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INTRODUCTION |
Within the bacterial domain of the kingdom prokaryotae exists a
general and ubiquitous response to nutritional and environmental stress, the stringent response (1). This general stress response is
mediated by high level accumulation of the transcription effector guanosine-3',5'-(bis)pyrophosphate (ppGpp). A major effect of elevated
ppGpp levels is an immediate and severe reduction of stable rRNA and
tRNA gene transcription (2). The cessation of stable RNA syntheses
halts the major energy consuming activities of the cell, transcription
and translation. This period of metabolic inactivity allows the cell to
utilize its remaining energy reserves to adapt to stressful growth
conditions through induction of specific "stress genes" (3). Once
adaptation is near completion, ppGpp levels decrease and growth
resumes. Failure to reduce ppGpp levels results in a severe reduction
of cell viability (4).
Several lines of evidence suggest that ppGpp exerts its effects by
directly binding to RNA polymerase
(RNAP).1 Certain
rifampicin-resistant mutants of the
-subunit of RNAP display
increased intracellular sensitivity to ppGpp (5, 6). Spontaneously
occurring mutants that confer survival under artificial and prolonged
exposure to toxic levels of ppGpp were mapped to the rpoB
gene, encoding the
-subunit of RNAP (7). Mutant strains devoid of
ppGpp, ppGpp0 strains, are incapable of surviving
nutritional deprivation; however, specific mutants of the
70-,
-, or
'-subunits of RNAP restores normal
survivability to ppGpp0 strains (8). Fluorescence quenching
studies of RNAP in the presence of increasing concentrations of a
fluorescently labeled ppGpp analogue
(1-aminonapthalene-5-sulfonate-ppGpp) is consistent with binding of
ppGpp to a single binding site on RNAP (9). Cross-linking analyses by
Chatterji et al. (10) using a radioactive photocross-linkable derivative of ppGpp,
8-azidoguanine-3',5'-(bis)pyrophosphate (8-azido-ppGpp),
demonstrated predominant cross-linking of ppGpp to the
-subunit. In
the same study (10), it was also observed that both N- and C-terminal
partial trypsin digestion fragments of the
-subunit were
cross-linked by 8-azido-ppGpp, suggesting a modular ppGpp binding site
analogous to that of the nucleotide binding site at the catalytic
center of RNAP (11). Despite extensive studies on RNAP-ppGpp
interactions, a precise localization of the ppGpp binding site on RNAP
is lacking.
An allosteric mechanism of ppGpp action on RNAP is generally invoked as
mediating its transcriptional effects, although this has not been
extensively studied. In this context, allostery refers to the
inducement of functionally relevant conformational changes of RNAP as a
result of ppGpp binding at a location other than the catalytic site.
Consistent with this notion, ribonucleoside triphosphates do not
compete with ppGpp for RNAP binding (12). In addition, circular
dichroism studies have revealed a small but significant change in total
-helical content of RNAP following addition of ppGpp (13). To date,
this single previous study (13) represents the only physical evidence
of induced conformational change of RNAP by ppGpp. Clearly, further
studies characterizing the nature of ppGpp allostery are warranted.
The aim of the present investigation is 2-fold: 1) to substantiate and
refine the location of the ppGpp binding site on E. coli
RNAP; and 2), to begin to elucidate the nature of ppGpp-induced conformational changes of RNAP. Toward the first of these goals, we
synthesized a new photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp). The
6-thio-ppGpp derivative is a zero-length photocross-linking
reagent, because the thiol group is directly photoactivatable and has a
van der Waals radius similar to the oxygen for which it is substituted (14). Photoaffinity labeling of RNAP with 6-thio-ppGpp indicates that
the N terminus of the largest subunit of RNAP,
', is the predominant
location of cross-linking. To begin to investigate the conformational
consequences of ppGpp-RNAP interactions, we assessed alterations in
trypsin sensitivity of RNAP in the presence and absence of ppGpp. Three
major ppGpp-dependent trypsin-resistant fragments of RNAP
were observed. Identification of these trypsin-resistant fragments and
their superimposition onto the three-dimensional structure of RNAP
indicates an overlap with and close proximity to the 6-thio-ppGpp
cross-linking site. These results have ramifications for both
definitive identification of the ppGpp binding site on RNAP as well as
for the allosteric consequences of ppGpp-RNAP interaction.
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EXPERIMENTAL PROCEDURES |
Enzymes and Chemicals--
-Escherichia coli RNA
polymerase was purified from E. coli K12 strain MG1655 using
the method of Burgess and Jendrisak (15) with the modifications of Lowe
et al. (16). Final
70 holoenzyme separation
from core enzyme was accomplished by heparin-Sepharose chromatography
(17). Crude RelA-ribosome preparations were prepared according to
Cashel (18). Chemicals and solvents were reagent grade, used without
further purification, and purchased from Sigma-Aldrich. All radioactive
nucleotides were obtained from ICN Pharmaceuticals Inc.
Synthesis of ppGpp and Derivatives--
Synthesis conditions for
ppGpp were as described (18). For synthesis of radioactive ppGpp, 0.5 mCi of [
-32P]ATP (4500 Ci/mmol) were diluted with 1 mM cold ATP and 0.5 mM GDP or
6-thioguanosine-5'-triphosphate (6-thio-GTP) in a 0.1 ml reaction
volume mixed with 60 A260 units of crude
RelA-ribosomes at room temperature for 12 h. Final purification of
ppGpp was on a QAE-Sephadex A-25 column as described (19). Nucleotides were typically eluted with a 0-0.5 M linear gradient of
triethylammonium bicarbonate, pH 7.5. Elution products were
visualized by autoradiography after separation by
polyethyleneimine-cellulose thin layer chromatography (20). It was
noted that 6-thioguanosine derivatives chromatographed with lower
mobility on polyethyleneimine-cellulose plates than the corresponding
guanosine containing compounds. The ppGpp-containing fractions were
pooled and concentrated through removal of the triethylammonium
bicarbonate solvent by lyophilization in a vacuum centrifuge at room
temperature. The product was resuspended in 50 µl of water followed
by precipitation with 2% NaI in acetone, the pellet was washed three
times with 1 ml of acetone and a final wash with 1 ml of ether.
The final product was resuspended in 50 µl of water. 6-Thio-ppGpp
exhibited an ultraviolet light spectrum typical for ppGpp with a
characteristic maximum at 253 nm and a shoulder peak between 265 and
280 nm at pH 7.0 (19).
Synthesis of
6-Thioguanosine-5'-triphosphate--
6-mercaptoguanosine was
converted into the corresponding triphosphate as described by Sergiev
et al. (14).
NMR Analyses of ppGpp and 6-Thio-ppGpp--
The proton and
phosphorus NMR spectra of purified ppGpp and 6-thio-ppGpp were obtained
at 500 MHz on a Varian INOVA 500 (Chemistry Instrumentation Center,
State University of New York at Buffalo) in 10% D2O
pH 4.0, spectra was collected for 27 h. 1H and
31P chemical shifts were compared and found to be nearly
identical between ppGpp and 6-thio-ppGpp. Assignments of proton shifts
were based on previous published spectra for 6-thio-GTP (14).
In Vitro Transcription--
RNA synthesis was carried out with a
mixture of argT and lacUV5 (10 nM
each) promoter-bearing linear templates in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 250 µM GTP, ATP, and
CTP, and 20 µM UTP with 15 µCi of
[
-32P]UTP (800 Ci/mmol) in 20-µl reaction volumes at
37 °C for 10 min. Increasing amounts of GDP, ppGpp, or 6-thio-ppGpp
were incubated as indicated with 100 nM RNA polymerase for
15 min at room temperature and then warmed to 37 °C for 2 min prior
to initiating the reaction by adding an equal volume of prewarmed
template/substrate mix. Reactions were terminated and processed
according to Hsu (21). RNA was fractionated on a 7 M
urea-6% polyacrylamide gel (19:1 acrylamide:bisacrylamide) in
Tris-boric acid-EDTA buffer.
Cross-linking--
All cross-linking experiments were carried
out in 25 mM HEPES, pH 7.9, 5 mM
MgCl2, 100 mM KCl, 25 µg/ml bovine serum
albumin, 5% glycerol. E. coli RNA polymerase (1 µM) was incubated with radioactively labeled 6-thio-ppGpp
in a 20-µl reaction volume. Samples were mixed, incubated for 15 min
at room temperature, and then transferred to ice and irradiated with
302 nm ultraviolet light (MacroVue UV-20 transilluminator; surface
intensity, 9000 µW/cm2) for 20 min at a distance of 4 cm
in an open Eppendorf tube covered with a polystyrene filter to remove
stray radiation of <290 nm, thus avoiding protein-protein
cross-linking. Photoaffinity labeling reactions were terminated by the
addition of 5× SDS sample buffer (0.625 M Tris-HCl, pH
6.8, 5% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 0.2%
bromphenol blue, 25% glycerol) to a 1× concentration. Samples were
heated to 65 °C for 5 min and then resolved by electrophoresis through a SDS-10% polyacrylamide gel (30:0.8 acrylamide:bisacrylamide) to separate all RNA polymerase subunits and through a SDS-4%
polyacrylamide gel to resolve
- and
'-subunits using Tris-glycine buffer.
Mapping of Cross-links--
Radioactive 6-thio-ppGpp
cross-linked
'-subunit was extracted from a denaturing SDS-4%
polyacrylamide gel following electrophoresis. The region of the
polyacrylamide gel containing
'-protein was excised. Protein was
eluted by diffusion out of polyacrylamide by overnight shaking of
macerated gel slices in 0.03% SDS at 37 °C. Eluted protein was
concentrated by freeze-drying and then resuspended in water to a final
concentration of 1-2% SDS (22). Partial cyanogen bromide (CNBr)
degradation of isolated
'-protein was performed as described
previously (23). Cleavage products were fractionated by electrophoresis
on a SDS-7-16% gradient polyacrylamide gels in a Tris-glycine buffer system.
Trypsin Digestion Studies--
RNA polymerase (0.25 µM) was incubated for 15 min at room temperature with 200 µM of either GDP or ppGpp in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM magnesium acetate,
5% glycerol, 0.1 mM EDTA. After a 2-min warming at
37 °C, samples were mixed with increasing amounts of
tosyl-L-phenylalanine chloromethylketone-treated trypsin (0, 0.1, 0.3, 0.6, 1.2, and 2.5 µM) in a
20-µl reaction volume and incubated at 37 °C for an additional 5 min. Trypsin digestions were stopped by the addition of
phenylmethylsulfonyl fluoride to a final concentration 10 mM, immediately followed by the addition of 5× SDS-sample
buffer to a 1× concentration and incubation at 105 °C for 2 min.
Total trypsin fragments were resolved in a SDS-12% polyacylamide gel
and visualized by colloidal Coomassie G-250 staining (24). Specific
trypsin fragments were identified by Western blot analyses using
monoclonal antibodies against N-terminal, middle, and C-terminal
epitopes of
' (7RC78, 7RC74, and NT73, respectively) and
(7RB145, 7RB135, and NT63, respectively) (Ref. 25 and courtesy of
Richard Burgess, University of Wisconsin-Madison). Reactive bands were
visualized by chemiluminescence using the Renaissance Western blot
Chemiluminescence Reagent Plus detection kit (PerkinElmer Life
Science Products)
Imaging and Quantification--
Radioactive samples were
visualized by autoradiography and, when necessary, imaged on a Bio-Rad
Molecular Imager and quantified using the Molecular Analyst
f software (Bio-Rad Laboratories Inc.).
Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass
Spectrometry (MALDI-TOF MS)--
Protein fragments were excised from
Coomassie-stained gels and analyzed at Borealis Biosciences Inc.,
Toronto Canada by MALDI-TOF MS.
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RESULTS |
Synthesis and Structure of 6-Thio-ppGpp--
The nucleotide
derivative 6-thioguanosine-5'-triphosphate (6-thio-GTP) was synthesized
from 6-mercaptoguanosine and used as a substrate in the enzymatic
reaction depicted in Reaction 1, to yield
6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp; Fig.
1). The reaction occurs in two steps,
using crude RelA-ribosomes (see "Experimental Procedures").

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Fig. 1.
Structure of
6-thioguanosine-3',5'-(bis)pyrophosphate. The chemical structure
of 6-thio-ppGpp is depicted with the thio group in the mercapto and
thio conformation, as indicated. NMR chemical shift in parts per
million (ppm) of the total applied magnetic field are
indicated for phosphorus (31P) and proton (1H)
spectra. The putative assignments of proton shift are indicated for
either a base (H8) or ribose (e.g. H1') proton followed by
the number of protons per signal, with apparent peak splitting
occurring for H1'. The proton and phosphorus NMR were collected at 500 MHz on a Varian INOVA 500 in 10% D2O, pH 4.0.
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(Step 1)
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(Step 2)
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The reactivity of 6-thio-GTP was comparable with GTP in the
RelA-mediated reaction (data not shown). The conversion of pppGpp to
ppGpp is mediated by the enzyme guanosine pentaphosphatase, which is
present in crude RelA-ribosome preparations (18). Highly purified
6-thio-ppGpp was separated and obtained as described by Cashel
(18).
The structure of purified 6-thio-ppGpp was verified by nuclear magnetic
resonance (NMR). Both phosphorus (31P) and proton
(1H) spectra of bona fide ppGpp and 6-thio-ppGpp
were comparable and indicative of very similar structures (Fig. 1).
Unlike the proton NMR spectra reported for 6-thio-GTP (14),
6-thio-ppGpp shows a strong chemical shift specific for a mercapto
group proton (Fig. 1). Thus, in solution at pH 4.0, most of
6-thio-ppGpp bears a mercapto group at position 6 of the guanine moiety
rather than a thio group as depicted in Fig. 1. The predominance of the
mercapto over the thio form of 6-thio-ppGpp may simply be
because of the acidic pH under which NMR was performed. This is
unclear, however, because the absolute pKa of the
thio group of 6-thio-ppGpp is unknown, and sufficient material was not
obtained to be able to determine this experimentally. Because the
photoreactive conformation of the 6-thioguanosine moiety is the thio
form, cross-linking to RNA polymerase is only possible if the bound
form of 6-thio-ppGpp is in the thio form and not the mercapto form, as
appears to be the case (Fig. 3).
Transcription Activity of 6-Thio-ppGpp--
To test the activity
of 6-thio-ppGpp compared with ppGpp, their ability to inhibit
transcription of the tRNA promoter, PargT, was assayed. The
activity of the argT promoter is negatively affected by
increased ppGpp levels in vivo (26). Here, we demonstrate for the first time a specific inhibition of PargT
in vitro by ppGpp under standard ionic and transcription
conditions. At low ionic conditions, <50 mM KCl, the
inhibitory effects of ppGpp were diminished (data not shown). A
4-6-fold inhibition of RNA synthesis from PargT was observed
at the highest concentration of ppGpp tested (Fig.
2). As an internal control, transcription
from a PlacUV5 template was monitored in an equimolar
mixed template reaction. The transcription from PlacUV5 was
also inhibited between 20-35%, as previously noted (27), but ppGpp
preferentially inhibited transcription from PargT. The
inhibition of transcription of PargT by 6-thio-ppGpp paralleled
that of ppGpp.

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Fig. 2.
Inhibition of in vitro
transcription by ppGpp and 6-thio-ppGpp. Multiple-round
in vitro transcription of an equimolar mixture of linear
templates bearing the argT and lacUV5 promoters
was performed in the presence of increasing concentrations of GDP,
ppGpp, or 6-thio-ppGpp, as indicated. The argT
promoter produced a terminated transcript of 230 nucleotides, and the
lacUV5 promoter produced a pair of run-off transcripts of
~250 nucleotides in length. The percentage of radioactivity in each
transcript was normalized to untreated samples and plotted against GDP,
ppGpp, or 6-thio-ppGpp concentrations. PargT transcription is
represented by open symbols and
PlacUV5 transcription by closed
symbols.
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Cross-linking of 6-Thio-ppGpp to RNA Polymerase--
Increasing
amounts of radioactive 6-thio-ppGpp were mixed with 1 µM
RNA polymerase and subjected to photoactivating ultraviolet light
irradiation. Electrophoretic analysis of radioactively labeled RNAP on
denaturing polyacrylamide gels revealed the presence of cross-linker on
all RNAP subunits. At limiting concentrations of cross-linker, however,
labeling appeared predominant for
- and/or
'-subunits (Fig.
3A). Further cross-linking
experiments were all performed at an equimolar concentration of RNAP
and 6-thio-ppGpp (1 µM ea). In competition experiments,
cross-linking of
'- and
-subunits was dramatically reduced in the
presence of a 200-fold excess of cold genuine ppGpp but not GTP (Fig.
3B). Electrophoresis of photoaffinity-labeled RNAP on low
percentage denaturing 4% polyacrylamide gels to resolve
'-
and
-subunits showed that the majority (~90%) of label was
associated with the
' subunit (Fig. 3C). Competition with
cold ppGpp had little effect on residual
-subunit cross-linking, but
it essentially eliminated cross-linking of the
'-subunits (Fig.
3D). No competition with 6-thio-ppGpp binding was observed
in experiments with either GTP or GDP (Fig. 3D). In
addition, we observed no effect on cross-linking in the presence of
nonspecific or promoter containing DNA, RNA, or rifampicin (data not
shown).

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Fig. 3.
Cross-linking of 6-thio-ppGpp to RNAP.
All cross-linking reactions were performed with 1 µM RNAP
and radioactively labeled 6-thio-ppGpp. A, increasing
concentrations of 6-thio-ppGpp were incubated with RNAP, as indicated,
followed by photocross-linking and fractionation on a denaturing 10%
polyacrylamide gel. B, 1 µM 6-thio-ppGpp was
competed with 200 µM GTP or ppGpp prior to
photocross-linking and fractionation as described in A. C,
six duplicate cross-linking reactions with 1 µM
6-thio-ppGpp and RNAP were performed followed by fractionation on a
denaturing 4% polyacylamide gel to resolve - and '-subunits.
D, 1 µM 6-thio-ppGpp was competed with 200 µM GTP, GDP, or ppGpp prior to photocross-linking and
fractionation as described for panel C.
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Mapping of 6-Thio-ppGpp Cross-linking on the
'-Subunit of
RNAP--
The labeled
'-subunit shown in Fig. 3C was
excised and extracted from the 4% polyacrylamide gel and subjected to
partial CNBr digestion. Following digestion, the fragments were
resolved on a gradient polyacylamide gel and visualized by
phosphorimaging. The actual partial CNBr digest is shown in Fig.
4 in comparison with idealized
computer-generated profiles of a single-hit partial CNBr digest of
'. Idealized digestions were generated assuming either extreme C- or
N-terminal labeling of
', respectively. The profile of the
experimental CNBr digestion fragments aligns well with the idealized
CNBr N-terminally labeled fragment ladder, particularly the three very
specific bands of 102, 130, and 152 amino acid residues (Fig. 4). The
pattern and intensity of bands is comparable with what has been
obtained previously for N-terminally labeled
' (28). These results
are consistent with 6-thio-ppGpp cross-linking between amino acid
residues 29 and 102 of the
'-subunit of RNAP, because no label was
found in association with the extreme N-terminal 29 amino acid residue
fragment.

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Fig. 4.
Partial cyanogen bromide digestion of
cross-linked '-subunit. Partial cyanogen
bromide digestion of the 6-thio-ppGpp cross-linked '-subunit
isolated from the gel of Fig. 3C was carried out as
described under "Experimental Procedures" under single-hit partial
digestion conditions. Digestion products were fractionated on a
denaturing 7-16% polyacrylamide gradient gel and visualized by
phosphorimaging. N-terminal (N) and C-terminal
(C) theoretical ladders of single-hit cyanogen bromide
digestion patterns are shown adjacent to the ' digestion.
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ppGpp-dependent Trypsin-resistant Fragments of
RNAP--
To begin to explore the allosteric consequences of ppGpp
binding to RNAP, resistance to trypsin proteolysis was used to probe for ppGpp stabilized domains of RNAP. Limiting proteolysis has been
used as a means of defining subtle alterations in the conformation of
RNAP upon promoter binding (29) as well as for the definition of major
RNAP structural domains (30). In contrast, we employed in this study an
excess of trypsin to assay for protection of small highly structured
domains of RNAP formed upon ppGpp binding. A range of trypsin
concentration between 0 and 2.5 µM was used to digest a
solution of 0.25 µM RNAP preincubated with 250 µM GDP or ppGpp. Trypsin fragments were resolved on
denaturing polyacylamide gels. Following Coomassie staining, three
prominent ppGpp-dependent trypsin-resistant fragments were
revealed with an apparent molecular mass of ~100, 40, and 28 kDa, respectively (Fig. 5A,
indicated by asterisks). The same fragments were also
obtained in the presence of GDP; however, they had a much lower
resistance to trypsin in comparison with ppGpp (Fig. 5A).
The binding of ppGpp does not, therefore, appear to induce formation of
unique conformers of RNAP. Instead, it seems to stabilize the existing
domain structure and/or sterically hinder trypsin accessibility. In
control experiments, bovine serum albumin trypsin sensitivity
was identical in the presence or absence of either GDP or ppGpp (data
not shown). Attempts to cleave RNAP cross-linked with radioactive
6-thio-ppGpp did not give a clear or consistent pattern of digestion;
we conjecture that this failure was the result of technical
problems, e.g. an interference with trypsin activity by the
presence of nonspecific UV-induced cross-links. Perhaps in future
experiments, the exact identification of the conformational change of
RNAP induced following 6-thio-ppGpp cross-linking may be achieved using
methods other than trypsin digestion to probe for changes in protein
conformation.

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Fig. 5.
Trypsin-resistant fragments of RNAP in the
presence of ppGpp. Increasing concentrations of trypsin, as
indicated, were incubated with 250 nM RNAP for 5 min and
fractionated on denaturing 10% polyacylamide gels. A,
Coomassie-stained gel of total protein. Known proteins and the
position of migration of relative molecular weight markers are
indicated. The major trypsin-resistant fragments are indicated by
asterisks. B, Western blot of the same total
protein samples as shown in A probed with monoclonal
antibody 7RC78, which recognizes an epitope between amino acid residues
115 and 236 of the '-subunit. C, Western blot of the same
total protein samples as shown in A probed with monoclonal
antibody NT63, which recognizes an epitope between amino acid residues
922 and 1090 of the -subunit.
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The identity of the tryptic fragments stabilized in the presence of
ppGpp was determined by Western blot analyses. The trypsinized samples
shown in Fig. 5A were processed for Western analyses and probed with a battery of monoclonal antibodies (mAb) against various epitopes of the
- and
'-subunits (see "Experimental
Procedures"). The
'-subunit-specific mAb, 7RC78, recognizes an
epitope located between amino acid residues 115 and 236. Western blots
of trypsin fragments with 7RC78 reveals that the 100- and 28-kDa
ppGpp-protected fragments contain the N terminus of the
'-subunit
(Fig. 5B). The
-subunit-specific mAb, NT63, recognizes an
epitope between amino acid residues 922 and 1099 and is the most
C-terminal epitope recognized by available RNAP mAbs (Burgess and
colleagues (15, 16, 25, 29, 37)). Western blot analysis with
NT63 is consistent with the 40kDa fragment containing the C terminus of
(Fig. 5C). Given the fact, however, that the
epitope of NT63 is a considerable distance from the actual C terminus
of the
-subunit, it was difficult to make a definitive C-terminal
assignment to this fragment. For this reason, MALDI-TOF MS analysis was
performed on the 40-kDa trypsin stable fragment. MALDI-TOF MS analysis
(see "Experimental Procedures") indicated that the 40-kDa
fragment spans amino acid residues 958-1328 ± 10. Thus, the
C-terminal assignment of the 40-kDa
-subunit band was confirmed.
Assignment of the approximate end points of the 28-kDa fragment by
MALDI-TOF MS was not successful because of contamination by identical
size fragments of the
-subunit. Fortunately, the close proximity of
the
'-specific mAb, 7RC78, allows confident N-terminal assignment to
the trypsin stable 28-kDa fragment.
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DISCUSSION |
The global stress regulator, ppGpp, a ligand and effector of RNAP,
has been studied since its discovery by Cashel and Gallant (31)
over 30 years ago, yet little is known about how it binds to and
modifies RNAP. Here, we report the synthesis of a novel photocross-linkable derivative of ppGpp,
6-thioguanosine-3',5'-(bis)pyrophosphate. This cross-linkable ppGpp
derivative has allowed us to obtain the first indication that ppGpp
binds near the N terminus of the large subunit of RNAP,
'. This
compound has the distinct advantage over preceding cross-linkable ppGpp
derivatives of carrying a zero-length cross-linking group. The chemical
structure of 6-thio-ppGpp in solution, confirmed by NMR spectroscopy,
reveals a predominance of the mercapto group (analogous to an enol
group in ppGpp) rather than the thio group (analogous to a keto group
in ppGpp) at the sixth position of the guanine moiety at pH 4.0 (Fig. 1). At this same pH level, a mercapto group was not noted or
reported for the precursor, 6-thio-GTP (14). Thus, the presence of the
3'-pyrophosphate on ppGpp may change the local chemistry of the thio
group on the guanine ring. No enol proton was detectable in bona
fide ppGpp, indicating that this property is unique for the
thiolated ppGpp. It is not clear which is the preferred form of
6-thio-ppGpp at pH 7.9, at which cross-linking and transcription
analyses were performed. Equilibrium competition experiments and
relative affinity measurements of 6-thio-ppGpp compared with ppGpp
could not be performed because of the nonequilibrium conditions
necessary for efficient 6-thio-ppGpp cross-linking (see "Experimental
Procedures"). If 6-thio-ppGpp at pH 7.9 is predominantly in the
mercapto conformation, however, it does not appear to affect
dramatically its efficacy in inhibiting transcription (Fig. 2).
Fundamental chemistry dictates that the thio and not the mercapto
conformation of the 6-thioguanosine moiety is subject to
photoactivation. With this in mind, the observed cross-linking of RNAP
by 6-thio-ppGpp (Fig. 3) is consistent with a preferred binding to RNAP
of the thio form of 6-thio-ppGpp. It is likely, therefore, that in
natural ppGpp the keto group at position 6 of the guanine moiety is
important for the binding of ppGpp to RNAP.
Predominant cross-linking of radioactively labeled 6-thio-ppGpp occurs
within the first 102 amino acid residues of the N terminus of the
'-subunit of RNAP (Figs. 3 and 4). This location overlaps with the
conserved region A of
' (
'A). In the recently solved crystal structure model of Thermus aquaticus RNAP, the
extreme N terminus of the
'-subunit, including
'A, is
disordered and lacks electron density (32). The crystal data together
with our finding that ppGpp binds to and induces trypsin resistance to
the N-terminal portion of
' (Fig. 5B) leads us to propose that ppGpp binding induces a higher order structure of this region.
The N terminus of
' is implicated as playing a crucial role in many
aspects of the transcription process. Region
'A contains a zinc finger, which is essential for stable DNA association of the
elongating transcription complex (33) and has been cross-linked to the
double-stranded DNA at the lagging end of the transcription bubble
(34). Additionally, within the transcription complex the extreme N
terminus of the
'-subunit cross-links along the entire length of the
"extruded" nascent RNA (34, 35). This close proximity of the
extruded RNA to the N terminus of
' is "replaced" in a paused
transcription complex by an alternate proximity of the RNA to the
so-called "flap" structure of the
-subunit (36). This
repositioning or "switching" of the path of RNA within the complex
is thought to be part of the mechanism of transcriptional pausing (36).
Finally, a primary determinant of
70 binding is found
between amino acid residues 260 and 309 of the
'-subunit (37). These
multifaceted functions associated with the N terminus of
' will be
considered below in light of the putative binding of ppGpp near this
region and the known effects of ppGpp.
The binding of ppGpp to RNAP has been proposed to destabilize
specifically the open complex of rRNA promoters (27, 38). Thus, ppGpp
binding and restructuring of the region of RNAP near the N terminus
could disrupt the function of the
'A zinc finger and/or
70 interaction and could lead to collapse of the
open complex. Another well documented effect of ppGpp is that it
decreases transcription elongation rates, primarily by increasing the
pause times at naturally occurring pause sites (12, 39-41). Because
the path of nascent RNA, extruded across the N terminus of
' in
elongating RNAP, is altered to putatively bring about transcriptional
pausing (36), ppGpp binding might directly influence this process.
Thus, the binding of ppGpp to this particular region of RNAP may be
linked mechanistically to many of the observed effects of ppGpp-RNAP interactions.
Previous data (see the Introduction) have indicated that ppGpp binding
is localized to the
-subunit of RNAP. However, in this study
we have determined that the N terminus of the
'-subunit is
in close proximity to bound ppGpp. Although our results seemingly conflict with these previous observations, upon closer examination they
actually corroborate earlier data concerning ppGpp-RNAP binding. We
have synthesized and used for the first time a zero-length cross-linking ppGpp derivative, 6-thio-ppGpp. Preceding studies (10)
used an azido group cross-linker, which replaces the proton at position
8 of the guanine moiety of ppGpp with an azido group (-N3). The thio group at position 6 of the guanine
moiety of 6-thio-ppGpp is 8-10 Å distant from the azido group of
8-azido-ppGpp. Given the considerable separation of these two
cross-linking groups, the thio group of 6-thio-ppGpp predictably would
be in a different location and thus provide unique cross-links compared
with 8-azido-ppGpp. Our trypsin resistance studies (Fig. 5) are
congruent with the formation of a highly structured N-terminal portion
of the
'-subunit and C-terminal portion of the
-subunit of RNAP
as a consequence of ppGpp binding. These results are consistent with
the fact that the N and C termini of
' and
, respectively, are
spatially close in the three-dimensional model of prokaryotic RNAP and
constitute an intertwined interface of the two subunits (32). These
observations lead us to propose that the binding of ppGpp is allosteric
and that the site of binding is modular and located close to the
intersubunit interface comprising the N- and C-terminal portions of the
'- and
-subunits, respectively.