From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030
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ABSTRACT |
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The Prokaryotic RNA polymerase
(RNAP)1 is a multimeric
enzyme consisting of the core subunits, Comparison of the amino acid sequences of members of the
subunit of RNA polymerase is a critical
factor in positive control of transcription initiation. Primary
factors are essential proteins required for vegetative growth, whereas
alternative
factors mediate transcription in response to various
stimuli. Late gene expression during flagellum biosynthesis in
Salmonella typhimurium is dependent upon an alternative
factor,
28, the product of the fliA gene. We
have characterized the intermediate complexes formed by
28 holoenzyme on the pathway to open complex formation.
Interactions with the promoter for the flagellin gene fliC
were analyzed using DNase I and KMnO4 footprinting over a
range of temperatures. We propose a model in which closed complexes are
established in the upstream region of the promoter, including the
35
element, but with little significant contact in the
10 element or
downstream regions of the promoter. An isomerization event extends the
DNA contacts into the
10 element and the start site, with loss of the
most distal upstream contacts accompanied by DNA melting to form open
complexes. Melting occurs efficiently even at 16 °C. Once open
complexes have formed, they are unstable to heparin challenge even in
the presence of nucleoside triphosphates, which have been observed to
stabilize open complexes at rRNA promoters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
' (E),
and a
factor (1). The
factor is responsible for directing the
recognition and binding of specific promoter DNA consensus sequences by
holoenzyme (
2
'
) and for facilitating
transcription initiation (2, 3). Many different
factors have been
identified, and they fall into two major categories, those similar to
Escherichia coli
70 and those similar to
54 (4). The
70 family can be further
subdivided into primary and alternative
factors (5, 6). Primary
factors are essential and required for the expression of housekeeping
genes in vegetative cells, whereas alternative
factors are
activated in response to environmental changes and in programming
developmental pathways (4, 6, 7). Promoters recognized by the
70 family consist of conserved hexamers at
35 and
10
with respect to the start site of transcription (8). Additional DNA
contacts are made at some promoters by the C-terminal domain of the
subunit of RNAP at an AT-rich region between
38 and
59 called the
UP element (9).
70 family reveals four highly conserved regions with
subdomains that have been implicated in specific functions (Fig.
1) (4). Region 1.1 inhibits
70 from binding to the DNA in the absence of the core
subunits (10) and is also required for efficient progression from the
earliest RNAP·DNA complex to a transcriptionally active complex
during initiation (11). Deleting regions 1.1 and 1.2 of
70 results in transcriptional arrest after initial
binding of RNAP to the promoter (11). Regions 2.1, 2.2, and 3.2 are
important for core binding (12-14), and region 2.3 has been implicated
in promoter melting (15-18). Both holoenzyme and the
factor
interact with non-template bases in the
10 element to stabilize the
open complex (19-21). Regions 2.4 and 4.2 are responsible for
contacting the -10 and -35 promoter recognition elements,
respectively, and for positioning holoenzyme for initiation (10,
22-26). Regions 1.2, 2, and 4 are found in almost all
factors, but
region 1.1 is found only in the primary
factors (4). Interestingly, both regions 1.1 and 1.2 are absent in the Salmonella
typhimurium alternative
factor required for flagellum
biosynthesis,
28, hinting at potential variation in the
structure of E
28·DNA complexes or in the mechanism of
transcription initiation.
View larger version (6K):
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Fig. 1.
Linear schematic diagram of
70 and
28. Primary
factors, such as
70, possess four highly conserved regions of amino
acids. Each shaded section represents further delineation
into subregions. Region 1.1 (black) is encountered
specifically on primary
factors. It controls DNA binding by free
and is required for efficient isomerization to the open complex
during initiation. Region 2.1 (gray) is involved in core
binding. Region 2.4 (white) interacts with the -10 promoter
consensus sequence, and region 4.2 (vertical stripes)
interacts with the -35 promoter consensus sequence. Regions 1.2, 2, and 4 are found in almost all
factors; however, for S. typhimurium
28, region 1.2 is absent.
Initiation can be described as a series of sequential steps, which have
been well characterized for both E70 and
E
32 from E. coli (27). By varying the
conditions, several intermediates can be visualized using footprinting
methods. RNAP (R) binds to the promoter (P) to form an initial closed
complex, RPC1, which protects the DNA from approximately
60 to
5 relative to the start point of transcription.
RPC1 isomerizes to a second closed complex
(RPC2) that maintains the upstream contacts and extends further downstream of the transcription start site to +20.
RPC2 then undergoes strand opening to form an open complex,
RPO, whereas the length of the footprint is unchanged.
There is evidence for more than one open complex, which is dependent on
the presence or absence of Mg2+ (28, 29). RPO
enters the initiation stage, or RPinit, in the presence of
initiating nucleotides. Short transcripts of 2-12 nucleotides in
length (abortive products) are synthesized while RNAP remains at the
promoter. RNAP then enters into the elongation phase of transcription
upon promoter clearance and
factor release. Several of these
intermediate complexes have been visualized by performing DNase I and
KMnO4 footprinting experiments over a range of
temperatures, with the rationale that temperature-dependent complexes represent time-dependent events (30-32).
The S. typhimurium flagellar operon can be divided into
three classes of genes (I-III) based on their transcriptional
hierarchy in flagellar assembly (33). 28, encoded by the
fliA gene, is expressed late in flagellum biosynthesis and
is required for transcription of all class III genes including fliC, encoding flagellin, the primary component of the
flagellar filament (34), and flgM, encoding the
28 anti-
factor (35, 36). In this report, we have
characterized transcription initiation by RNA polymerase carrying the
S. typhimurium
factor,
28, on variants of
the flagellin (fliC) promoter. We detected intermediate complexes formed by E
28 during initiation that are
distinct from those characterized for other holoenzymes. Initial
binding to the promoter does not require the
10 element, but further
extension of the RNA polymerase-DNA contacts and isomerization to the
open complex demand the presence of the
10 sequence. We propose an
alternative mode of promoter recognition and binding as compared with
other well characterized holoenzymes.
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EXPERIMENTAL PROCEDURES |
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Overproduction and Purification of 28 and
Reconstitution of Holoenzyme--
The fliA gene, encoding
28, was inserted into plasmid pET15b (Novagen, Inc.) to
generate pKH439 (a gift from K. Hughes), which resulted in the addition
of six histidines at the amino terminus. Hexahistidine-tagged
28 was overproduced and purified using the method
described by Wilson and Dombroski (11). Holoenzyme was reconstituted by
adding 1.0 pmol of E. coli core RNA polymerase (E)
(Epicentre Technologies Corp.) to 5.0 pmol of
28 in
protein dilution buffer (10 mM Tris-HCl (pH 8.0), 10 mM KCl, 10 mM
-mercaptoethanol, 1 mM EDTA, 0.4 mg/ml bovine serum albumin, and 0.1% Triton
X-100) and incubating on ice for 15 min. Because of the amino acid
sequence conservation among the core subunits, heterologous holoenzymes
have been used to assess the behavior of alternative
factors
(37-40). Additionally, we compared the amino acid sequences of the
and
subunits of E. coli and S. typhimurium
RNA polymerases (11) (data not shown). There is 100% identity between
the
subunits and 98% identity between the
subunits.
Constructions and Generation of Promoter
Fragments--
pfliC35 was constructed by annealing two
oligonucleotides (Integrated DNA Technologies) of 122 bases in length
(41). The DNA was designed to incorporate HindIII and
BamHI restriction sites near the 5'- and 3'-ends,
respectively. The double-stranded DNA was digested with
HindIII and BamHI, ligated into the same sites in
pBluescriptII KS+ (Stratagene), and transformed into
E. coli strain DH5
(Life Technologies, Inc.). The clones
were sequenced to confirm the deletion using the
fmolTM DNA sequencing system (Promega).
pfliC10 was constructed using an oligonucleotide with a
deletion of the
10 element as a polymerase chain reaction primer with
plasmid pMC72, containing the wild-type fliC promoter, as the template. The resulting DNA fragment was ligated into pCR2.1 and
transformed into Inv
F' One Shot competent cells from the original TA
cloning kit (Invitrogen). The clones were sequenced to confirm the
deletion using the fmolTM DNA sequencing system.
32P-5'-End-labeled primers were generated for use in
synthesizing labeled fliC promoter DNA (10). Oligonucleotide
primers were from BioServe Biotechnologies, Genosys Biotechnologies,
Inc., or Integrated DNA
Technologies.2 Plasmid pMC72
(a gift from K. Hughes) and the two plasmids containing pfliC10 and pfliC
35, as described above,
were used as the template DNA to generate the fliC promoter
DNA and derivatives. Both radiolabeled and unlabeled fliC
promoters were synthesized using the polymerase chain reaction to
generate a 230-base pair fragment. Each polymerase chain reaction
contained 10× Taq Buffer A (Fisher), 50 pmol of each
primer, 40 ng of template DNA, and 2.5 units of Taq
polymerase (Fisher) in a final volume of 100 µl. A Perkin-Elmer
Thermocycler was set for 35 cycles with 95 °C for denaturation,
50 °C for annealing, and 72 °C for extension. The products were
purified using the Qiaquick polymerase chain reaction DNA purification
kit (QIAGEN Inc.).
DNase I Footprinting-- 32P-End-labeled DNA promoter fragment and DNase I buffer (20 mM sodium Hepes (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 200 µg/ml bovine serum albumin) were combined in a volume of 50 µl. Holoenzyme was added in 10-fold excess over the DNA unless noted otherwise. Additional processing of these samples has previously been described (40). Several variations of the DNase I footprinting are described below.
Heparin Competition--
The E28-promoter
complexes were allowed to form at 37 °C for 15 min. In some cases,
nucleoside triphosphates (NTPs) were added to a final concentration of
0.2 mM for 1 min, and then heparin was added (25 µg/ml
final concentration), followed by incubation for another minute. The
complexes were treated with 0.5 units of DNase I (Promega) for 30 s and processed as already described.
Temperature Variation--
The E28-promoter
complexes were allowed to form at 0, 4, 16, 25, and 37 °C for 15 min. The complexes formed at 0 and 4 °C were subjected to DNase I
digestion (2 units of DNase I) for 35 and 30 min, respectively.
Complexes formed at 16 °C were digested for 4 min (1.5 units of
DNase I), and the 25 and 37 °C samples were digested for 1 min (1.0 unit of DNase I).
KMnO4 Footprinting--
E28 and 0.1 pmol of end-labeled DNA promoter fragment were combined in
KMnO4 buffer (20 mM sodium Hepes (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 0.1 mM EDTA, 0.2 mM dithiothreitol, and 200 µg/ml bovine serum albumin) to a final volume of 50 µl and then treated with 2.5 µl of 50 mM KMnO4 (Sigma) for 2 min
at the temperatures indicated. Additional processing has been described
(42).
Nucleotide Stabilization Assay in Vitro--
The nucleotide
stabilization assay was performed as outlined by Wilson and Dombroski
(11) with the following modifications. The E28-promoter
complexes were allowed to form for 15 min, and then 0.1 mM
ATP, CTP, and GTP were added to the preformed complexes for 30 s,
prior to filtration and washing with 0.8 M NaCl.
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RESULTS |
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Promoter Binding and Transcription in Vitro--
RNAP was formed
by mixing hexahistidine-tagged 28 with purified E. coli core RNAP. Because of the high degree of similarity between
the core subunits of E. coli and S. typhimurium
RNAPs, a heterologous system was used as reported by others to assess the behavior of alternative
factors (37-40). Runoff transcription assays were performed to analyze the overall efficacy of transcription initiation by E
28. The promoter chosen for this
analysis, pfliC, drives the expression of flagellin, one of
the late flagellar gene products. A transcript of the expected size was
observed. No difference in behavior was noted in the presence or
absence of the hexahistidine tag (data not shown). Deletion of the
AT-rich sequence just upstream of the
35 element had a slight effect
on transcription (2-fold reduction), but was not attributable to the
presence of an UP element since holoenzyme containing a truncation of
the carboxyl-terminal domain of the
subunit resulted in the same
transcriptional behavior as the wild-type enzyme, independent of the
upstream DNA sequence (data not shown).
DNase I footprinting was used to characterize the interaction of
E28 with pfliC (Fig.
2). A DNA fragment of 230 base pairs in
length containing the fliC promoter was 5'-end-labeled on
the template strand with 32P. Increasing amounts of
reconstituted E
28 were added, followed by DNase I
digestion (Fig. 3). Continuous protection
was observed from
24 to +17, with partial protection from
46 to
24 relative to the transcription start site. Additional weak
interactions between +17 and +20 could be discerned. Overall, this
footprint is shorter in the upstream region than those typical for
E
70 or E
32 under similar conditions.
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Intermediate Complexes Formed during Transcription
Initiation--
We characterized the intermediate
E28·DNA complexes during the process of transcription
initiation that can be visualized by manipulating the temperature of
incubation. E
28 was incubated with 5'-end-labeled
pfliC (template strand) for 15 min, at 0, 4, 16, 25, and
37 °C. Protection was observed from
65 to
54 and from
48 to
19 at 0 and 4 °C (Fig.
4A). At 16 °C, a
hypersensitive band appeared at
46, and partial protection began to
extend downstream toward the
10 region and the start site (
46 to
+17), with the reappearance of bands at
33 and -24. By 25 °C,
E
28 fully occupied the region from
46 to +17. At
37 °C, the upstream contacts from
65 to
54 disappeared, but
strong protection from
46 to +17 remained. Thus, E
28
appeared to initially bind primarily to the upstream region of the
promoter since
10 protection was absent at lower temperatures and
then shifted downstream to contact the
10 element up to the +17
region as the temperature was increased. We cannot rule out the
possibility that some minor contacts in the upstream region are
maintained. However, it is clear that a major rearrangement takes place
as a function of temperature. The same pattern of contacts was observed
with footprinting performed using DNA labeled on the non-template
strand (data not shown).
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Open Complex Formation--
Extended DNase I footprints at
37 °C are usually indicative of open complexes for
E70 (27, 43-46). We used KMnO4 sensitivity
to determine which E
28-promoter complexes in the DNase I
temperature series were open complexes. KMnO4 chemically
modifies thymine residues in single-stranded DNA, which renders
modified positions sensitive to cleavage upon piperidine treatment
(47). E
28-promoter complexes were formed for 15 min at
0, 4, 16, 25, or 37 °C and subjected to KMnO4 treatment
and piperidine cleavage (Fig. 4B).
From 0 to 4 °C, the DNA remained base-paired as demonstrated by lack
of reactivity to KMnO4. Open complexes were fully
established at 16, 25, and 37 °C with reactivity to
KMnO4 at +1, 6,
7, and
9. Thus, similar to
RPO for E
70 and E
32, the
E
28 DNase I footprints that extend into the
10 region
and start site represent open complexes. Surprisingly, however,
E
28 formed open complexes at temperatures as low as
16 °C, whereas E
70 and E
32 form
primarily extended closed complexes (RPC2) under these
conditions (27, 30-32, 46, 48). We did not observe an
RPC2-type complex for E
28 under any conditions.
Promoter Variants Lacking the 10 or
35 Element--
If
E
28 initially binds to the
35 region, releases
upstream interactions, and shifts its contacts to include the
10 and
downstream regions, then removal of the
35 element should preclude
any binding by E
28. Likewise, removal of the
10
consensus sequence should permit binding of only E
28 in
the
35 region. We constructed deletions of the TAAA sequence at
35
(pfliC
35) and the GCCGATA sequence at
10
(pfliC
10) (Fig. 2) and analyzed the DNase I footprints
over a range of temperatures. Deletion of the
10 element still
allowed normal binding of E
28 at 0 and 4 °C, as
expected based on the presence of the predicted initial binding site at
35 (Fig. 5A). However, upon
raising the temperature, binding to pfliC
10 was gradually
abolished, consistent with the idea that polymerase shifts its contacts
downstream during open complex formation to interact with the
10
element and downstream sequences. In the case of pfliC
10,
these sequences have been removed, and E
28 is unable to
remain stably bound to the upstream sequences and results in
dissociation.
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Also as expected, E28 was unable to bind to
pfliC
35 even at 0 °C, presumably due to lack of the
initial binding site in the
35 region and upstream portions of the
promoter (Fig. 5B). KMnO4 footprinting showed no
open complexes at either pfliC
10 or pfliC
35 at any temperature (data not shown). Taken together, these results support the idea that initial binding occurs from
65 to
19, followed by isomerization to relinquish major contacts between
65 and
54 and establishment of new contacts extending to +20 with
concomitant DNA melting.
Stability of E28-Promoter Complexes--
Previous
studies have shown that E
70 typically forms a
heparin-stable open complex (48-51). One exception is the ribosomal
operon promoter, rrnBP1, which requires the addition of NTPs
to confer heparin stability (52). We used heparin challenge as a tool to examine the stability of E
28·DNA complexes. The
binding of E
28 to pfliC was sensitive to 25 µg/ml heparin, even in the presence of initiating NTPs (Fig.
6). Thus, E
28 open
complexes (RPO and RPinit) appear to be
generally less stable than E
70 open complexes. RNAP with
the primary
factor from Bacillus subtilis,
E
A, also forms heparin-sensitive complexes in the
presence or absence of NTPs (53).
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RPinit complexes for E70 can be
distinguished from RPO complexes by resistance to challenge
with high salt in the presence of NTPs (11, 50, 54). We used an NTP
stabilization assay to determine whether RPinit complexes
formed by E
28 on pfliC were stable to salt
challenge. E
28·fliC complexes were formed
in the absence and presence of NTPs and were then filtered through
nitrocellulose and subjected to a 0.8 M NaCl wash. In the
presence of NTPs, 52% of the complexes were retained on the filter, as
expected since RPinit is in equilibrium with
RPO, whereas in the absence of NTPs, only 5% were
retained. Therefore, E
28 RPinit complexes on
pfliC, like E
70·DNA complexes, are stable
to 0.8 M NaCl, indicating a equivalent conformational
change and stabilization upon NTP binding.
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DISCUSSION |
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Transcription initiation has been characterized as a multistep
process in studies in which intermediates on the pathway to formation
of an initiated complex have been detected by manipulating the
temperature of incubation and varying the level of Mg2+ for
the RNAP-promoter interactions (30, 31, 43, 46, 50, 51). There is
evidence for two different closed complexes (RPC1 and
RPC2), which differ in the extent of their contacts with
the promoter DNA. RPC1, which extends from approximately
60 to
5, is only observed at low temperatures (0 °C) for
E
70 (30) and E
32 (31, 32). However, we
have recently shown that derivatives of
70 with region
1.1 or both regions 1.1 and 1.2 removed slow the initiation process
such that RPC1 can be detected at 37 °C (11).
We have investigated the mechanism of transcription initiation by
holoenzyme carrying 28, the alternative
factor
required for flagellum biosynthesis in S. typhimurium. Our
analysis was conducted using the promoter for fliC, encoding
flagellin, a late gene in this pathway. The identity and stability of
transcription complex intermediates that we identified during
transcription initiation by E
28 are distinctive from
those previously identified for E
70 and
E
32.
Based on our results, we suggest the following pathway for the
mechanism of E28 transcription initiation (Fig.
7). E
28 forms a short
complex with pfliC at 0 °C that protects primarily the
upstream region of the promoter (
65 to
19) from DNase I digestion.
This suggests that the initial binding of E
28 is
mediated mainly through interactions in the vicinity of the
35
consensus sequence. E
28 then isomerizes to make
additional contacts that extend to +20. During this progression, some
upstream contacts are lost as evidenced by the reappearance of bands
from
65 to
54 on the DNase I footprints at 25 and 37 °C. Thus,
E
28 appears to initially bind in the
35 region and
then shifts downstream to contact the
10 region, start site, and
downstream sequences to +20 while relinquishing some of the original
upstream contacts. This represents a notable difference in binding
intermediates from those observed for E
70 or
E
32.
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The identity of RPC versus RPO
complexes was assessed by KMnO4 sensitivity, which probes
for DNA strand separation. Typically, significant open complex
formation for E70 or E
32 requires
temperatures above 16 °C (27, 30, 31). One distinctive characteristic of E
28·fliC complexes is
that they are optimally strand-separated between -9 and +1 even at
16 °C. Thus, the extended DNase I protection observed at 16 °C is
representative of RPO, rather than an extended closed
complex like RPC2. Thus, one major difference between
E
28 intermediates and those found for E
70
or E
32 from E. coli is the absence of a
detectable RPC2-like complex under the conditions that we
used. E
28 appears to form a single closed complex
characterized by a short DNase I footprint at 0-4 °C and then
progresses to RPO. If an RPC2 complex forms, it
may be too rapid or unstable to be detected with the methods employed
in this study.
Because E28 is functionally similar to the B. subtilis holoenzyme that is required for flagellin biosynthesis,
E
D, we compared the transcription initiation complexes
formed by these two enzymes at their respective flagellin promoters.
The flagellin gene promoter (phag) from B. subtilis is very similar to pfliC, except it contains
an UP element between
60 and
40 that provides additional contact
sites for RNAP through the
subunit (57). E
D forms
complexes that are more similar to E
70 and
E
32 because, at 4 °C, the DNase I footprint occupies
from
73 to +1 (55). This protection then extends to +9 and +21 at 23 and 37 °C, respectively. Disappearance of the upstream contacts,
which is observed for E
28, does not occur. Thus, DNA
binding primarily in the
35 and upstream regions in the earliest
detectable complex, with lack of contact in the
10 region, appears to
be novel for E
28.
With respect to open complex formation, however, E28 and
E
D share the ability to generate strand-separated
promoter regions at low temperatures. At 16 °C, E
28
displays the same degree of open complex formation that is observed at
37 °C, but forms only closed complexes at 4 °C. E
D
begins to show DNA distortions in the
10 region at temperatures as
low as 4 °C; however, the region of strand melting propagates unidirectionally as a function of temperature, forming three distinct open complex intermediates (55), whereas the reactivity of bases in the
E
28 open complex remains the same from 16 to 37 °C,
with no indication of directional movement.
We used challenge with the polyanionic competitor heparin as a tool to
assess the relative stability of open (RPO) and initiated (RPinit) complexes. E28·fliC
open complexes are unstable to even low levels of heparin in the
presence or absence of initiating NTPs. In contrast, many E
70 open complexes are stable to heparin even in the
absence of NTPs (11, 48-51). E
28 appears to form
transcriptionally competent complexes that remain dissociable
throughout the initiation process. We cannot rule out that
E
28 may form heparin-resistant complexes on other
28-dependent promoters or that heparin is
actively destabilizing these complexes. However, the information from
these studies is useful in comparing the relative stability of
holoenzymes carrying different
factors.
In the case of 70, it has been shown that amino-terminal
region 1.1 is involved in inhibition of DNA binding by
(10, 56) and
is required for efficient open complex formation by holoenzyme. Region
1.2, which is typically present in all primary and alternative
factors, may also be involved in open complex formation for E
70 (11).
28 is a very unusual member of
the
70 family of proteins since it lacks any homology to
region 1.2, leading to some speculation regarding how this difference
in structure may translate into a difference in function. The flagellar
biosynthesis
factor from B. subtilis,
D,
retains homology to region 1.2. E
D forms two closed
complexes and isomerizes through several open complex intermediates
(55).
28, on the other hand, lacks both regions 1.1 and
1.2. E
28 forms an unusually short RPC1, and
we did not observe an RPC2-like intermediate. Additionally,
many E
70 open complexes are stable to heparin either in
the absence or presence of NTPs, and open complexes do not form
efficiently at low temperatures, whereas the opposite is true in both
cases for E
28. The composition of the amino terminus of
the
factor may affect the nature of the RNAP·DNA complexes that
can be discerned during initiation.
28 appears to
facilitate transcription initiation at pfliC very efficiently by utilizing a mechanism with few intermediate
complexes, but with reduced stability of the open complex. Whether this
mechanism is a general phenomenon for all
28-dependent promoters or whether it is
characteristic for the flagellin gene promoter remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Dr. K. Hughes for plasmids
containing the fliA and fliC genes. We are
grateful to Dr. W. Ross for sharing footprinting protocols. We thank
Drs. R. Gourse and T. Gaal for core RNA polymerase with a C-terminal
deletion of the subunit. We thank the other members of the
laboratory, N. Baldwin, B. Johnson, and C. Wilson Bowers, for helpful
discussions and critical reading of the manuscript. We are grateful to
M. Schaubach for assistance with computer graphics.
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FOOTNOTES |
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* This work was supported by Research Grant NP-902 from the American Cancer Society and Research Grant GM56453-01 from the National Institutes of Health.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: Dept. of Microbiology
and Molecular Genetics, University of Texas Health Science Center, 6431 Fannin, Houston, TX 77030. Tel.: 713-500-5442; Fax: 713-500-5499;
E-mail: dombros{at}utmmg.med.uth.tmc.edu.
2 The sequences are available upon request.
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ABBREVIATIONS |
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The abbreviations used are:
RNAP, RNA
polymerase;
E28,
28 holoenzyme.
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REFERENCES |
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