From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
Received for publication, January 8, 2003, and in revised form, February 20, 2003
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ABSTRACT |
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Transcription of stable RNA genes is known to be
dramatically reduced in the presence of guanosine tetraphosphate
(ppGpp), the mediator of the stringent response. Using in
vitro transcription systems with ribosomal RNA P1 promoters, we
have analyzed which step of the initiation cycle is inhibited by the
effector ppGpp. We show that formation of the ternary transcription
initiation complex consisting of RNA polymerase holoenzyme, the
promoter DNA, and the first initiating nucleotide triphosphate is the
major step at which ppGpp exerts its regulation. Neither primary
binding of RNA polymerase to the promoter nor isomerization to the open binary complexes or the subsequent promoter clearance steps contributes notably to the observed inhibition. The effect of
ppGpp-dependent inhibition in the formation of the ternary
transcription initiation complex could be mimicked by nucleotide
derivatives known to bind to the RNA polymerase active center. Using
these model compounds, almost identical inhibition characteristics were
observed as seen with ppGpp. The results support the previously
published model, which suggests that ppGpp-dependent
inhibition is based on competition between the inhibitor
molecules and NTP substrates for access to the active center of RNA polymerase.
Bacterial cells adapt their growth very efficiently in response to
changes in the environmental conditions. The cellular growth is
generally determined by the capacity for protein synthesis, which
itself is tightly linked to the number of ribosomes. During amino acid
deprivation bacterial cells react, among other metabolic adaptations,
by an immediate transcriptional shut-down of ribosomal RNAs
(rRNAs),1 the key molecules
for the constitution of the protein synthesis apparatus. This global
regulation is triggered by the rapid accumulation of the effector
molecule guanosine tetraphosphate (ppGpp) and termed the stringent
response (1). Although gradual progress in understanding this
remarkable network has been made in recent years, the molecular details
of the regulatory mechanism(s) are still obscure (2-10). The
observations that some promoters (e.g. amino acid
biosynthesis operons) are activated during the stringent response and
that in vivo and in vitro studies have often
yielded contradictory results have further aggravated our understanding of the mechanism of ppGpp-dependent regulation (11).
Moreover, ppGpp-dependent regulation is not restricted to
the steps of transcription initiation but has also been shown to act
during elongation where the pausing properties of transcribing RNA
polymerase are specifically changed (6, 12).
There is consensus today that RNA polymerase is the obvious target for
the effector molecule ppGpp (7, 8, 13). Binding has been shown to occur
close to the active center formed modularly by both the Whether a gene is stringently regulated or not is clearly encoded by
its promoter structure. It is known for some time that promoters under
negative stringent regulation share a common GC-rich sequence element
downstream of the Materials--
RNA polymerase holoenzyme was purified from
Escherichia coli DG156 cells (19) as described
previously (20, 21), and the activity of the enzyme was assessed by a
quantitative transcription assay (22). Templates for RNA polymerase
binding and in vitro transcription were isolated by
restriction hydrolysis of plasmid DNAs. The following fragments were
used for in vitro transcription: a 256-bp rrnB P1
promoter fragment (positions
ppGpp was prepared and purified as described previously (6). Care was
taken to keep residual LiCl concentrations in the final reaction
mixture below 2 mM. Ultrapure NTPs (Amersham Biosciences, HPLC-purified) were used for in vitro transcription.
Dinucleotides (ApA, ApC, ApU) were purchased from Sigma.
Multiple-round in Vitro Transcription--
Multiple-round
in vitro transcription reactions were performed at 30 °C
with linear templates (1 nM) as described recently (4, 23)
in 50 µl of transcription buffer (50 mM Tris acetate, pH
8.0, 80 mM potassium glutamate, 10 mM MgAc, 1 mM dithiothreitol, 0.1 mM EDTA, 10 µg/ml
acetylated bovine serum albumin). The reactions were incubated for 8 min after the addition of RNA polymerase (3 nM) and
incubation for 8 min. A nucleotide mix was added (final concentrations:
65 µM each ATP, CTP, and GTP, 84.5 pM
[ Single-round in Vitro Transcription--
Reactions were
performed as described (23). Briefly, RNA polymerase (3 nM)
was incubated with template DNA (1 nM) in 20 µl of
transcription buffer for 8 min at 30 °C. 5 µl of a NTP solution containing a final concentration of 500 or 50 µM ATP and
radioactively labeled CTP (5 µCi, 5 µM) was incubated
for 15 min. Reactions were then chased for 8 min in the presence of 433 µg/ml heparin and 433 µM all four NTPs. The reactions
were stopped by addition of formamide sample buffer and separated as
described above.
Formation of Ternary Initiation Complexes--
RNA polymerase (3 nM) together with template DNA (1 nM) was
incubated in presence of the first two initiation nucleotides (each 50 nM) in 20 µl of transcription buffer for 8 min at
30 °C to form ternary initiation complexes. Heparin was then added (final concentration: 200 µg/ml) to prevent reinitiation. For the
elongation reaction the lacking NTPs (each 50 µM, one of
which was radioactively labeled (5 µCi)), were added and incubated
for 15 min. Reactions were chased for 8 min and stopped by addition of
formamide sample buffer prior to gel electrophoresis.
Determination of Ternary Complex Stabilities--
The
time-dependent decrease in the concentration of preformed
ternary complexes was determined by single-round in vitro
transcription reactions under conditions that precluded reinitiation.
Reactions consisted of 5 nM template DNA with the
rrnB P1 promoter and 15 nM RNA polymerase in
transcription buffer. Samples were incubated in the absence or presence
of 300 µM ppGpp for 8 min at 30 °C. For ternary
complex formation, the starting nucleotides ATP (50 µM)
and 20 µCi of [ KmnO4 Footprinting--
The structure of the
transcription bubble in ternary initiation complexes was tested by
KMnO4 footprinting (23, 24). The ternary initiation
complexes were preformed by incubation of 15 nM RNAP, 5 nM rrnB P1 DNA, 50 µM ATP, and 5 µM CTP in transcription buffer in absence and presence of
ppGpp (300 µM) for 30 min at 30 °C. Heparin was added
(final concentration: 200 µg/ml) to prevent reinitiation, and samples
were treated with 37 mM KMnO4 for 1 min at
30 °C. To this mixture Gel Retardation Assays--
The kinetics of ternary complex
formation was analyzed by gel retardation (23). Briefly, RNA polymerase
(3 nM) and radioactively labeled DNA promoter fragments (1 nM) were incubated in transcription buffer for 8 min at
30 °C. Complex formation was started by addition of the first two
nucleotides necessary for initiation at the concentrations indicated.
At different time intervals, aliquots (10 µl) were withdrawn and
complex formation was stopped by transferring samples to fresh tubes
containing heparin (final concentration: 200 µg/ml). Initiation
complexes were separated from free DNA by native gel electrophoresis
after the addition of glycerol (5%).
Effect of ppGpp on Overall Transcription from Different
Promoters--
To compare the inhibitory effect of ppGpp on different
promoters, we performed multiple-round in vitro
transcription reactions with DNA fragments containing either one of the
following promoters: rrnB P1, rrnD P1, or, as
unregulated control promoter, PtacI (see Fig.
1A). Standard reactions were
performed with 3 nM active E. coli RNA
polymerase holoenzyme and 1 nM DNA template with increasing ppGpp concentrations (0-1 mM) as described under
"Experimental Procedures." RNA polymerase, template DNA, and ppGpp,
when present, were preincubated for 8 min before the reaction was
started by adding the NTP mixture (50 µM each ATP, GTP,
CTP; 0.85 nM (2.5 µCi) [ Effect of ppGpp on the Different Stages of Transcription--
The
different steps of the transcription cycle may formally be broken down
into initiation, elongation, and termination. Initiation itself is a
multistep process, and a simplified description of the reactions from
the first encounter of RNA polymerase with a promoter to the elongation
complex is outlined in Fig. 1B. The schematic pathway is
divided in four substeps (I-IV), which lead to characteristic
intermediates on the way to productive transcription. Those
intermediates have been characterized as the closed complex (RPc), the binary open complex (RPo), the
ternary open complex or initiating complex that contains at least one
bound substrate NTP (RPinit), and finally the elongating
complex (EC), which has moved away from the promoter and generally lost
the sigma subunit. In principle, the formation of each of the complexes
might be rate-limiting and thus represent a target for
ppGpp-dependent regulation.
To determine whether ppGpp exerts its effect before or after formation
of the ternary initiation complexes (RPinit), we took advantage of the fact that open binary rRNA P1 promoter complexes (RPo) are notoriously unstable and decompose with a very
short half-life if not stabilized by the addition of the initiating NTPs (17, 18, 25). Hence, to find out whether ppGpp acts before or
after formation of the ternary initiating complexes, we measured the
inhibitory effect when ppGpp was either present from the start of the
reaction (before substrate NTPs were added) or only after the
initiating nucleotide substrates had been added. Reactions were
performed under single-round in vitro transcription conditions with preformed initiation complexes in the presence of only
two NTPs (ATP and CTP for rrnB P1; GTP and UTP for
rrnD P1). Samples were prepared in duplicate with either
ppGpp present before or after the addition of the initiating NTPs.
After 8 min at 30 °C, heparin was added (final concentration: 200 µg/ml) to prevent reinitiation and elongation was allowed for 15 min
after addition of the remaining NTPs (50 µM each,
including 5 µCi of [ Effects of the Starting NTP Concentration on
ppGpp-dependent Inhibition--
Because the presence of
the starting NTPs appears to be necessary for rRNA promoters to form
stable initiation complexes, we wished to understand the role of the
initiating nucleotides for the ppGpp-dependent inhibition
for this step of transcription. Therefore, we performed transcription
experiments with the rrnB P1 promoter with different
concentrations of the starting nucleotide ATP in the absence and
presence of ppGpp.
According to earlier studies it has been reported that the first
nucleotide of rrn promoters has a very high apparent
Km, which limits formation of ternary complexes at
rRNA P1 promoters. This step has therefore been considered as
rate-limiting during transcription initiation (see, e.g.,
Ref. 26). To test the ppGpp-dependent effect on this step
of rrnB P1 initiation, we used multiple-round in
vitro transcription experiments with increasing concentrations of
the starting nucleotide ATP in absence or presence of 300 µM ppGpp. The range of ATP concentrations was varied
between 0.1 µM and 1 mM. The results are
summarized in Fig. 4. It can be seen that
ppGpp inhibits transcription at all ATP concentrations, however, with
different efficiencies. Inhibition is maximal within a medium concentration range between 10 and 100 µM ATP. At ATP
concentrations below or above this range, the
ppGpp-dependent inhibition is less pronounced. The results
indicate that the inhibitory effect of ppGpp on ternary complex
formation can be released at high initiating NTP concentrations,
suggesting competition between the inhibitor and the substrate NTPs,
whereas at very low substrate NTP concentrations transcription is too
inefficient to allow accurate determination of the effects on
inhibition.
ppGpp Effects on Promoter Escape--
It is known for several
promoters that the slowest step in transcription initiation is not
represented by RNA polymerase binding or open initiation complex
formation but occurs during passage of RNA polymerase through the
initial transcribed region. Those promoters are considered to be
limited at the promoter escape or promoter clearance step (27).
Usually, before RNA polymerase escapes from the promoter, a
characteristic set of short RNA products (between 2 and 12 nucleotides
in length) is synthesized and released as abortive transcripts. During
abortive initiation RNA polymerase cycles repeatedly at the promoter
without dissociation. At some point during abortive cycling, triggered
by an unknown mechanism, a decision is made in favor of productive
transcription and RNA polymerase changes into elongation mode. We
wished to know whether this step is affected by ppGpp. The question
could in principle be answered by a quantitative assessment of the
accumulation of abortive products after formation of the initiation
complexes (28). Hence we quantified the abortive products derived from rrnB P1 initiation complexes formed in the absence or
presence of ppGpp. Preformed initiation complexes were elongated under single- and multiple-round transcription conditions, and aliquots were
taken at different time intervals. Although the multiple-round reactions were started in the presence of all four NTPs, single-round conditions were maintained by starting the reaction in the presence of
only ATP and CTP to allow initiation complex formation. The remaining
NTPs were added 10 min after the initiation reaction was started
together with 200 mg/ml heparin. It should be noted that the formation
of abortive products is not affected by heparin. Reactions were stopped
and separated on 20% denaturing polyacrylamide gels. Results are
presented in Fig. 5. It can be seen that
the presence of ppGpp does not cause any significant qualitative or quantitative change in the abortive product pattern. Note, however, that significant overall inhibition is apparent by a marked reduction in the yields of full-length run-off transcripts. The rate of abortive
transcription as well as the decision between abortive cycling and
formation of productive elongation complexes therefore seem not to be
influenced notably by the presence of ppGpp. The results are consistent
with the conclusion that ppGpp does not exert its inhibitory effect by
changing the efficiency of abortive initiation or slowing down promoter
escape.
Effect of ppGpp on the Structure and Stability of Ternary
Initiation Complexes--
It is known that promoters under negative
stringent control generally do not form stable open binary complexes
(see, however, Ref. 29). Although the lifetime of binary open complexes
is affected by ppGpp at almost any promoter, the specific inhibitory effect of ppGpp has been attributed in a recent study to the
intrinsically short lifetime of sensitive promoters (2, 3). Here we
asked whether ppGpp has any effect on the structure, stability, and lifetime of the ternary initiation complexes. Therefore we analyzed the
structure of ternary complexes of the rrnB P1 promoter by footprinting, monitoring the accessible nucleotides of the template and
non-template strand within the single-stranded region of the initiation
complex. Footprint modification was performed with KMnO4 as
described (23, 30), and the patterns were compared for complexes that
had been formed in the presence or absence of ppGpp. Modified positions
were identified by primer extension analysis, and the results are
displayed in Fig. 6A. It is
evident that the presence of ppGpp did not affect the KMnO4
modification pattern for the single-stranded template regions,
indicating that the conformation of the DNA in the environment of the
active center is not affected to a measurable extent. Still, the
possibility remained that the stability or lifetime of the ternary
complexes might be influenced by the presence of ppGpp. To test this we employed two independent methods suitable to determine the stability of
preformed ternary complexes. First, we measured the
time-dependent decrease in the capacity of the complexes
for RNA product formation after challenging preformed ternary complexes
with heparin (200 µg/ml). For this purpose aliquots of ternary
complexes that were formed either in the presence or absence of ppGpp
were subjected to conditions for run-off transcription by adding a
mixture of all four substrate NTPs including
[ Effect of ppGpp on the Rate of Ternary Complex Formation--
It
is known that the binary open complexes of rrn promoters
(RPo, Fig. 1B) are very unstable and are hardly
accessible for a quantitative analysis (17). We therefore measured the
formation of the stable ternary complexes (RPinit) after
the addition of the appropriate starting NTPs to RNA polymerase, which
had been preincubated with promoter DNA in the presence or absence of
ppGpp. The rate of RPinit formation was followed in
different ways. In one approach the time-dependent
formation of ternary complexes was analyzed directly by gel retardation
as described previously (23). In this experiment we either employed
32P-labeled promoter DNA or non-labeled DNA, which was
complexed in the presence of 32P-labeled starting NTPs.
Separation of free DNA and RNA polymerase complexes was performed on
5% nondenaturing polyacrylamide gels. The respective amounts of
complexes formed were determined by autoradiography. In an alternative
approach, we determined the increase of KMnO4 modification
of position ppGpp-dependent Inhibition Is Related to Substrate
Inhibition--
Together the experiments described above all point to
a mechanism of ppGpp-dependent inhibition consistent with
competition of the effector for the step of ternary initiation complex
formation. For this step it is essential that the substrate NTP
representing the first nucleotide in the growing chain of the
transcript is recognized by sequence complementarity to the template
position +1 and bound into the catalytic site of the RNA polymerase,
where it must be arranged such that its 3' hydroxyl group is ready for the acceptance of a phosphodiester bond with the 5' phosphate of the
incoming second NTP. If the mechanism of ppGpp-dependent inhibition is related to substrate competition, then any potential other substrate able to reach the active center of RNA polymerase but
not suitable to produce productive RNA chains should principally show a
similar inhibition characteristic as ppGpp. We therefore tested a set
of substances known to be accepted in the active site of the RNA
polymerase but unable to produce transcriptionally competent ternary
complexes. The dinucleotides ApA, ApC, or CpC, for instance, are such
potential substrates. It is known that they are accepted in the active
center of RNA polymerase, where they have been shown to act as
acceptors for growing RNA chains, e.g. 5' primers for RNA
transcription. On the other hand, because of the lack of a 5'
triphosphate group, they cannot be incorporated as substrates for 3'
chain elongation via phosphodiester bond formation and pyrophosphate
release. Their dinucleotide character might otherwise be essential for
a retarded passage through the nucleotide entrance channel formed by a
cavity of the RNA polymerase
To characterize at which step(s) the dinucleotide competitors exert
their inhibition, we performed single-round transcription initiation
and elongation experiments in presence or absence of the competitor
compounds as described before when ppGpp served as inhibitor (compare
with Fig. 3). The results obtained were again remarkably similar to
those in the experiments where ppGpp acted as inhibitor. A summary of
the data is presented in Fig. 9A. Both rrn
promoters, rrnB and rrnD P1, are significantly
inhibited when the dinucleotide effectors (300 µM) were
present before ternary complex formation. The activity of the
non-regulated Ptac promoter is again not affected. As shown
with ppGpp before, addition of the competitor substances after the
ternary complexes had been formed did not cause any inhibition for all
promoters tested. Fully consistent with the observation that ppGpp does
not specifically inhibit the Ptac promoter, we found a
slight increase in transcription efficiency for this promoter in
presence of ApC both before and after ternary complex formation. The
results demonstrate that the dinucleotide competitors behave very
similar to ppGpp. They show the same promoter specificity, and specific
inhibition occurs at a concentration range similar to the range that
has been determined for ppGpp.
Next, we wished to know whether the inhibitory effect of the
dinucleotides could also be ascribed to the change of the rate at which
ternary transcription initiation complexes are formed (see Fig. 7). To
answer this question, we analyzed the rrnB P1 promoter and
used ApA as inhibitor substance at a concentration of 150 µM. In addition we tested, in the same way as we did
before with ppGpp as inhibitor substance, whether the presence of
increasing concentrations of the starting nucleotide triphosphates
(ATP) would reduce or overcome the extend of inhibition. The results summarized in Fig. 9B demonstrate that the rate of ternary
complex formation at the rrnB P1 promoter is clearly reduced
in the presence of moderate concentrations of the dinucleotide ApA. As
shown for the regulator ppGpp, this inhibition is reduced or partly
counteracted when higher concentrations (500 µM instead
of 50 µM) of the starting NTP are present during the
reaction. We infer from this result that the mechanism of inhibition
must be very similar for ppGpp and the competitor dinucleotides tested.
Moreover, the results further support the notion that the inhibition
can most easily be described as substrate competition during the
formation of the stable ternary transcription initiation complexes.
In this study we have attempted to solve some of the questions
that are still pending with respect to the mechanism of the stringent
control. This type of regulation is mediated by the small effector
molecule ppGpp, which affects transcription of many genes, in
particular stable RNA genes. The concentration of this global regulator
changes rapidly in response to a variety of environmental signals, for
which amino acid starvation is the most notable example. Despite
numerous attempts to understand the mechanism of this fundamental
regulatory process of bacterial physiology, we still lack important
details. Many conclusions from former studies are controversial,
although some findings are consistent and in general agreement. There
is consent, for instance, that RNA polymerase is the target for ppGpp
(7, 8, 13), and, although not all the criteria are completely
understood, it is known that the respective promoter structures
determine whether a gene is under stringent control or not (5, 33-35). Moreover, both activation and repression of transcription are mediated
by ppGpp, yet activation may not be a direct effect of the regulator.
For instance, ribosomal RNA promoters represent the most outstanding
examples for negative stringent control, whereas for example the
universal stress protein UspA or amino acid biosynthesis proteins are
known to be induced at elevated ppGpp concentrations. Much of the
recent data suggest that the latter group of genes is probably
regulated by passive mechanisms (2), although direct effects have also
been reported (36-38).
To explain the mechanism of the stringent control, various steps of the
transcription cycle have been proposed to be crucial for negative
regulation. For instance, formation of the open promoter complex has
been suggested as the relevant kinetic step for the negative control of
rrn promoters (39, 40). A different conclusion, namely
trapping in the non-productive closed complex followed by efficient
inhibition at promoter clearance, was reached by a kinetic analysis of
stringently and non-stringently controlled rrnB P2 promoter
variants (4). The model is consistent with the partition model,
proposed earlier, which suggests that RNA polymerase can exist in two
interconvertible forms with different properties. Interconversion of
the enzyme is triggered by bound ppGpp. RNA polymerase without ppGpp
preferentially transcribes stable RNA promoters, whereas the enzyme
with bound ppGpp prefers mRNA promoters (41). Based on the
observation that stable RNA promoters form intrinsically unstable open
complexes, models for both positive and negative stringent control have
been proposed recently (3, 42). According to those models ppGpp has a
direct effect only on promoters under negative stringent control
(e.g. stable RNA promoters), whereas positive stringent
regulation is explained as passive effect of increasing RNA polymerase
concentration, which results from transcriptional shut-down of the
stable RNA genes. Recent studies describe clear evidence in
vivo and in vitro for transcriptional inhibition by
ppGpp at the lambda PR promoter (29, 43). Interestingly,
the PR promoter forms rather stable open complexes
indicating that ppGpp-dependent regulation is apparently not restricted to promoters with short-lived open complexes. Moreover, based on earlier studies, several passive models for stringent control
have been proposed previously (44, 45). The many different models, and
the fact that none by itself can explain all the existing data, may
reflect that there is probably not one single mechanism for the
stringent control. This view is further strengthened by the finding
that ppGpp is able to affect steps during transcription initiation but
also during elongation (6, 12, 46).
In this study we have performed experiments in vitro,
designed to define which step(s) of the initiation pathway are
primarily affected by ppGpp and to explore the molecular mechanism by
which ppGpp exerts it regulatory function. We selected the ribosomal RNA P1 promoters from the rrnB and rrnD operons,
which differ in their starting nucleotide (ATP for rrnB and
GTP for rrnD), bearing in mind the NTP-sensing model, which
proposes that the starting NTP concentrations in the cell determines
the sensitivity of stringent regulated promoters (18). For comparison
we employed the synthetic PtacI promoter, which does not
contain a stringent discriminator sequence and which is known not to be
affected by ppGpp. Consistent with the expectation, we found only a
very small inhibition in multiple-round transcription experiments. In
contrast, the two stable RNA promoters were strongly inhibited under
the same conditions. At the same ppGpp concentration, inhibition of the
two rrn promoters was not identical, however, and the
rrnB P1 promoter exhibited a stronger inhibition (92%
compared with 82% for rrnD). Whether this slight difference
is caused by the minor structural deviations of the core promoter
sequences (rrnB P1 deviates in only one, and rrnD
P1 in three positions from the ideal consensus sequence; see Fig.
1A) or whether the difference reflects the alternative
starting nucleotides (ATP versus GTP) remains unclear.
Our study clearly demonstrates that the major effect of ppGpp is
exerted before or during the formation of the ternary transcription initiation complexes. This can be concluded from the following findings. 1) Strong inhibition is only observed when ppGpp is added
before or at the formation of the ternary complex (see Fig. 3). 2)
Effects later during the initiation cycle (promoter clearance, abortive
initiation or pausing) do not contribute significantly to overall
inhibition (see Fig. 5). In addition we can show that the concentration
of initiating nucleotides is crucial for the extent of inhibition. High
starting NTP concentrations abrogate ppGpp-dependent
inhibition (see Fig. 4). We could further demonstrate that the DNA
structure within the open complex is very likely not altered in
presence of ppGpp. We take this as indication that the conformation of
ternary transcription complexes are not changed dramatically by ppGpp
(see Fig. 6A). Consistent with previous studies
(e.g. Ref. 3), we obtained evidence that the difference in
stability of the ternary complexes in presence or absence of ppGpp is
insufficient to explain the degree of inhibition (see Fig.
6B). Most interestingly, we find that the rate of ternary complex formation is reduced in presence of ppGpp. This effect can be
compensated by an increase in the concentration of the initiating NTPs
(see Fig. 7).
Together these results are consistent with a previously published
model, suggesting that ppGpp competes with the initiating NTP
substrates (25). The model makes the assumption that the obligatory
path for substrate NTPs to enter RNA polymerase active center is the
secondary channel, a structure formed by elements of the Our conclusions are in an apparent contrast to a previously published
study (51) demonstrating that ppGpp lowers the overall elongation rate
of RNA polymerase by enhanced pausing of the transcription elongation
complex. The authors, by performing classical kinetic analyses,
concluded that ppGpp-dependent inhibition did not appear to
be competitive with the ribonucleoside triphosphate substrates. There
are several possible explanations for this discrepancy. First, the
mechanism of ppGpp-dependent inhibition on elongation may
be substantially different from that on ternary initiation complex
formation where the promoter structure is certainly involved. The
latter studies, however, were performed at elongation with transcription complexes initiated at the non-stringently controlled T7
A1 promoter. Moreover, because overall elongation rates and not
individual pauses were studied, it is not completely clear whether the
kinetic formalism correctly applies to the rather complex situation
with individual pauses dependent on a sequence-specific context. The
observed overall inhibition may be the result of more than one
inhibition mechanism, and competitive inhibition my be masked under
such a complex situation.
Whether substrate competition is actually involved in the mechanism of
ppGpp-dependent inhibition was further investigated by
substituting the effector molecule ppGpp with nucleotide analogues. We
selected dinucleotides (ApA, ApU, and CpC) that are known to be
accepted as primers and thus are able to enter the active center of RNA
polymerase. As can be seen in Figs. 8 and 9, these competitor substances not only inhibited transcription of the stable RNA promoters
but showed almost the same characteristics of inhibition and were
effective at the same concentration range as ppGpp. Moreover, in all
cases the PtacI promoter was not inhibited. As shown for ppGpp, the dinucleotide competitors acted exclusively before or at the
step leading to formation of the ternary initiation complex. Moreover,
they exhibited the same dependence on the concentration of the starting
NTPs as observed with the regulator ppGpp; finally, they reduced the
rate at which the ternary complexes are formed. We believe that all
these observations are certainly not an accidental coincidence.
Therefore, we take these findings as a strong indication that substrate
competition plays a major role in the mechanism of ppGpp-mediated
stringent control.
Of course there is also the possibility that dinucleotide substrates
might mimic other mechanisms of ppGpp-dependent inhibition. For instance, such substrates might directly interact with a putative ppGpp binding site and invoke allosteric effects in much the same way
as might be the case for ppGpp. In a recent in vitro
transcription initiation analysis employing the dinucleotide ApU at the
lambda PR promoter, it was shown that ppGpp-mediated inhibition was
abrogated (29). As the simplest interpretation of this finding, the
authors concluded that ppGpp inhibits formation of the first
phosphodiester bond and this step is by-passed by the dinucleotide. It
was thus concluded that the effect of ppGpp is less drastic in the
presence of the dinucleotide ApU. The alternative explanation as
outlined above might, however, also apply to this observation. To
further test the model and to explore the exact type of inhibition or competition, more experiments with competitor analogues are under way.
There is independent evidence for the regulation of NTP access to the
active site of RNA polymerase, and blocking the secondary channel for
free passage of NTP substrates has been proposed recently in quite a
different context. For the structure that is central for the secondary
channel, different conformations have been described. Within the RNA
polymerase, the bridging helix formed by The proposed mechanism of competition outlined above does not rest on a
static RNA polymerase structure. To the contrary, more complex
scenarios can be envisaged involving dynamic changes of the
transcription complex. Many observations are in favor of the view that
RNA polymerase activity can be regulated by allosteric mechanisms, as
in the case of the swing-gate mechanism, where the conformation of the
F helix is modulated by factors acting elsewhere in the molecule
binding to stringent promoters or the interaction with an effector
molecule like ppGpp might change the architecture of distant domains
within the multi-subunit enzyme. Allosteric mechanisms could
principally explain a different phenomenon of the stringent control,
which appears to be independent from substrate competition. For
instance, several reports have provided evidence that ppGpp affects the
relative competitiveness of sigma factor interaction with core RNA
polymerase (9, 15, 48). In this case ppGpp could be an allosteric
effector that interacts at some site within the secondary channel (or
elsewhere) and triggers a conformational change, which alters the
complex interacting surface of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
'
subunits. The presence of ppGpp renders the activity of the enzyme in
such a way that transcription from stringent promoters (e.g.
rRNA and tRNA promoters) is strongly repressed. However, mutations in
the RNA polymerase sigma subunits have also been reported which
apparently obstruct
70-core polymerase interaction and
thereby cause altered initiation properties of RNA polymerase at
elevated ppGpp concentrations (14). This phenomenon has been observed
for other sigma factors like
32,
38, and
54 as well (9, 15).
10 promoter recognition element (16). This sequence
is termed the discriminator, and it has been shown to be a necessary,
although not sufficient, structural element for stringent regulation.
It is also clear, however, that other properties of a promoter, in
addition to the discriminator sequence, are involved in defining
whether a transcription unit is under stringent control or not (5).
Studies in the past have revealed that promoters under negative
ppGpp-dependent control are characterized by a striking
instability of their binary open complexes. For instance, the
stringently regulated rRNA promoters will only form stable open
complexes in presence of their corresponding starting nucleotides (3,
4, 17, 18). In the absence of starting nucleotides, the lifetime of
open binary complexes is very short and such complexes have a high
tendency to dissociate. Hence, at low NTP concentrations, formation of
the ternary complexes will be the critical, rate-limiting step for
productive transcription initiation. How, and at which step of the
initiation pathway, can ppGpp affect transcription from such promoters?
To answer this question we have systematically analyzed the effect of
ppGpp on the individual steps leading to productive transcription
initiation at several rRNA P1 promoters. Studies were performed
in vitro, and the unregulated PtacI promoter
served as a control. We show here that ppGpp affects the negatively
regulated rRNA promoters at the transition from the binary open
complexes to the ternary initiation complexes. The concentration of the
starting substrate NTPs play a crucial role in this inhibition,
suggesting that competition between the substrate NTPs and ppGpp for
the catalytic site is involved. This conclusion is supported by almost
identical inhibition characteristics when ppGpp is replaced by NTP
substrate analogues known to bind to the active site of RNA polymerase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
183 to +63, relative to the
transcription start site) obtained from plasmid pUC18-1 (5), a 575-bp
rrnD P1 promoter fragment (positions
259 to +316, relative
to the transcription start site) obtained from pHD1D,2 and a 403-bp
PtacI promoter fragment (positions
223 to +180, relative
to the transcription start site) from PtacW (5). Overhanging DNA ends were filled in by a Klenow reaction. Fragments employed for
gel retardation were labeled by Klenow reaction and
[
-32P]dATP.
-32P]UTP), and incubation continued for additional 15 min. Reactions were chased in the presence of all four NTPs (433 µM each) and 433 µg/ml heparin for 6 min at 30 °C.
The transcripts obtained were precipitated with ethanol, resolved, and
denatured in formamide sample buffer. Products were identified by
autoradiography after separation of transcripts on denaturing 15%
polyacrylamide gels.
-32P]CTP (5 µM) were
added and incubation was continued for another 30 min. Heparin was then
added (final concentration: 200 µg/ml) to prevent reinitiation. From
this mixture aliquots were withdrawn at defined time intervals and
subjected to a chase reaction for 8 min. Reactions were stopped by the
addition of formamide sample buffer and analyzed by gel electrophoresis
as described above.
-mercaptoethanol was added at a final
concentration of 10%. The modified DNA was ethanol-precipitated, resolved in 50 µl of TE-buffer, and purified using the Qiaquick PCR
purification kit (Qiagen, Germany) according to the protocols from the
manufacturer. KMnO4-modified nucleotides were identified by
primer extension as described (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP) for
the rrnB P1 and PtacI promoters. For the
rrnD P1 promoter, the reaction mixture contained 50 µM each ATP, GTP, and UTP and 0.85 nM (2.5 µCi) [
-32P]CTP. The reactions were stopped after 15 min by addition of heparin (433 mg/ml) followed by a 6-min chase with
433 µM amounts of all four NTPs. For the quantitative
analysis of transcription products, a radioactive DNA fragment that
differed in size from the expected transcripts was added as recovery
standard and samples were precipitated with EtOH prior to loading on a
sequencing gel. Fig. 2 shows the
autoradiogram of a typical inhibition experiment after gel
electrophoretic separation. The quantitative evaluation of the
transcription products according to densitometry is presented as an
inset (Fig. 2D). Strong inhibition can be seen
for the rrnB (92%) and rrnD (82%) P1 promoters,
reaching a maximum at ppGpp concentrations above 300 µM.
As expected, only a weak and nonspecific inhibition is apparent for the
PtacI promoter under the same conditions. This
multiple-round in vitro transcription experiment provides information on the overall inhibition, including
the various steps of the initiation cycle, as well as promoter
clearance and elongation. To address the question at which defined
step(s) of the transcription pathway ppGpp exerts its inhibitory
effect, it was necessary to dissect the individual steps of the
transcription reaction.
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Fig. 1.
A, the sequences of the core promoter
structures rrnB P1, rrnD P1, and PtacI
are presented. The 35 and
10 recognition sequences are
boxed, and the transcription start site is denoted by
+1. The starting nucleotide is given in bold
type, and nucleotides matching the discriminator consensus
are underlined. B, kinetic scheme indicating the
four main steps of transcription initiation. I indicates the
first binding of RNA polymerase (R) to a promoter
(P) forming a closed binary RNA polymerase-promoter complex
(RPC), II indicates isomerization to
the binary open complex (RPO), and
III formation of a ternary initiating complex after binding
of the first NTPs (RPinit). Finally formation of
an elongating complex (EC) under release of the promoter is
shown schematically. The latter step involves the loss of the sigma
subunit (
), which may be reused for the next initiation cycle.
Before the elongating complex is formed and productive transcription is
started, the initiating complex may undergo multiple rounds of abortive
cycling upon which short transcripts (Abortive
products) are repetitively released.
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Fig. 2.
Multiple-round transcription analysis in the
presence and absence of ppGpp. A-C, gel
electrophoretic separation of run-off products from multiple-round
in vitro transcription reactions from the rrnB P1
(A), rrnD P1 (B), and PtacI
(C) promoters. Bands corresponding to the respective run-off
products are indicated. Standard denotes the position of a
radioactive DNA fragment added as recovery standard. The effector
concentrations present during the reaction are given on top
of each lane. D, quantitative evaluation of the
band intensities shown in A-C. Symbols are
defined in the inset.
-32P]UTP for rrnB P1
and 5 µCi of [
-32P]CTP for rrnD P1). The
reaction was stopped after an 8-min chase, and samples were mixed with
loading buffer prior to separation on a denaturing polyacrylamide gel.
For each promoter-specific template, a control reaction was performed
in exactly the same way, except that ppGpp was omitted during the
complete procedure. Transcription products were quantified by
densitometry, and the results are presented in Fig.
3. From this experiment it can be concluded that both rRNA promoters (rrnB and rrnD
P1) are inhibited by ppGpp before the stable ternary
initiation complexes were formed. The degree of inhibition roughly
matches the overall inhibition presented in Fig. 2, with a somewhat
stronger inhibition for the rrnB compared with the
rrnD P1 promoter. Most remarkably, no significant inhibition
can be seen for both rRNA promoters for the reactions analyzed
after the addition of the initiating NTPs which enable stable ternary complex formation. As expected, the PtacI
promoter is not specifically affected before or after ternary complex
formation. Note that formation of the open binary initiation complex
for PtacI does not require the presence of starting NTPs for
stabilization. From the results it can be concluded that the major
effect of ppGpp-dependent inhibition must occur before RNA
polymerase forms the ternary transcription initiation complex. Taken
together the results demonstrate that ppGpp-dependent
inhibition occurs during the early initiation phase, before ternary
initiation complexes are formed, and steps after formation of the
stable ternary complex (non-productive, abortive transcripts, promoter
clearance, and elongation) do not contribute appreciably to overall
inhibition.
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Fig. 3.
Effects of ppGpp addition at different steps
during the transcription cycle. Results from single-round in
vitro transcription reactions with the rrnB P1,
rrnD P1, and PtacI promoters are summarized with
ppGpp present before (left panel) or after
(right panel) ternary initiation complex
formation. Relative transcription yields in the absence ( ) of ppGpp
(gray bars) are normalized to 1. Transcript
yields in the presence of ppGpp (+) are indicated by black
bars.
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Fig. 4.
Effect of the starting NTP concentration on
ppGpp-dependent inhibition. Relative transcription
products from the rrnB P1 promoter are presented as a
function of the starting nucleotide concentration (ATP). Results were
obtained from multiple-round transcription reactions. Dark
squares indicate transcripts obtained in the absence of
ppGpp; open circles indicate transcripts obtained
in the presence of ppGpp.
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Fig. 5.
Analysis of in vitro
transcription products from single and multiple-round
transcription reactions in the absence and presence of ppGpp.
Products obtained from the rrnB P1 promoter were separated
on a 20% polyacrylamide gel. The absence or presence of ppGpp (300 µM) is indicated and the reaction times are given in
minutes on top of the lanes. Bands characteristic
for the run-off transcripts, and abortive or pausing products are
indicated at the margin. The position of the recovery
standard to normalize differences in loading is also shown. Samples in
the lanes indicated by 23 min were obtained after
the reaction has been chased.
-32P]CTP. The run-off products were then quantified
after gel electrophoresis by densitometry. A summary of the results is
presented in Fig. 6B. Consistent with the high stability of
ternary initiation complexes, only a small reduction of the
transcription products can be observed within the time course of the
experiment. Moreover, it is evident that the presence of ppGpp has only
a slight effect on the complex stability. Although in the absence of
ppGpp approximately 5% of the starting complexes were inactivated
within 30 min, this inhibition increased only by a factor of 2 (10%)
in the presence of ppGpp. Very similar results were obtained when the
lifetime of the ternary complexes was analyzed in a similar way as
described for the footprint analysis, namely by the KMnO4
reactivity of single-stranded T nucleotides in the template strand of
the transcription bubble (data not shown). The small decrease in
stability of the ternary initiating complexes is clearly not sufficient
to explain the measured inhibition of transcription in the presence of
ppGpp. We conclude from this results that the observed inhibition
mediated by ppGpp cannot be explained by destabilization of the ternary initiation complexes.
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Fig. 6.
KmnO4 modification of ternary
transcription complexes. A, a primer extension analysis
after KMnO4 modification of the ternary transcription
complex at the rrnB P1 promoter is shown. Products for the
template and the non-template strand were separated by gel
electrophoresis. Samples were run next to nucleotide-specific
sequencing ladders (A, C, G, T). The relevant sequence given at the
margin. K and K+ denote protein-free
DNA controls with (+) and without (
) KMnO4 modification,
respectively. Lanes marked with 0 or
300 indicate the absence or presence of 300 µM
ppGpp. Numbers at the right margin
indicate the modified nucleotide positions in the open complex.
B, time-dependent decrease of
transcription-competent ternary complexes formed at the rrnB
P1 promoter. The decrease in complex concentration was determined by
assessing the relative amounts of transcripts that could be formed
after addition of heparin. Reactions were followed in the absence
(dark squares) and presence of (300 µM) ppGpp (open squares).
T10 in the template strand (see above), and finally we
measured the time-dependent increase in synthesized RNA
transcription products. Measuring the RNA products has the potential
advantage that only productive ternary complexes capable of elongating
transcription are evaluated and transcription-incompetent complexes,
dead-end complexes, or moribund complexes (31) will remain
unconsidered. All methods gave almost identical results, and therefore
only findings from the latter experiments are presented in Fig.
7. It can be assumed that at the initial
phase of the reaction the rate of RNA products formed should be
directly proportional to the concentration of the ternary initiation
complexes. As shown in Fig. 7, comparison of the initial rates of
ternary complex formation yields notable differences in the absence and
presence of ppGpp. Interestingly, this difference depends on the
concentration of the starting nucleotides. Although there is
approximately a 10-fold difference in the initial product rates at low
ATP (50 µM) concentrations, inhibition is significantly
reduced at higher amounts of the starting NTP (500 µM),
and now only approximately a 1.2-fold reduction is apparent. It can
also be seen by the plateau values presented in Fig. 7 that the final
yield of RNA transcripts is not significantly different at high
versus low ATP concentrations. The results are consistent with the conclusion that the presence of ppGpp reduces the rate of
RPinit formation. This inhibition can be overcome largely
by increasing the concentration of starting nucleotides. We take this
finding as an indication that competition for the substrates might be
involved in the ppGpp-dependent inhibition mechanism.
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Fig. 7.
Kinetics of ternary transcription initiation
complex formation. The time-dependent formation of
ternary transcription complexes in the absence or presence of 300 µM ppGpp is shown for the rrnB P1 promoter.
The kinetics of complex formation was followed at high (500 µM) and low (50 µM) concentrations of the
starting nucleotide ATP. The upper part of the
figure shows the run-off transcription products obtained from the
ternary complexes as visualized by autoradiography. In the
lower part of the figure, the data are quantified
in a diagram. Open symbols denote the presence of
300 µM ppGpp, closed symbols its
absence during complex formation. Results obtained at 500 µM starting nucleotide concentration are shown by
circles, whereas the data obtained at 50 µM
are shown by squares.
/
' subunits (32). Hence, we used
these compounds as potential analogs that might mimic the inhibitory
effect of ppGpp. To avoid problems resulting from interference as RNA
primers, we selected different dinucleotide phosphates that did not
match the start sequence of the different promoters analyzed (see Fig.
1). For the control Ptac promoter, we chose CpC and ApC. For
the rrnD promoter ApA, and for the rrnB promoter
ApA and ApU, were tested as potential effectors for transcription. The
chosen compounds were then analyzed in the same way as shown for the
effector compound ppGpp before. First we determined the effect on
overall transcription in multiple-round in vitro
transcription reactions. Experiments were performed as described
before, except that dinucleotides were added as effectors instead of
ppGpp. As shown in Fig. 8
(A-C), it turned out that the effector compounds resulted
in a very similar overall inhibition of the transcription reactions. A
very strong inhibition, essentially comparable with the effect induced
by ppGpp, is apparent when ApA is used as a competitor with both rRNA
promoters rrnB and rrnD P1 (Fig. 8, A
and B). Inhibition of the rrnB P1 promoter with
ApU is significant, although markedly reduced in comparison with ApA
(Fig. 8A). Moreover, when the dinucleotide CpC was used as
competitor (Fig. 8C), the Ptac promoter showed only a very weak and apparently nonspecific response that had already
been observed with ppGpp (compare with Fig. 2). Note that the same
dinucleotide ApA could not be analyzed with the Ptac promoter because it serves as starting primer. ApU could also not be
used for this promoter because it provoked a different start site (data
not shown). It is interesting to note that inhibition by the
dinucleotide competitors occurred at a concentration range similar to
the range that has been found for ppGpp-mediated inhibition.
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Fig. 8.
Effects of competitor nucleotides on
multiple-round transcription reactions from different promoters.
A, diagram summarizing the effects of ppGpp
(black bars), and the dinucleotides ApU
(gray bars) and ApA (white
bars) on relative transcription from the rrnB P1
promoter. B, relative amount of transcripts from the
rrnD P1 promoter at increasing concentrations of ppGpp
(black bars) in comparison to the dinucleotide
competitor ApA (white bars). C,
relative amount of transcripts from the PtacI promoter at
increasing concentrations of ppGpp (black bars)
in comparison to the dinucleotide competitor CpC (white
bars).
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Fig. 9.
Effects of competitor dinucleotides on the
different steps of transcription initiation. A, diagram
showing the effects of the dinucleotides ApA and CpC, respectively, on
the relative amount of transcription from the rrnB P1,
rrnD P1, and PtacI promoters when present before
or after ternary transcription initiation complexes were formed.
Relative amounts of transcripts in the absence of competitors are
normalized to 1. B, kinetics of ternary transcription
complex formation in the presence and absence of the dinucleotide
competitor substance ApA. Relative amount of transcripts are shown for
reactions performed at low (50 µM, squares)
and high (500 µM, circles) concentrations of
the starting nucleotide ATP in presence (open
symbols) or absence (filled symbols)
of 150 µM ApA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
' domains F
and G (32). The diameter of this nucleotide entry channel is broad
enough to allow passage of only one NTP at a time but also to
accommodate ppGpp. It is feasible, therefore, that at high ppGpp
concentrations free movement of substrate NTPs to the active site is
restricted. This does not matter much if the Km
values for substrate binding are low and the respective complexes are
stable. If, however, the requirements for substrate NTPs are high (high
apparent Km values), as is the case for initiation
complexes at stable RNA promoters, then the competition situation will
take effect. Stringent sensitivity can thus be explained by the
promoter structure, which through its conformation and specific
interaction with RNA polymerase, determines the stability of the
initiating complex and the apparent Km for the first
substrate NTP. The effector molecule ppGpp, which accumulates to
millimolar concentrations during the stringent control, may cause a
competition situation. According to the simplest explanation, ppGpp
effectively competes with the substrate NTPs for access to the active
center of RNA polymerase. As a consequence less or no productive
initiation complexes are formed.
' domains F and G
(alternatively bent or straight) are believed to provide a mechanism
for RNA polymerase translocation. The bent
' subunit F bridge
involves a clash competing with the incoming nucleotides at the active
site of RNA polymerase. The alternating movement pushes template bases
from the i+1 site (translocation) and concomitantly blocks
the entry for nucleotides into the active site. This switch mechanism,
termed "swing-gate" by the authors, explains translocation of the
transcript and simultaneously regulates the entry of substrate nucleotides (47).
/
' subunits that is known to
make contacts with the alternative sigma factors (49, 50).
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-211-811-4928;
Fax: 49-211-811-5167; E-mail:
r.wagner@rz.uni-duesseldorf.de.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300196200
2 A. Hillebrand and R. Wagner, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: rRNA, ribosomal RNA; R, RNA polymerase; P, promoter; RPc, closed complex; RPo, binary open complex; RPinit, ternary open complex or initiating complex that contains at least one bound substrate NTP; EC, elongating complex.
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