensus
(25). To investigate the effects of a
10 extension on the pattern of
cleavage, we also compared the KAB-TG and KAB-TTcon promoters. These
promoters, which are also derived from the galP1 sequence,
have similar activities. KAB-TG carries an extended
10 motif and
canonical
10 (4/6 fit to consensus) and
35 (5/6 fit to consensus)
hexamer sequences. KAB-TTcon lacks the extended
10 motif but carries
an improved
35 (6/6 fit to consensus) hexamer.
70 tagged with FeBABE at positions 581, 422, 454, 458, 459, and 461. For each holoenzyme, the pattern of cleavage is
similar at all three promoters, with some small variations in the
positions and intensities of cleavage. For example, some upstream
cleavages at the galP1 (19T8A9A) promoter are reduced.
This is consistent with this promoter's lack of homology to the
consensus within the
35 hexamer, leading to weaker interactions with
upstream sequences. However, the overall similarity in cleavage
patterns suggests that the organization of the different parts of
70 is the same, irrespective of the precise promoter
sequence. In contrast, changes in the position of FeBABE in
70 result in marked variations in the positions of
cleavage. Cleavage by FeBABE tethered at positions 581 (region 4.2) and
422 (region 2.3) of
70 have been studied previously in
open complexes at the lacUV5 promoter (37). The data here
are in agreement with those previously published. FeBABE attached to
581C cleaved promoter DNA at positions
44/
45,
34 to
37, and
24 to
26. FeBABE positioned on 422C cleaved promoter DNA with very
low efficiency at position
13/
12. The FeBABE positioned within
region 2.5 (at residues 454, 458, 459, and 461) resulted in cleavage
around
20 for all the
70 derivatives, with additional
cleavages at other positions being dependent on which
70
derivative was studied. The predominant position of cleavage resulting
from FeBABE tethered at 458 is ~
20 for all three promoters; however, additional upstream cleavage around
36 is seen for complexes at the KAB-TG promoter. Cleavage by FeBABE attached to 459C is limited
to DNA around position
20. FeBABE located at 454 cleaves the
nontemplate strand at position
20 but also at
17/
16. Cleavage at
17/
16 is not observed for other
70 derivatives.
FeBABE positioned at 461 again cleaves at
20, but additionally, there
is cleavage from
13 to
1.
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Fig. 5.
Detailed FeBABE-mediated DNA cleavage at
three promoters nontemplate strand (a) and template strand
(b). The positions of FeBABE attachment to
70 are indicated. The promoters studied are:
1, KAB-TTcon; 2, KAB-TG; 3,
galP1(19T8A9A); M, Maxam-Gilbert G+A tracks
(shown as calibration markers).
70. Differences in the positions
and intensities of DNA cleavage were observed compared with the
nontemplate strand. A single region of promoter DNA at positions
13
and
14 is sensitive to hydroxyl radical attack by FeBABE when
attached to all four positions in region 2.5. In addition FeBABE
tethered at 458 and 459 cleaves at positions
21/
22, and 458C
generates more distal cleavage at
38 to
40. FeBABE at 454C also
gives unique cleavage at
18/
19 and, in common with 461C, cleaves
downstream at
7/
8. There is increased cleavage at
7/
8 by FeBABE
positioned at 461. Faint upstream cleavage for FeBABE attached at 461 was seen at
21/22 (common to 458C and 459C). Promoter-specific
differences were seen in the cleavage pattern from FeBABE at 454, with
an increase in the intensity of bands at
8 corresponding to an
alteration of promoter bias toward the
10 element. As previously
observed, FeBABE positioned at 581 in conserved region 4.2 cleaves
promoter DNA from
38 to
41 and at
28/
29, whereas 422C in
conserved region 2.3 gives weak cleavage at
16/
17. Fig.
6 shows the cleavage pattern from both
strands of promoter KAB-TG in schematic form.
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Fig. 6.
Summary of cleavage pattern at KAB-TG.
Boxes above each horizontal line represent
nontemplate strand cleavage. Boxes below the line indicate
template strand DNA cleavage. The "gray scale" reflects
the observed cleavage efficiency of DNA, black being more
efficient. The position of single cysteine replacement in
70 for FeBABE tagging is shown next to each
line. Numbers below the tick marks
provide a reference for the positions of strand cleavage along the DNA
template.
DISCUSSION
70 subunit of RNA
polymerase are not essential for transcription initiation. Thus, it is
possible to introduce cysteine residues in
70 and to
attach the DNA cleavage agent FeBABE and still retain transcriptional
competence. In this work, FeBABE attached to the
70
subunit of RNA polymerase was used to probe open complexes at a set of
related promoters. The first important point to note is that the
overall structure of RNA polymerase bound at promoters carrying
different sequence elements appears to be very similar; the cleavage
data was similar for all three variants of the galP1 promoter (Fig. 1) as well as the pX and pbla
promoters. FeBABE attached to positions 422 in region 2.3 and 581 in
region 4.2 were used as controls in our experiments, and the data are
similar to those observed in a previous study (37). This is consistent with the view that RNA polymerase uses a variety of protein-DNA interactions to form essentially the same open complex at different classes of promoters. Note that the principal information gained from
this technique concerns location, and the relative intensities of
different bands cannot be interpreted to give detailed information about binding mechanisms. For this reason, we chose to work with strong
promoters, where open complex formation would not be hindered by the
bulky substitution of FeBABE.
70 is close to promoter DNA sequences just
upstream of the
10 hexamer. One aim of this work was to propose a
model for the interaction of region 2.5 of
70 with
promoter DNA. Interpretation of the data is complicated by the fact
that the holoenzymes carrying FeBABE-modified
70
proteins are interacting with DNA that is known to be both bent and
unwound. However, Fig. 7 shows possible
models of how regions 2.4 and 2.5 of
70 may interact
with promoter DNA, based on the data obtained from this study and
previous genetic work as discussed below. The similarity of cleavage
pattern observed for FeBABE tethered at 454 and 461 and for FeBABE
tethered at 458 and 459 is consistent with region 2.5 being
-helical. The cleavage pattern seen for FeBABE at 459 indicates that
the reagent contacts the double-stranded promoter DNA around position
20. In the model, consistent with the length of the FeBABE spacer
arm, the
carbon of Thr-459 is approximately 1.2 nm away from the
proposed position of radical release. The FeBABE attached to 459C may
be constrained, allowing it to sit in only one position relative to the
DNA. We propose that position 459 is buried by the helix of region 2.5. FeBABE positioned at 458 results in the same cleavage pattern seen for
459C, but with additional weaker upstream DNA cleavage around the
35
hexamer, which may be because of bending and wrapping of upstream DNA
sequences (33). The cleavage patterns observed with FeBABE positioned at 454 and 461 are more complex. We suggest that this complexity arises
from the location of these side chains in an exposed position in the
region 2.5 helix and the fact that promoter DNA is melted downstream
from position
12 (12, 38).
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Fig. 7.
Modeling of possible interactions between
promoter DNA and 70 subunit of RNA polymerase.
a shows the possible orientation of helix 14 with respect to
the
10 element of promoter DNA. Residues thought to be important for
promoter melting, Tyr-430, Trp-433, and Trp-434 are shown in
purple. The distance of these residues from the site of
nucleation of melting is shown by dotted lines. Residues
Gln-437 (blue) and Thr-440 (green) are shown
within hydrogen-bonding distance (dashed line) of the base
edge of the thymine residue at position
12 of the nontemplate strand.
Basic residues Arg-446 and Arg-441, which may interact with the
negatively charged phosphate backbone of DNA, are shown in light
blue. A schematic representation of the
-carbon backbone of
region 2.5 of
70 (454-461) shows a
-helix docked in
the major groove with Glu-458 (red) in hydrogen-bonding
contact with the base edges of adjacent template strand adenine (
15)
and cytosine (
14). b shows a graphical interpretation of
the cleavage pattern observed by attaching FeBABE to cysteine residues
within region 2.5 of
70. This space-filling model of
region 2.5 shows wild-type amino acid side chain positions that were
substituted by cysteine for FeBABE conjugation: Val-454
(gold), Thr-459 (green), and Asn-461
(red). Glu-458 (blue) is buried and not visible.
On the basis of the cleavage patterns observed, the proposed locations
of free radical generation (i.e. the position of the iron
within FeBABE) are shown as star bursts.
Starbursts are colored to reflect their origins and are
placed 1.2 nm from the
-carbon of the respective amino acid side
chain. The diffusion path of hydroxyl radicals is indicated by
arrows, with the efficiency of cleavage reflected by the
thickness of the line. More distal DNA cleavage
around the
35 hexamer by FeBABE tagged to a cysteine side chain at
position 458 are not shown but are referred to by two horizontal
blue arrows. Helix 14 (29) is shown schematically in close contact
with the nontemplate strand, behind the template strand. The suggested
arrangement of unwound promoter DNA provides an explanation for the
observed cleavage pattern.
The model presented in Fig. 7b shows a conformation for open
complex promoter DNA consistent with the cleavages observed from FeBABE
at positions 454, 458, and 459, and 461. The modeling of region 2.5 as
an -helix is consistent with the data where FeBABE at positions 458 and 459 (100° apart on the
-helix) only cleave promoter DNA
upstream on the TG motif, and FeBABE cleavages from positions 454 and
461 span the TG motif. The FeBABE cleavage upstream of the TG is best
modeled with double-stranded DNA. In contrast, the complex pattern
downstream of the TG motif cannot be modeled on double-stranded DNA,
because the DNA was probed in the open complex. Modeling of region 2.5 as an
-helix in contact with the TG motif has important consequences
for the orientation of
70 and, in particular, region
2.4. Thus, in addition to the results presented here, we used previous
genetic data to orientate both regions 2.4 and 2.5 with respect to the
promoter (12, 30, 31). The aromatic residues Tyr-430 and Trp-433 are
implicated in DNA melting and believed to interact at the
10/
11
positions on promoter DNA. The Gln-437 and Thr-440 residues interact
with the base pair at
12. The Glu-458 residue is involved in the
binding of the extended
10 motif at
14/
15 by region 2.5 of
70. Hence the structures shown in Fig. 7, a
and b, represent proposed orientations of regions 2.4 (helix
14) and 2.5 that can account for both the genetic and the biophysical
data. Note that the orientation of helix 14 proposed in Fig. 7 is
different to that suggested by Owens et al. (37) on the
basis of cleavage patterns generated by FeBABE, located at positions
132, 376, 396, and 422 in
70. Our present data is
insufficient to prove either proposal (because of flexibility both in
the DNA and in
70 just downstream of helix 14). For
example, increased DNA distortion could fit the data presented by Owens
et al. to the models shown in Fig. 7. Similarly our data
could be fitted to the Owens et al. model if a sharp kink is
introduced into
70 between helix 14 and region 2.5.
According to the model presented here (Fig. 7b), the seven
amino acids immediately downstream of helix 14 form a junction between
separate domains of 70 (39). Flexibility of the loop
would allow movement of region 2.4 relative to 2.5 during open complex
formation. In the closed complex, this loop constrains the helix of
region 2.4 relative to promoter DNA in the orientation shown (Fig.
7b). This model shows how region 2.5 can serve as an anchor,
providing a scaffold on which the open complex may be built. These
observations support the idea that the TG motif sets a limit on the
conformational fluctuation of the
10 region (34, 40, 41). This is
consistent with analysis of the temperature dependence of promoter
opening at galP1 and supports a mechanism of open complex
formation whereby melting nucleates around
10 (42, 43). Such a
feature would be of particular importance at extended
10 promoters
that lack an identifiable
35 hexamer.
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ACKNOWLEDGEMENTS |
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We are most grateful to the Wellcome Trust for generous funding of this work with a project grant and to the Royal Society for an Anglo-Japanese Scientific Collaboration Award. We thank Virgil Rhodius for help with the molecular modeling.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.:
+44-121-414-5438; Fax: +44-121-414-7366; E-mail:
s.d.minchin{at}bham.ac.uk.
The abbreviations used are: RNAP, RNA polymerase; FeBABE, p-bromoacetamidobenzyl-EDTA·Fe.
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REFERENCES |
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