Sequences within the DNA Cross-linking Patch of 
54
Involved in Promoter Recognition, Isomerization, and Open Complex
Formation*
Matthew
Chaney
,
Melinda
Pitt, and
Martin
Buck§
From the Department of Biology, Imperial College of Science,
Technology, and Medicine, London SW7 2AZ, United Kingdom
Received for publication, March 14, 2000, and in revised form, April 21, 2000
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ABSTRACT |
The bacterial RNA polymerase holoenzyme
containing the 
54 subunit functions in
enhancer-dependent transcription. Mutagenesis has been used
to probe the function of a sequence in the 54 DNA
binding domain that includes residues that cross-link to promoter DNA.
Several activities of the and holoenzyme are shown to depend on the
cross-linking patch. The patch contributes to promoter binding by
54, and holoenzyme and is involved in
activator-dependent isomerization. As part of the
54-holoenzyme, some residues in the patch limit basal
transcription. Other cross-linking patch sequences appear to limit
activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme
unable to respond to activator but capable of activator-independent
(bypass) transcription in vitro. Overall results are
consistent with the cross-linking patch directing interactions to the
12 promoter region to set basal and activated levels of transcription.
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INTRODUCTION |
factors play a pivotal role in bacterial transcription.
Through their association with core RNA polymerase they function in
promoter-specific initiation. DNA contact by is required for
promoter recognition and can contribute to the DNA opening event in
open promoter complex formation. Two classes of factor exist,
apparently unrelated by sequence (1, 2). Members of the class related
to the 70 of Escherichia coli form
holoenzymes that are active for transcription in the absence of
accessory proteins (3, 4). In contrast, the 54-type
factor forms a holoenzyme silent for transcription until acted upon by
an enhancer binding activator protein. In a reaction requiring
nucleoside triphosphate hydrolysis by the activator protein, the
54-holoenzyme bound in a closed complex isomerizes, and
start site proximal DNA is melted out to form a stable open promoter
complex (5-9). Thus, one core RNA polymerase can participate in two
distinct mechanisms of transcription initiation.
Key functional activities of 54 are arranged in discrete
sequences across three regions identified by sequence alignment (Fig. 1). The amino-terminal Region I includes
sequences that are essential for enhancer responsiveness, whereas
Region II sequences assist in promoter recognition (9-12). Region III
contains determinants for core polymerase binding at its amino end and
a domain containing motifs for DNA binding toward the carboxyl-terminal
end (13, 14). The 54-holoenzyme recognizes promoters
with conserved sequences at 24 and 12 relative to the transcription
start site, and contacts these sequences (15). Determining
the amino acids in that direct promoter binding is important for
understanding how the initial specific closed complex is formed.
Additionally, whether -DNA contacts contribute to post binding steps
such as DNA melting can be evaluated. Biochemical and genetic
experiments with Klebsiella pneumoniae and E. coli 54 has shown that the major DNA-contacting
surfaces of 54 are located in a carboxyl-terminal
domain of approximately 150 amino acids (14-17). Within the
holoenzyme-promoter closed complex, DNA sequences that must be melted
to form open complex are contacted by 54 (14, 15, 17,
18). The DNA binding domain is likely to be functionally involved in
open complex formation.
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Fig. 1.
K. pneumoniae
54 is divided into three regions
(I-III) based on sequence alignments (2). The amino-terminal
Region I is required for response to activator and masks
single-stranded DNA binding activity in the
54-holoenzyme (9, 39, 40). Region II is acidic and
variable among members of the 54 protein family. Region
III includes the core RNA polymerase binding sequences and many DNA
binding determinants: the sequence that cross-links to promoter DNA
(X-L), a helix-turn-helix motif (HTH), and the
conserved eight-residue sequence termed the RpoN box and sequences that
modulate DNA binding (MOD) (17, 21, 41, 42). The amino acid
sequence of the cross-linking patch is shown. Conserved residues that
have been mutated are underlined
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Cross-linking methods are useful for identifying -DNA interacting
domains. Native promoter DNA can be specifically cross-linked to
54 and its holoenzyme by a "zero length" UV
light-catalyzed reaction (19). The DNA cross-linking patch of
54, composed of residues 329-346 (17), sits at the
amino terminus of the DNA binding domain and is linked by a predicted
surface-exposed element to an adjacent domain that enhances DNA binding
activity (20, 21). We have shown that the invariant residue Arg-336, within the cross-linking patch, is important for maintaining the holoenzyme as a closed complex, silent for transcription before activation (22). Deletion of Region I (
I) or mutation of Arg-336, each, alter the interaction of holoenzyme with 12 promoter sequences and allow the holoenzyme to isomerize independent of activator (9, 22,
23). Region I binds core polymerase, and deletion of Region I alters
the conformation of the carboxyl-terminal DNA binding domain of
54 as part of the holoenzyme, suggesting a linked
interaction between amino and carboxyl parts of 54 (13,
24).
Identification of 54 sequences involved in interaction
with promoter DNA provides a basis for understanding the mechanism of transcription initiation by enhancer binding activators. To further explore the involvement of the DNA cross-linking patch in
54 function, conserved residues within the patch were
mutated. Single amino acid substitutions were obtained that led to
defects in promoter binding, isomerization, and
activator-dependent transcription. We also deleted the
adjacent surface-exposed element, amino acids 310-328, which links the
cross-linking patch with a domain that enhances DNA binding (20, 21).
Deletion of the linking element resulted in holoenzyme unable to form
open complex but able to initiate transcription in the absence of
activator from supercoiled DNA.
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MATERIALS AND METHODS |
DNA Manipulations--
The K. pneumoniae rpoN gene
codons 328 (Trp), 338 (Asp), and 342 (Arg) were changed to CGA (Ala);
codon 340 (Leu), in line with other substitutions of Leu in
54 (10), was changed to AGC (Ser); and codons 310-328
were deleted using the Sculptor in vitro mutagenesis system
(Amersham Pharmacia Biotech) to mutate a carboxyl-terminal-coding
SacI-HindIII fragment from pMM70 (25), cloned in
M13mp18. Replacement cloning of a carboxyl-terminal-encoding pMM70
BamHI-HindIII fragment and DNA sequencing were
used to confirm the structure of the altered rpoN genes. For
overexpression, BamHI-HindIII fragments were
replacement-cloned into pWVC93025 (14).
Growth and 
-Galactosidase Assays--
Mutant rpoN
genes were recloned into the low copy-number plasmid pHSG576
(43) and transformed into the rpoN deletion strain of
K. pneumoniae UNF2792 (hisD2,
rpoN71::kan, recA56,
sbl300::Tn10) (26). For the growth assay, colonies
were replicated from Luria agar plates onto nitrogen-free Davis minimal
agar (NFDM) plates (44) supplemented with 1 mg/ml
arginine or ammonium sulfate and grown overnight at 37 °C. The
-galactosidase assays were performed by inoculating 4.0 ml of NFDM
containing appropriate supplements with 0.1 ml of overnight nutrient
broth culture and grown under anaerobic conditions for 18 h at
30 °C. Chromosomal ntrC and nifA expression
was activated by supplementing NFDM with 100 µg/ml aspartic acid or
repressed with 200 µg/ml ammonium sulfate. Assays were performed in
duplicate. Test promoters were provided on the following plasmids (see
also Table II): K. pneumoniae nifH by pMB1, K. pneumoniae nifH049 by pWVC88049, K. pneumoniae nifH050 by pWVC88050, K. pneumoniae nifH053 by pWVC88053,
and K. pneumoniae nifH054 by pWVC88054 (27).
Immunoblotting--
Mutant plasmids were transformed into
K. pneumoniae UNF2792, and transformed cells were grown
overnight. Cells (1 ml) were collected by centrifugation and
resuspended in 60 µl of 10 mM Tris, 0.1 mM
EDTA, pH 7.9; 10 µl of concentrated cells was lysed with 10 µl of
2× SDS sample buffer and heated to 95 °C, and 6 µl of each sample
was loaded. Proteins were resolved on denaturing SDS-7.5%
polyacrylamide gel electrophoresis mini-gels and blotted on
polyvinylidene difluoride membrane. Anti-54 (a gift from
A. Ishihama (28)) and alkaline phosphatase-conjugated anti-rabbit IgG
(Promega) antibodies were used for detection.
Protein Preparations--
All mutant 54 proteins
except W328A were overproduced and re-folded from urea as described
previously (15), except that induction was at 42 °C. Re-folded
protein was chromatographed on DEAE-cellulose at pH 7.0 in 20 mM imidazole buffer and eluted with a NaCl gradient. Purified protein was stored in 50% glycerol, 100 mM NaCl,
10 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol,
0.1 mM EDTA at 20 °C. The L340S protein did not bind
to DEAE-cellulose and was chromatographed on heparin (Hi-Trap, Amersham
Pharmacia Biotech).
In initial overexpression trials, only W328A was found as soluble
protein. The W328A protein was expressed in the tryptophan auxotroph
E. coli CY15077 grown in Luria broth at 30 °C. Cells were
disrupted by French pressure cell treatment, and the protein was
purified at 4 °C by the same procedures used for wild type 54 (14). Briefly, the cell-free extract was subjected to
streptomycin sulfate precipitation to remove nucleic acid and then
ammonium sulfate fractionation to recover the W328A protein as a
precipitate. This was followed by DEAE-Sepharose chromatography,
heparin (Hi-Trap, Amersham Pharmacia Biotech), and finally strong anion
exchange chromatography. The protein was stored as above except NaCl
was at 50 mM.
Open Complex Assay--
The DNA used in the assay was a 650-base
pair fragment containing the Sinorhizobium meliloti nifH
promoter and NifA upstream activation sequences, prepared by
polymerase chain reaction amplification of the promoter in pMB210 (27)
and gel-purified. Reactions contained 5 nM
[32P]DNA, 50 nM E. coli
core RNA polymerase (Epicentre Technologies), 25-400 nM
54, 3.8 µM PspFHTH (29), and 1 mM GTP in STA buffer (25 mM Tris acetate, pH
8.0, 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% w/v polyethylene glycol 6000).
Core and 54 were added to the DNA and incubated at
30 °C for 5 min before the addition of activator and GTP. At 15 min,
the reaction was stopped by the addition of 3 µl of stop mix (50%
glycerol, 600 µg/ml heparin, and 0.05% w/v bromphenol blue) and
gently mixed. Reactions were directly loaded onto 4% native
polyacrylamide Bio-Rad mini-protean II gels and run at room temperature
at 60 V in 25 mM Tris, 200 mM glycine buffer,
pH 8.6, for 2 h. [32P]DNA was detected by a
phosphorimaging device. Reactions were replicated to enhance
reliability of the data.
Transcription Assays--
S. meliloti nifH promoter
DNA as plasmid pMKC28 was used as the template in transcription
experiments (22). Assays (at 30 °C) contained holoenzyme assembled
from 100 nM E. coli core RNA polymerase and a
6-fold excess of 54. Pre-incubation of promoter DNA (20 nM) and holoenzyme was for 10 min in STA buffer with ATP (4 mM) and activator protein PspFHTH (4 µM)
unless otherwise stated in the figure legend. A mixture of the
remaining 3 NTPs (GTP and CTP 0.2 mM, UTP 0.05 mM) and 1.5 µCi of [
-32P]UTP plus
100 µg/ml heparin was then added for a further 10 min. Reactions were
stopped by mixing in formamide-sequencing loading buffer and heated to
90 °C, and half the sample was directly run on 6% denaturing
sequencing gels. Reactions were replicated to enhance reliability of
the data. Transcripts were detected by phosphorimaging device.
Transcription from nifH on pMKC28 yields a 469-base product.
Gel-shift Assays--
These were conducted using linear
end-labeled S. meliloti nifH promoter fragments as described
previously (9, 30). Reactions were replicated to enhance reliability of
the data. Either an 88-mer homoduplex or heteroduplex molecule from
60 to +28, the latter with the 10 to 1 or 12/11 non-template
strand sequence mismatched, was used. One strand was
[32P]DNA, and the other unlabeled strand was
annealed at a 2-fold excess. Binding reactions with DNA (2.5 nM) were in STA buffer at 30 °C, and bound and unbound
DNA was resolved on 4-4.5% native polyacrylamide Bio-Rad mini-protean
II gels run at room temperature at 60 V in 25 mM Tris, 200 mM glycine buffer, pH 8.6.
DNA Footprints--
These were conducted on linear DNA fragments
generated by primer extension of single-stranded DNA derived from
M13mp19 clones of the S. meliloti nifH promoter (9, 23).
End-labeled universal sequencing primer was extended by Klenow
polymerase. Footprints were as before and used either S1 nuclease (9)
or ortho-copper phenanthroline
(o-Cup)1 (23).
Protein-Promoter Cross-linking--
This was conducted as
described previously (17). Briefly 54 (4 µM) and labeled 21-base pair (30 to 10) S. meliloti nifH promoter duplex (100 nM) were incubated
together in STA buffer with 0.34 µg/µl salmon sperm DNA at 30 °C
for 10 min, chilled on ice, and irradiated with a 254-nm light source
for 20 min on ice. The cross-linking reactions were warmed to 37 °C
and resolved on denaturing SDS-7.5% polyacrylamide gel electrophoresis
mini gels. Reactions were replicated to enhance reliability of the data. Cross-linking products were detected by a phosphorimaging device.
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RESULTS |
Mutations in 54--
Enzyme and chemical cleavage
of 54 suggested that the patch of 54 that
can be UV-cross-linked to promoter DNA resided between or included
residues Trp-328 to Cys-346 (14, 17). To further study the importance
of this patch for 54 function, single amino acid
substitutions of conserved residues W328A, R336A, D338A, L340S, R342A,
and deletion of an adjacent surface-exposed region 310-328 were
created by site-directed mutagenesis. Preparation of R336A has been
described previously (22). In each case the integrity of the mutated
rpoN was established by DNA sequencing. Immunoblots using
polyclonal anti-54 antibody confirmed that all mutants
were expressed as intact protein (data not shown).
In Vivo Function and Transcription Activities of the
54 Mutants--
Initially the ability of each mutant
54 to complement an rpoN
K. pneumoniae mutant for growth on arginine as a nitrogen source was tested (Table I). The 310-328
mutant did not allow growth, whereas L340S allowed approximately 25%
growth compared with wild type. The mutants W328A, D338A, and R342A
were largely unimpaired for growth. Using the
rpoN K. pneumoniae strain, each
54 mutant was tested for transcription activity using
54-dependent reporter promoters fused to
lacZ. Five test promoters were used to evaluate any
promoter-specific effects: the wild type K. pneumoniae nifH
and mutants nifH049, nifH050,
nifH053, and nifH054, all of which respond
to activator NifA. The nifH049, -050,
-053, and -054 promoters are mutants of the
K. pneumoniae nifH promoter (see Table
II) with changed affinities for
54 (18). Bacteria were grown under anaerobic
nitrogen-limiting conditions to induce synthesis and activity of
chromosomal nifA. Overall, the in vivo
transcription results mirror the initial growth test. 310-328 gave
no detectable transcription activity, whereas L340S produced
significantly lower transcription activity compared with wild type
54-holoenzyme at all promoters (Table II, data in
parentheses). At the K. pneumoniae nifH promoter, all
mutants gave reduced levels of activity compared with wild type (Table
II, column 2, data in parentheses). At the K. pneumoniae
nifH049 promoter, all point mutant transcription activity
increased with D338A and R342A, producing close to wild type level
(Table II, column 3, data in parentheses). At the nifH049
promoter, the activity of L340S, although low, was 4-fold that seen at
the K. pneumoniae nifH promoter. As discussed previously
(22), the "T-tract" in the nifH049 promoter may be
compensating for a lost or reduced contact made by 54 as
part of the holoenzyme (18). Greater affinity for the promoter could
increase occupancy and potentially favor initiation by holoenzymes, which form open complexes slowly.
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Table I
Complementation for growth on arginine in an rpoN K. pneumoniae mutant
+++ indicates growth judged comparable with cells containing the wild
type rpoN. + indicates poor growth, and indicates no
growth.
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Table II
-Galactosidase activity from the K. pneumoniae nifH mutant promoter
series
The mean activity of wild type and each mutant 54-holoenzyme
at the wild type K. pneumoniae nifH promoter was set at 1. -Galactosidase activity at the mutant promoters is shown to the
nearest fold difference. The actual mean values for -galactosidase
activity in Miller units at the wild type K. pneumoniae nifH
promoter were: wild type, 19852; W328A, 14529; D338A, 18392; L340S,
536; R342A, 13955. The 17 to 11 sequence of each promoter is
indicated with the mutated base in bold. Values for each mutant at the
wild type K. pneumoniae nifH and mutant promoters, compared
with the mean -galactosidase activity of wild type
54-holoenzyme taken as 100%, are shown in parentheses.
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To further explore the stimulatory effect of the T-tract we used a
K. pneumoniae nifH mutant promoter series in which each C
from 17 to 15 has individually been replaced by T (27) (Table II).
These data show that for wild type and all mutant
54-holoenzymes, a C to T change at 17 or 15 produces
at least a 2-fold increase in transcription activity and is at least as stimulatory for transcription as the 17 to 15 CCC to TTT change (Table II). Except for L340S, which showed a 3-fold increase in activity, the C to T change at 16 was not stimulatory for
transcription. The L340S 54-holoenzyme, which has the
lowest activity compared with wild type 54-holoenzyme
(Table II, data in parentheses), was highly stimulated by the C to T
change at 15, producing a 10-fold increase above its activity at the
wild type nifH promoter. In vivo experiments using a consensus promoter sequence where the consensus 12 core promoter sequence (TTGC) was randomly mutated suggested that the 15
consensus T was important for maintaining promoter function (31). The
results suggest that the L340S mutation may cause a disruption in
correct recognition and/or binding to the K. pneumoniae nifH-12 promoter region, which is to some degree rescued by the C
to T change at 15.
Overall the in vivo analysis revealed that substitutions in
the cross-linking patch and deletion of adjacent residues leads to
three classes of mutant: class I, no transcription activity (310-328); class II, reduced transcription activity (L340S); and
class III, significant levels of activity stimulated further by a high
affinity promoter. To facilitate further analysis of any defects in the
mutant s, proteins were overexpressed and purified. Each
54 was judged to be >90% pure by SDS-polyacrylamide
gel electrophoresis.
Core Binding--
An essential early step in transcription is the
binding of to core to form the holoenzyme. To determine whether any
mutation grossly altered the interaction of with core, a native gel
holoenzyme assembly assay was conducted. With the exception of L340S,
each mutant shifted all core at a molar ratio of 1:1
(54:core), indicating that core binding was intact and
could not readily account for the defects in transcription seen
in vivo (data not shown). In contrast to the other mutants
and wild type 54, L340S and its holoenzyme did not enter
the native gel, indicating this protein has altered physical
properties. All other mutant holoenzymes appeared to have a mobility
indistinguishable from wild type holoenzyme. This suggests that, with
the exception of L340S, the conformation of the holoenzyme is not
grossly changed by the cross-link patch substitution or deletion of
residues 310-328. The native gel also showed that free-running W328A
54 (not associated with core) has increased mobility
compared with wild type 54, suggesting that the W328A
substitution has altered the conformation of the 54
protein (data not shown).
54 Interactions with Native
DNA--
54 binds the S. meliloti nifH
promoter in the absence of core polymerase (18). To evaluate the
effects of mutations on 54 DNA binding, gel-shift assays
using S. meliloti nifH promoter DNA were conducted.
Titration of the mutant 54 showed that mutations W328A
and R342A each reduce DNA binding, whereas 310-328 and D338A were
essentially wild type for DNA binding in this assay (Fig.
2A). Wild type
54 shifted half the DNA at 1.5 µM, whereas
W328A and R342A shifted approximately half the probe at 4 µM. L340S did not detectably bind the DNA; however, it
could not be properly assayed because of its aberrant gel mobility. The
lack of DNA binding by L340S may therefore be related to structural
defects (as indicated by native gel mobility) rather than loss of
an essential DNA contact.
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Fig. 2.
A, DNA binding activity of mutant
54 compared with wild type (WT)
54 on the S. meliloti nifH promoter in a gel
mobility shift assay. B, UV cross-linking of mutant and wild
type 54 to a 21-base pair S. meliloti nifH
promoter fragment. The arrow indicates the lack of a lower
mobility product in the W328A cross-linking reaction. C,
ortho-copper phenanthroline footprint of 54
protein at the S. meliloti nifH promoter. The
arrow indicates loss of the hypersensitive site at 5 in
the W328A 54 footprint. All 54 proteins
were present at a concentration judged to bind 50% of the DNA in the
gel mobility shift assay (A). Lane 1, DNA alone.
Lane 2, wild type 54. Lane 3,
W328A
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The reduced DNA binding of W328A and R342A indicates that they are
functionally important residues within the DNA cross-linking patch of
54 that contribute to DNA binding. We directly confirmed
this by conducting a DNA cross-linking assay (Fig. 2B). The
formation of two products in the cross-linking assay probably reflects
stable cross-linking of 54 to either both strands or one
strand of the DNA (17). Interestingly R342A, which showed reduced DNA
binding in the gel-shift assay, gave wild type levels of cross-link
product, indicating that residue Arg-342 is less important for the
cross-linking reaction (Fig. 2B, compare lanes 1 and 6). W328A consistently produced only the lower band
cross-link product (Fig. 2B, lane 3). Trp-328 may
be important for a DNA interaction necessary to form the slower running cross-link product, or the altered conformation of the W328A mutant, as
suggested by its native gel mobility, may result in the loss of the
slower running cross-link product. It is notable that the 310-328
54 protein, which lacks residue Trp-328, was unlike the
W328A mutant and not defective for DNA binding or UV-cross-linking
(Fig. 2B, lane 2). The effect of the W328A
mutation upon DNA binding appears hidden in the 310-328 protein.
This might be because deletion of residues 310-328 reveals further DNA
binding activity in the 54 protein (21), or via
310-328, a change in Region I position has occurred to unmask DNA
binding (11).
The interaction of the mutant 54 with the S. meliloti nifH promoter was further investigated by DNase I, S1,
and o-Cup footprinting. In the DNase I and S1 assays, all
mutant 54 gave footprints indistinguishable from wild
type 54 (data not shown). In the o-Cup
footprinting assay, D338A and R342A 54 gave similar
footprints to wild type 54 (data not shown).
o-Cup footprints of W328A and 310-328 54
did not show the typical cut at 5 on the top strand, suggesting that
Trp-328 is involved in a start site proximal promoter interaction (Fig.
2C).
Taken together, the gel-shift, DNA-cross-linking, and o-Cup
footprinting assays establish that residues W328A and R342A both contribute to the DNA binding activity of 54 and that
each mutant has a different qualitative and quantitative effect on the
54 DNA interaction.
54 Interactions with Melted DNA--
Artificially
melted DNA at 12/11 simulates the nucleation of promoter DNA
melting seen within the natural holoenzyme closed complex (23). The
12/11 position was identified as a nucleation site by
o-Cup footprinting and by gel mobility assays with DNA fork
junction probes (23, 32). Heteroduplex S. meliloti nifH DNA,
where the top strand bases at 12 and 11 are mismatched, was used to
investigate the interactions of the mutant 54 with the
12 promoter region early melted DNA. As with fork junction DNA, where
the bottom strand thymine residue is unpaired compared with
double-stranded DNA, the 12/11 early melted DNA was preferred for
wild type binding (32). Like wild type 54, mutants
W328A, D338A, and R342A all showed a preference for binding the early
melted DNA (Fig. 3). In contrast, the
310-328 54 showed no preference for binding the
early melted DNA and bound it less well than native double-stranded DNA
(Fig. 3).
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Fig. 3.
DNA binding activity of mutant
54 compared with wild type
(WT) on the S. meliloti nifH promoter
using native 88-mer, early melted (12/11) and late melted
(10/1) heteroduplex 88-mer DNA. Values shown are for percent
DNA bound with 54 proteins at 1 µM.
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A second assay to examine whether the 54 interaction
with melted DNA changed was performed by measuring the ability of to bind heteroduplex DNA that is mismatched from 1 to 10, DNA which is melted in the later stages of open complex formation (9, 22). Wild
type and mutant 54 including 310-328
54 showed a preference for binding the 10/1
late-melted DNA (Fig. 3). It seems that 54 has a modest
preference for binding to melted DNA structures that are normally only
created when holoenzyme is bound to the promoter. This suggests that,
normally as part of the holoenzyme, 54 is likely to be
intimately involved in the initiation and maintenance of the DNA strand
separation needed for open complex formation (9, 22, 32). Apparently
the cross-linking patch mutants assayed here, unlike 310-328
54, do not change the binding preference of
54 but do contribute to overall binding activity.
54 Isomerization--
When activator and
hydrolyzable nucleoside triphosphate are added to bound to early
melted DNA, a new 54-DNA complex results (the
supershifted band) with an extended DNase I footprint indicating
that activator interacts with and causes it to isomerize and
increase its interaction with
DNA.2 All cross-linking patch
mutant s were tested for isomerization. For activation we used a
deletion mutant form of the activator PspF that lacks its DNA binding
domain, PspFHTH (29), to simplify the gel-shift assay. In the
isomerization assay, mutants D338A and R342A 54 behaved
as wild type (data not shown). The 310-328 54 mutant
did not respond to activator to produce a supershift (data not shown),
whereas W328A produced only a small amount of supershift (Fig.
4). The lack of any supershifted band for
310-328 is found also with 54 lacking Region I and
correlates with an inactivity for transcription in
vivo.2 The defective response of W328A to activator in
the supershift assay was investigated further. Titration of the W328A
protein did not increase the amount of supershifted complex (Fig. 4), nor was the amount of supershifting DNA increased by longer incubation times (data not shown). Replacing PspF with the activator NifA, increasing the amount of PspF, or increasing the amount of DNA probe in
the reaction did not result in an increased conversion of DNA-bound
54 to the isomerized form (data not shown). Exposing the
W328A protein to PspF and nucleotide before adding the DNA did not
detectably change the percentage of supershifted product (data not
shown). Overall, these results show that the W328A protein is unable to be rescued for 54 isomerization. Whether its altered
conformation or its changed DNA interaction per se, detected
by o-Cup footprint (Fig. 2C), reduces isomerization is not clear. Nonetheless, Trp-328 contributes to
54 isomerization.
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Fig. 4.
Gel-shift mobility assay for
54 isomerization on the 88-mer early melted
(12/11) heteroduplex. Plots indicate the percentage of DNA
bound with normal mobility (54-DNA) and the percentage
of DNA bound with lower mobility
(ss54-DNA) in response to the
activator and nucleotide. The free DNA is not shown. Conditions
were as for other gel-shift assays except PspFHTH (4 µM) and dGTP (4 µM) were also added.
54 proteins were at 1 to 6 µM.
WT, wild type.
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Holoenzyme Interaction with Native DNA--
Gel-shift assays using
the S. meliloti nifH promoter 88-mer were conducted to study
the effect of the 54 mutants on holoenzyme DNA binding.
The mutant W328A that showed decreased 54 DNA binding
activity formed holoenzyme that bound DNA with approximately the same
affinity as wild type holoenzyme, suggesting that core was still able
to stabilize the on DNA (Fig. 5). In
contrast, the R342A holoenzyme showed a decrease in DNA binding
compared with wild type. At 80 nM, the wild type holoenzyme
bound 39% of the DNA, whereas the R342A holoenzyme bound 17% (Fig.
5). This result suggests that residue Arg-342 makes an important
contribution to the DNA binding energy of the holoenzyme in the closed
complex, consistent with the reduced R342A 54 DNA
binding (Fig. 2A). The 310-328 and L340S holoenzymes
bound approximately 2-fold more of the DNA probe than wild type
holoenzyme, approximately equivalent to the DNA binding activities
observed for deleted Region I 54 and deleted Regions I
and II 54 (11). This suggests that these mutants may
have allowed the holoenzyme to form a conformation equivalent to that
of the deleted Region I 54-holoenzyme, which has an
extended DNA contact (9). Because L340S 54 alone does
not bind DNA and has altered physical properties (aberrant mobility on
native gel), the above result suggests that on binding to core to form
the holoenzyme, L340S is reconfigured and revealed to have much of its
DNA binding activity intact. The interaction with promoter DNA
apparently stabilizes a more native conformation of the L340S
holoenzyme. It seems L340S alters 54 conformation but in
a way readily rescued in the holoenzyme DNA complex. On association
with core RNA polymerase to form the holoenzyme, the conformation of
54 is altered (20, 24). The mutants W328A, L340S, and
310-328 may be changed for core-dependent DNA
binding.
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Fig. 5.
DNA binding activity of mutant holoenzyme
compared with wild type holoenzyme on the S. meliloti nifH
promoter 88-mer DNA. Data shown is for holoenzymes
(E54) at 40 nM.
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Holoenzyme Interaction with Melted DNA--
Closed promoter
complexes formed with the 54-holoenzyme are very
heparin-sensitive, but holoenzyme binding to early melted DNA results
in an increased stability toward heparin (30). Stability may reflect
altered 54-core interactions, which are normally
disrupted by heparin (13). Heparin stability of holoenzymes on the
early melted DNA was tested to evaluate the contribution of the
cross-linking patch to stability. All mutant holoenzymes except
310-328 bound the DNA with an affinity similar to wild type
54-holoenzyme with between 70 and 85% of the DNA bound
with 100 nM holoenzyme (Fig.
6, top). The 310-328
holoenzyme bound about 56% of the probe (Fig. 6). The
holoenzyme-DNA complex was then challenged for 5 min with the polyanion
heparin. Of the initial complex formed for wild type, W328A, D338A, and
R342A holoenzymes, 37-50% of the complex was heparin-stable after 5 min (Fig. 6). For the 310-328 and L340S holoenzymes, only 7 and
23% of the complexes were heparin-stable after 5 min. This suggests
that the 310-328 and L340S 54 disrupt holoenzyme
interactions in the 12/11 promoter region, which leads to heparin
stability. For 310-328, the defect may be in DNA binding to early
melted DNA, for L340S in core interactions (see above). In both cases,
defects in stability on the early melted DNA correlate with an
activator-independent transcription activity (Ref. 32 and see
below).
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Fig. 6.
Heparin stability of mutant holoenzyme on
early melted (12/11) S. meliloti nifH 88-mer
heteroduplex DNA. The top section shows the amount of DNA probe
bound before () and 5 min after (+) the addition of heparin. The top
band in the L340S () lane has the mobility of DNA probe
bound by free core (E). The graphed data shows
the percentage of DNA still bound by holoenzyme
(E54) after the 5-min heparin challenge.
WT, wild type.
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Holoenzyme binding to late melted (10/1) DNA was assayed. Wild type
holoenzyme is able to form a heparin-stable interaction with this
premelted DNA template when activated. The 310-328, as shown for
deleted Region I 54 and the R336A holoenzyme (10, 22),
was able to form a heparin-stable complex on premelted DNA in the
absence of activation (data not shown). This result suggests the
310-328 54, like deleted Region I 54
and R336A 54, may result in activator-independent
holoenzyme isomerization (9, 22). The unactivated L340S holoenzyme on
the late melted DNA gave slightly increased stability toward heparin
compared with wild type holoenzyme (data not shown), suggesting an
improved interaction with single strand DNA and a low level of
holoenzyme isomerization. The mutants W328A, D338A, and R342A
holoenzymes behaved like wild type holoenzyme and did not form
heparin-stable complexes on the late melted DNA in the absence of
activator and NTP (data not shown).
Holoenzyme Footprinting--
As previously reported for holoenzyme
with 54 lacking its Region I sequence and the R336A
holoenzyme, there is a correlation between activator-independent
heparin-stable binding of holoenzyme to late melted DNA (10/1
bubble), activator bypass transcription, and altered holoenzyme-DNA
interactions at start site and 12 proximal promoter sequences as
detected by S1 and o-Cup footprints (9, 22). In the S1
footprint assay, only 310-328 holoenzyme gave the extended
footprint to +20 (Fig. 7A,
lane 4), equivalent to that made by activated wild type
54-holoenzyme or the deregulated deleted Region I
54 and R336A holoenzymes (Fig. 7A,
lanes 3 and 6) suggested to be related to
holoenzyme isomerization and single-stranded DNA binding activity (9,
22). The L340S holoenzyme did not give the extended footprint but was
different from wild type holoenzyme in that it did not protect the DNA
from cutting at 10 (Fig. 7A, lane 8).
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Fig. 7.
A, S1 footprint of holoenzymes at the
S. meliloti nifH promoter. The extended footprint of the
310-328 (lane 4), R336A (lane 6), and Region
I deleted (I) (lane 3) holoenzymes is indicated by the
dotted line. I and R336A holoenzymes are included as
positive controls for the extended footprint. The arrow
indicates sensitivity at 10. Lane 0, DNA alone. Lane
1, core RNA polymerase. B, ortho-copper
phenanthroline footprint. The arrow indicates the loss of
the hypersensitive site at 12 in the 310-328 (lane 4)
and L340S (lane 8) holoenzyme footprints. I (lane
3) and R336A (lane 6) holoenzymes are positive controls
for loss of the hypersensitive site. Lane 0, DNA alone.
Lane 1, core RNA polymerase. WT, wild type.
E, core RNA polymerase.
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When the holoenzymes were footprinted with o-Cup, both
310-328 and L340S holoenzymes gave footprints in which the
hypersensitive site around 12 was absent (Fig. 7B,
lanes 4 and 8) equivalent to that made by deleted
Region I 54 and R336A holoenzymes (Fig. 7B,
lanes 3 and 6). The W328A holoenzyme and the
other mutants produced footprints similar to the wild type holoenzyme.
The 12 site is suggested to be where nucleation of DNA melting occurs
(23) and is important for maintaining the inhibited state of the
holoenzyme by preventing polymerase isomerization (32). With wild type
54-holoenzyme, reactivity at 12 is diminished upon
open complex formation (23). The L340S holoenzyme would appear to be
similar to the 310-328, deleted Region I 54, and
R336A holoenzymes in that it has lost the o-Cup
12-sensitive site but different in that its S1 footprint (and binding
to late melted DNA) shows it has not isomerized to the same extent.
This suggests that some interactions with the promoter 12 sequence can be separated from other interactions that, when altered, result in
efficient activator-independent isomerization of the holoenzyme.
In Vitro Transcription--
For the 54-holoenzyme
closed complex to be converted into an open complex, the closed complex
must interact with an activator of the 54-holoenzyme in
a reaction that requires nucleotide hydrolysis. Single round in
vitro transcription assays were conducted to study the effect of
the mutants on activator-dependent and -independent transcription initiation. Using supercoiled DNA, the level of 54-dependent transcription from the S. meliloti nifH promoter was measured with PspFHTH as activator
(Fig. 8A). We also measured any potential of the mutant 54 to support transcription
without activator by preincubating holoenzyme and template DNA with GTP
before heparin challenge (Fig. 8B). The initiating sequence
of the S. meliloti nifH promoter is GGG (+1 to +3); thus,
the addition of GTP before heparin could allow open complexes to
initiate and form heparin-stable complexes. Transcripts are then
extended from the heparin-stable activator-independent initiated
complex that has formed. In activator-dependent assays, ATP
and PspFHTH were added before the heparin challenge. Hence transcripts in this assay arise from heparin-stable uninitiated open
complexes, since ATP is not an effective initiating nucleotide at the
S. meliloti nifH promoter.
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Fig. 8.
A, in vitro
activator-dependent transcription activity at the
supercoiled S. meliloti nifH promoter. Changes in
transcription activity as a function of activator PspFHTH
concentration (nM). Holoenzyme was present at 100 nM. Reactants were preincubated with ATP (4 mM)
before the addition of heparin. B, in vitro
activator-independent (bypass) transcription assay in which reactants
were preincubated with GTP (1 mM) before the addition of
heparin. Reactions were performed as standard with (+) or without ()
activator. I and R336A are included as positive controls for bypass
transcription. WT, wild type.
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Using saturating levels of PspFHTH, the
activator-dependent assay revealed a trend for
transcription activity levels closely following the pattern seen in the
in vivo assays (see Table I). The 310-328 holoenzyme
essentially produced no activated transcript, whereas W328A, D338A, and
R342A gave levels closest to wild type activity. The L340S mutant
produced approximately 18% of the transcripts made by wild type
holoenzyme. With holoenzyme present at 100 nM, the amount
of PspFHTH required for half-maximal transcription was measured as
an indicator of the apparent affinity of each closed complex for
activator. Mutants W328A, D338A, and R342A showed similar half-maximal
PspFHTH requirements as the wild type holoenzyme. The L340S
holoenzyme required approximately 2-fold more activator, similar to
results previously reported for R336A (22). The apparent reduced
affinity for activator of the L340S mutant may be indirect and due to
its aberrant structure rather than loss of any specific activator
contact. As previously reported for R336A holoenzyme (22), a time
course of exposure to heparin before the addition of the NTP extension
mix showed no significant difference in stability of the
transcription-competent open complexes between L340S and wild type
54-holoenzyme (data not shown). This together with the
closed complex (Fig. 5) and open complex gel mobility assay data (Fig.
9) suggests that L340S holoenzyme is
defective at some step where the closed complex is converted to the
open complex but that Leu-340 is unimportant for the stability of the
resultant open complex. Activator-dependent transcription
was also tested on a linearized DNA template. All mutants were active
for transcription on the linear DNA, suggesting no obvious defect in
DNA melting (data not shown). However, mutants D338A and R342A were
more active than the wild type 54-holoenzyme for
activated transcription from the linear DNA template. With PspFHTH
at 800 nM, the D338A and R342A
54-holoenzymes were 42% and 24%, respectively, more
active for transcription than wild type 54-holoenzyme
(data not shown). This suggests that the D338A and R342A
54-holoenzymes could be enhanced for a DNA-melting
activity that is not detected when transiently melting supercoiled DNA
is used as template.
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Fig. 9.
Gel mobility shift assay for heparin stable
open complex formation on linear S. meliloti nifH
promoter DNA homoduplex. 54 was titrated
against core (50 nM) with a saturating level of the
activator PspFHTH (4 µM) and 1 mM GTP. The
maximum activity for wild type (WT) was taken as
100%.
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In the activator-independent assay (GTP addition before heparin), among
the mutants investigated here only 310-328 and, to a much lower
extent, L340S holoenzyme (both of which showed altered interaction in
the heteroduplex and footprinting assays) produced discernable levels
of transcripts (Fig. 8B, lanes 5 and
9). The native gel mobility data indicate that L340S is
structurally disrupted. 310-328 has lost a potential
surface-exposed loop connecting the carboxyl-terminal DNA binding
domain with the rest of 54. In both cases the activator
bypass activity and correlating holoenzyme activities (instability on
early melted DNA, stability on late melted DNA, and altered footprints)
might be due to local or more extensive structural disruptions in
54, interfering with correct functioning of Arg-336 or
the Region I of 54, both of which are known to function
to restrict the holoenzyme to the transcriptionally silent closed
complex (9, 22, 32).
Stable Initiated Complex Formation--
In the single round
transcription assay, abortive initiation and promoter escape activities
can influence the amount of transcript produced. Therefore, to
investigate the ability of holoenzyme to form heparin-stable initiated
complexes on linear DNA, we used a gel mobility shift assay. To ensure
that the polymerase was saturated with 54, increasing
amounts of were added to a constant concentration of core (33). For
activation, we used PspFHTH with GTP as the hydrolyzable nucleotide
to allow open complexes to initiate. The 310-328 holoenzyme was
unable to efficiently form heparin-resistant complexes and, as
suggested by the in vivo transcription assays and DNA
binding assays, appears to form a closed complex unable to respond to
activator. Although the L340S holoenzyme was very active for closed
complex formation, it appears that little of the closed complex
detected can be converted to heparin-stable initiated complex (Fig. 9).
This correlates with the poor transcription activity observed for this
mutant in vivo and in vitro (Table I and Fig.
8A). The W328A holoenzyme was approximately 60% as active
as wild type for stable initiated complex formation, whereas D338A and
R342A 54-holoenzymes, as suggested by transcription on
linear DNA, were in repeated experiments consistently improved compared
with wild type for stable complex formation (Fig. 9). The increase in
initiated complex formation as the ratio of to core polymerase is
increased must reflect that D338A and R342A 54 have an
altered affinity for core within the assay. It is notable that R342A
holoenzyme, which formed low levels of closed complexes, was not
obviously defective for stable complex formation. Thus, the mutation
R342A appears important for 54 and holoenzyme DNA
binding but unimportant for the post DNA binding step of stable
initiated complex formation. Closed complexes that form with R342A may
convert more efficiently to initiated complexes than wild type.
The reduced activity of the W328A holoenzyme for initiated complex
formation might be due to a defect in DNA strand separation. A repeat
of the initiated complex assay at 15 °C showed that stable initiated
complex formation by W328A holoenzyme was not especially temperature-dependent (data not shown). Cold-sensitive DNA
opening is evident for mutants in 70 that are believed
to lack single-stranded DNA binding activity (34). The insensitivity of
the W328A holoenzyme activity to lower temperature suggests residue
Trp-328 is not involved in DNA opening in the way that aromatic
residues in 70 are.
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DISCUSSION |
The 54-holoenzyme closed complex is converted to an
open complex via a reaction employing activator nucleoside triphosphate hydrolysis (5-9). In vivo and in vitro
transcription assays identified three classes of mutant in the patch of
54 that can be cross-linked to DNA. The 310-328
54 was inactive for transcription; L340S, like R336A
(22), gave reduced transcription activity; and the remaining mutants
gave close to wild type transcription levels. The cross-linking patch contains residues both important for promoter binding (Trp-328 and
Arg-342) and isomerization (Trp-328) as well as restricting basal
transcription (amino acids 310-328 and Leu-340) and setting the level
of open complex formation (Trp-328, Asp-338, Leu-340, and Arg-342). DNA
footprints suggest that the cross-linking patch directs interactions
with promoter DNA near 12. The 12 promoter sequence has a crucial
role in 54-dependent transcription,
providing interactions that set basal transcription and which start DNA
melting (23, 31, 32). Mutations in the 12 region produced promoters
that increased basal transcription or diminished activation (31). Some
sequences in the cross-linking patch apparently contribute to
interactions with the 12 promoter region important for control of
54-holoenzyme transcription activity. Interactions of
the three classes of mutant and their holoenzymes with both native
and heteroduplex promoter DNA templates are discussed in the context of
the 54 transcription initiation mechanism.
The class I mutant 310-328 holoenzyme was inactive for
activator-dependent transcription in vivo and
in vitro due to the inability of its closed complex to
respond to activator and form an open complex. The 310-328
holoenzyme did allow activator bypass transcription in vitro
(Fig. 8B, lane 5) and formed a heparin-stable complex on late melted DNA (data not shown), showing the conformation of the holoenzyme had changed. The 310-328 and wild type
54 had similar affinities for native promoter DNA (Fig.
2A). Like the deleted Region I 54-holoenzyme
(11) the 310-328 holoenzyme was enhanced for DNA binding (Fig. 5).
Also, as for deleted Region I and R336A holoenzymes (9, 22), S1
footprinting showed the 310-328 holoenzyme has isomerized to give
an extended footprint (Fig. 7A, lane 4), and o-Cup footprinting revealed an altered interaction at 12
(Fig. 7B, lane 4). The 310-328
54 was defective for binding to early melted DNA (Fig.
3), and its holoenzyme was unstable on this template (Fig. 6).
Possibly, this critically relates to the inability to be activated
because the initial nucleation of DNA melting may not have occurred.
Furthermore, residues 310-328 are likely surface-exposed (20) and
could be a site for activator contact (35). Removal of these residues could change the holoenzyme conformation such that it is unable to
respond to activator and isomerize properly. Interestingly, single-stranded DNA binding activity has been implicated for a region
of 54 between amino acids 329 and 364 (21). Deletion of
residues 310-328 may cause a local change in conformation, which
reveals this single strand DNA binding activity. Thus 310-328
appears to behave like deleted Region I 54. Both are
unable to respond to activator and form an unstable holoenzyme
interaction with early melted DNA. Instead, a stable interaction with
late melted DNA is favored. In contrast, wild type holoenzyme can form
an activator-independent heparin-stable complex on early melted DNA but
cannot on late melted DNA (9, 30). Mutant s such as 310-328 and
deleted Region I 54, which exhibit a reverse activity
with heparin-stable holoenzyme complex forming on late melted DNA are
also capable of activator bypass transcription (see Fig. 8B,
lanes 3 and 5). The bypass activity of the
310-328 54-holoenzyme supports the idea that
disrupting 54 recognition of early melted DNA near the
12 promoter region directs the holoenzyme to form a more stable
complex with melted downstream promoter sequences and which, under
specialized conditions, can allow activator-independent transcription
(9, 22, 32).
The class II L340S holoenzyme was shown to be defective in conversion
from closed to open complex (compare Fig. 5 with Figs. 8A
and 9). The purified L340S 54 protein was physically
disrupted and could not be directly assayed for DNA or core binding.
However, when the L340S 54 was mixed with core and
promoter DNA, a closed complex with normal gel mobility was observed.
Interaction between , core, and DNA apparently caused the to
become restructured and to form a closed complex capable of
transcription. The L340S holoenzyme showed a 10-fold increase in
transcription activity at the K. pneumoniae nifH promoter
when the C at 15 was changed to T (Table II). Replacing the K. pneumoniae nifH 15 C with a T is stimulatory for binding of
54 and its holoenzyme
TOP
ABSTRACT
INTRODUCTION
