From the Department of Molecular Biology, Umeå
University, 901 87 Umeå, Sweden and the
Department of Cell
and Molecular Biology-Microbiology, Göteborg University, Box 462, 405 30 Göteborg, Sweden
Received for publication, September 10, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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Some promoters, including the DmpR-controlled
Escherichia coli holoenzyme RNA polymerase
is composed of a core enzyme
(E,1 subunit composition
Promoters of the In addition to the specific DmpR-mediated control mechanism described
above, transcription from the Down-regulation of transcription from stringent promoters is believed
to occur through the effects of binding of (p)ppGpp at the interface of
the The seven Strains and Culture Media--
Strains used are listed in Table
I and were routinely cultured and assayed
in Luria broth (LB; Ref. 38) supplemented with appropriate antibiotics
for selection. Where indicated, assays were also performed using rich
defined medium consisting of M9 minimal medium (38) supplemented with
22 amino acids (50 µg/ml, Sigma kit), serine at 1 g/liter as the
carbon source, all five nucleotides (20 µg/ml), trace metals, and
thiamine (0.05 mM). E. coli
MG1655 Plasmids and DNA Manipulations--
Plasmids and primers are
listed in Tables II and III. Plasmids
were constructed by standard recombinant techniques and the fidelity of
all PCR-derived DNA confirmed by sequencing. Plasmids pVI466 and pVI684
are equivalent dmpR-Po-luxAB luciferase reporter plasmids carried on either an RSF1010-based vector pVI466 (copy numbers
16-20) or a p15A-based vector pVI683 (copy numbers 18-22) generated
by replacing the EcoRI-to-NaeI kanamycin
resistance gene and promoter region (bp 93-1440) of pPROLar.A331
(Clontech) with an
EcoRI-to-SmaI PCR-amplified
spectinomycin/streptomycin gene generated from
pUT-mini-Tn5-Sp/Sm using primers Sp1/Sp2. The
NotI dmpR-Po-luxAB fragment of pVI466
was inserted into the unique NotI site of pVI683 to generate
pVI684.
The dmpR-Po-dmpB plasmid pVI686 used as a
reporter for P1-linkage analysis was assembled from previously cloned
regions of the dmp cluster of Pseudomonas CF600
and consists of a 4-kb HindIII-to-SmaI dmpR-Po-spanning fragment fused to a 2.3-kb
SmaI-to-HpaI dmpB encoding fragment on
the RSF1010-based cloning vector pVI398. The pVI687
dmpR-Po-tet selection plasmid has the same Po
reporter fusion point as pVI686. Plasmid pVI687 was generated by first cloning a 2.4-kb NruI-to-SmaI
dmpR-Po-spanning fragment into the EcoRV site of
pBluescript SK+ followed by insertion of a
HindIII-to-KpnI fragment carrying the
promoterless tetracycline resistance gene, generated from pBR322 using
primers Tc1/Tc2.
The in vitro transcription template plasmid pVI695 is based
on pTE103 and carries an EcoRI-to-BamHI Po
promoter fragment generated using primers Po1 and Po2 (Table III). This
Po promoter fragment spans from
The RpoN expression plasmid pVI688 carries the E. coli
rpoN gene as an EcoRI-to-KpnI fragment
spanning from 34 bp upstream of the ATG start to 7 bp downstream of the
termination codon. The PCR-amplified fragment was generated using
primers N1/N2 and cloned between the
EcoRI-to-KpnI region of pEXT21. Plasmids pVI690 to pVI694, for expression of wild type and mutant Selection of Second Site Suppressor Mutants--
Independent
cultures of E. coli CF1693 Localization and Identification of Mutations--
Given the
predominance of rpoBC mutants in a previous prototrophy
rescue screen (40), isolates were first screened for alterations in
rpoBC by using a non-isotopic RNA cleavage assay (NIRCA) kit (MutationScreenerTM) supplied by Ambion, Inc. and/or P1
transduction. For NIRCA analysis, each ~5-kb gene was amplified by
colony PCR from each mutant strain using rpoB B1/B2 and
rpoC C1/C2 primers and Expand High Fidelity Taq
polymerase (Roche Molecular Biochemicals). Using these products as
templates, five overlapping regions (of ~1000 bp) of each gene were
amplified using primers that incorporated the T7 promoter region at the
5' end (BT7-1A/B to BT7-5A/B and CT7-1A/B to CT7-5A/B; Table III).
These products were then mixed with the corresponding section amplified
from wild-type E. coli MG1655, transcribed to form RNA, and
the hybridized products subjected to limited RNase cleavage. The sizes
of RNase-cleaved bands resulting from mismatches were used to estimate
the location of the mutation, which was subsequently confirmed by DNA
sequencing. For the majority of isolates, assignment was made to the
rpoBC region as soon as a mutation was detected during
sequential analysis from the 5' region of rpoB through to
the 3' region of rpoC. In the case of the three rpoB and three rpoC alleles further analyzed in
this study, NIRCA analysis of the entire rpoBC region of
each strain revealed the single genetic changes listed in Table I.
However, even after introduction into a clean genetic background,
because NIRCA analysis does not detect all mutations (see below), there
is a small possibility that these derivatives may also contain
additional closely linked but undetected mutations that contribute to
their phenotype.
The NIRCA analysis assigned mutations in 53 isolates to
rpoBC. Mutations in an additional 12 isolates were assigned
to the rpoBC region by P1 linkage analysis. The assay system
used to determine linkage frequencies utilized the
thi-39::Tn10 marker and
CF1693
The remaining four isolates with mutations unlinked to rpoBC
were subjected to NIRCA analysis for mutations in both rpoD
(using primers D5/D6 and DT7-1A to DT7-2B) and rpoN (using
primers N3/N4 and NT7-1A to NT7-2B) (Table
III). This analysis identified two suppressors, sup40 (with a mutation in rpoD) and sup35 (with
mutations in both rpoD and rpoN). DNA sequence
analysis of the entire rpoN and/or rpoD genes was
used to confirm the genetic changes listed in Table I. In addition to
changes that result in two substitutions within RpoN, the sup35 strain
also possessed eight additional silent mutations in the wobble
positions of codons within rpoN. The original sup-35 strain
has also lost KmR associated with the
Purified Proteins--
Purification of E. coli
Synthesis and Purification of ppGpp--
Preparative-scale
synthesis and purification of ppGpp was performed using two different
methods that resulted in indistinguishable preparations that gave the
same results in in vitro assays. Synthesis using native
ribosome-associated RelA, prepared from LB-grown E. coli
MG1655 pALS10 cells cultured in the presence of 1 mM
isopropyl In Vivo Luciferase Transcription Assays--
Cultures for
in vivo luciferase transcription assays were grown overnight
in the test media, diluted and grown to early exponential phase, and
then diluted once more prior to initiation of the experiment by the
addition of 0.5 mM of the DmpR effector 2-methylphenol. For
cultures of strains harboring the Ptrp-rpoD
system, cultures were grown as described above in LB supplemented with
0.2 mM IAA, concentrated by centrifugation, and then used
to inoculate the media indicated. Luciferase activity of LuxAB within
whole cells was assayed with a 1:2000 dilution of decanal as described
previously (44).
In Vitro Transcription--
Assays (20 µl) were performed at
37 °C in transcriptional buffer containing 50 mM
Tris-HCl, pH 7.5, 50 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
EDTA, and 0.275 mg/ml bovine serum albumin. Core RNA polymerase (10 nM) was allowed to associate with variable amounts of wild
type or mutant Western Blot Analysis--
Crude extracts of cytosolic proteins,
SDS-polyacrylamide gel electrophoresis, transfer to nitrocellulose
filters, and Western blot analysis with polyclonal rabbit
anti- Mutations in RNA Polymerase Restore Transcription from Po in the
Absence of (p)ppGpp--
In wild type E. coli,
transcriptional output from the Po promoter carried on a luciferase
reporter plasmid (pVI466 dmpR-Po-luxAB) is
induced by more than 3 orders of magnitude by the presence of aromatic
effectors. This genetic system reproduces the exponential/stationary phase transition induction observed in the native system (Fig. 1A, and Ref. 44). The
dependence of the Po promoter on (p)ppGpp leads to an ~10-fold
decrease in maximal transcription from Po in (p)ppGpp-deficient strains
(Fig. 1A, and Refs. 25, 26, and 44). We utilized this
10-fold effect on Po transcription to genetically select mutations that
restored transcription to Po in the absence of (p)ppGpp (Fig.
1B).
To generate mutants we used a selection plasmid carrying the
DmpR-controlled Po promoter driving transcription of a promoterless tetracycline resistance gene (pVI687,
dmpR-Po-tet). This plasmid confers aromatic
effector-dependent tetracycline resistance in the E. coli (p)ppGpp+ strain, but is incapable of conferring
tetracycline resistant in a (p)ppGpp0 counterpart.
Spontaneous mutants were selected by asking for growth of
(p)ppGpp0 E. coli on LB containing tetracycline
and an aromatic effector of DmpR. P1 transduction and/or a non-isotopic
RNA cleavage assay were used to determine the location of the mutations
in the independent isolates as described under "Experimental
Procedures." Of the 69 mutants that proved amenable to analysis, 65 harbored mutations in rpoBC (
By screening a selection of the mutants using the
dmpR-Po-luxAB luciferase reporter plasmid pV1466,
we found that the degree of restoration of Po transcription in
(p)ppGpp0 CF1693 Mutant
As a first step in the analysis, we added increasing amounts of the
individual
To assess relative competitiveness of the different
Based on the results above, we determined the relative competitiveness
of all the mutant ppGpp Has No Major Influence on E
To test whether ppGpp has any direct effect on
The results outlined in this and preceding sections suggest that,
although both Effect of Modulating
The maximal Po transcription level in the parental
(p)ppGpp-proficient MG1655 Modulating
When rpoD is under control of its native promoter, ~2-fold
higher
The E. coli Rsd protein has been proposed to act as an
anti- Here we report on the function of (p)ppGpp as a master determinant
of the outcome of The data outlined above clearly support a recently proposed model for
(p)ppGpp as a master regulator of alternative The role of (p)ppGpp in The in vivo impact of In the holoenzyme, The four mutant rpoD alleles employed in this study provided
an important mechanistic tool to dissect the role of N-dependent Po promoter, are
effectively rendered silent in cells lacking the nutritional alarmone
(p)ppGpp. Here we demonstrate that four mutations within the
housekeeping
D-factor can restore
N-dependent Po transcription in the absence
of (p)ppGpp. Using both in vitro and in vivo
transcription competition assays, we show that all the four
D mutant proteins are defective in their ability to
compete with
N for available core RNA polymerase and
that the magnitude of the defect reflects the hierarchy of restoration
of transcription from Po in (p)ppGpp-deficient cells. Consistently,
underproduction of
D or overproduction of the
anti-
D protein Rsd were also found to allow
(p)ppGpp-independent transcription from the
N-Po
promoter. Together with data from the direct effects of (p)ppGpp on
N-dependent Po transcription and
-factor
competition, the results support a model in which (p)ppGpp serves as a
master global regulator of transcription by differentially modulating
alternative
-factor competition to adapt to changing cellular
nutritional demands.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
'
) associated with one of seven sigma
(
)-factors that program the complex to engage and initiate transcription at different sets of promoters (1). Thus, the levels and
binding properties of alternative
-subunits together with factors
that modulate their ability to associate with core RNA polymerase are
critical for the relative composition of the multiple holoenzymes
available for transcription of the distinct promoter classes within the
prokaryotic genome. The seven different
-factors of E. coli fall into two groups. The larger of these comprises six
factors that share notable sequence and functional similarities to the
major
D (
70)-factor that is responsible
for transcribing "housekeeping" genes (2). Recent structural
studies have shown that the
D-like proteins comprise
three globular domains that encompass previously identified
conserved regions (
2 (1.2 to 2.4),
3 (3.0 to 3.1),
4 (4.1 to 4.2)) that are tethered by flexible
linkers (3-6). In contrast, the alternative
N
(
54)-factor is in a class on its own, bearing little
sequence homology to other
-factors and determining recognition of
the well conserved but unusual
24,
12
(TGGCAC-N5-TTGC) promoter sequences
(7). In addition, there are significant differences in the action of the cognate holoenzymes at promoters. Unlike E
D, which
can undergo transition from the initial closed complex to the
transcriptionally competent open complex without any other regulatory
factor, the E
N closed complex is kinetically and
thermodynamically stable and E
N cannot melt promoter DNA
on its own. Transition to the open complex is dependent on interaction
with, and nucleotide hydrolysis by, a member of the bacterial family of
enhancer binding proteins (reviewed in Ref. 8).
N-dependent
24,
12
class direct transcription of genes involved in a variety of
physiological processes responsive to nutrient limitation such as
nitrogen assimilation and fixation, substrate-specific transport
systems, and utilization of alternative carbon and energy sources.
Appropriate environmental signals lead to activation of the cognate
regulator by diverse mechanisms that result in a common active form of
the regulator (9). For DmpR, which controls transcription of the
N-Po promoter of an operon encoding the enzymes for
metabolism of (methyl)phenols in Pseudomonas CF600, the
activation mechanism is direct. Binding of aromatic phenolic pathway
substrates to its N-terminal regulatory A-domain alleviates interdomain
repression to give the active form of the protein (10, 11), and DmpR mediated aromatic effector- and ATP-dependent transcription
from Po can be fully reconstituted in vitro (12, 13).
N-Po promoter is dependent
on the unusual nucleotides guanosine tetraphosphate (ppGpp) and
guanosine pentaphosphate (pppGpp), collectively referred to as
(p)ppGpp. These molecules are heralds of metabolic stress and were
originally identified as the mediators of the classical stringent
response to down-regulate superfluous stable RNA synthesis upon amino
acid starvation (reviewed in Refs. 14 and 15). Synthesis of these
molecules by ribosome-associated RelA (p)ppGpp synthetase is activated
by the arrival of an uncharged tRNA on the ribosome, whereas the dual
function SpoT protein (which is primarily responsible for (p)ppGpp
degradation) catalyzes (p)ppGpp synthesis in response to glucose
starvation (16). Since their discovery, however, these signaling
molecules have also been implicated in the up-regulation of
transcription from many classes of promoters including those dependent
on
D (e.g. Refs. 16 and 17), the stationary
phase
-factor
S (e.g. Refs. 18-22), the
heat shock
-factor
H (e.g. Refs. 22-24),
as well as
N (e.g. the Po and Pu promoters;
Refs. 25-27).
- and
'-subunit of RNA polymerase (28, 29). Binding of
(p)ppGpp destabilizes E
D-promoter complexes, and,
because open complexes formed by E
D at rRNA P1 promoters
are intrinsically very unstable, it has been proposed that these
promoters are likely to be particularly sensitive to (p)ppGpp
destabilization (30). Support for this mechanism comes from analysis of
suppressor mutations within
and
' (isolated by rescue of the
polyauxotrophic growth defect of (p)ppGpp-deficient strains) that also
destabilize E
D-promoter open complexes in
vitro (31-33). One possible consequence of down-regulation of
transcription from the powerful stringent promoters is an increase in
the pool of core RNA polymerase that is normally sequestered in
producing these abundant transcripts. Thus, accumulation of (p)ppGpp
and, by analogy, suppressor mutations has been proposed to increase the
amount of E
D available for (p)ppGpp-stimulated
D-promoters that are difficult to saturate and have
E
D recruitment as the rate-limiting step (33-35).
-factors of E. coli exhibit quite different
affinities for core RNA polymerase (reviewed in Ref. 36), and (p)ppGpp has the potential to play multiple roles in their function. In addition
to the potential to directly alter the promoter recognition and
kinetics of transcriptional initiation at promoters, (p)ppGpp could
also plausibly modulate the
-association properties of the core RNA
polymerase. Models involving the influence of (p)ppGpp on
-factor
competition for limiting available core have been put forward to
explain poor induction of
S- and
H-dependent promoters in (p)ppGpp-deficient
cells (20, 21, 37). Most recently evidence for a direct effect of
(p)ppGpp on the competitive abilities and levels of
S
and
H associated with core RNA polymerase has been
presented (22). Primarily based on the ability of two mutations in
D to restore transcription to a
N-dependent promoter in the absence of
(p)ppGpp, we have previously postulated that modulation of
-factor
competition may at least in part underlie the (p)ppGpp dependence of
the Po promoter that requires the action of the structurally and
functionally distinct
N protein (25). Here, we dissect
the potential of (p)ppGpp to directly affect
N-dependent Po transcription and to modulate
N competition by (i) identifying new suppressor
mutations that restore transcription from Po in (p)ppGpp-deficient
cells and (ii) performing in vivo and in vitro
-factor competition in the presence and absence of (p)ppGpp. We
present evidence that (i) the levels of alternative
-factors have a
large impact on the output from the Po promoter and (ii) the
requirement for (p)ppGpp can be suppressed by four mutations in
D that exhibit defects in competition against
N, or by underproduction or sequestering of
D. These results strongly support a model in which
(p)ppGpp serves as a master regulator of transcription through
-factor competition for limiting core RNA polymerase and brings the
performance of the structurally and functionally distinct
N protein within the global control of the stringent response.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
lac and CF1693
lac strains carrying
Ptrp- rpoD were generated by transferring
(CAM
) Ptrp- rpoD from E. coli MC4100 (CAM
) Ptrp- rpoD into these strains
by P1-mediated transduction using the tetracycline resistance
aer-3075::Tn10 marker and testing for
chloramphenicol resistance and/or indole-3-acrylic acid (IAA) dependence. Prior to analysis, all the rpo alleles were
introduced in clean genetic backgrounds by P1-mediated transduction
exploiting the thi-39::Tn10 marker for
rpoBC alleles,
aer-3075::Tn10 for rpoD
alleles, and zhc-6::Tn10 for the
rpoN allele. DNA sequencing was used to monitor
co-transduction of the mutant alleles. Suppressor phenotypes were
assessed using a Po-luxAB reporter and the ability of the
strains to grow on M9 minimal media plates supplemented with 10 mM glucose and 100 µg/ml thiamine.
Bacterial strains
Plasmids
408 to +26 relative to the
transcription start site and thus encompasses the DmpR binding sites
(upstream activating sequences
170 to
127), the IHF binding site
(-72 to
37), and the
24,
12 Po promoter (39).
D,
were generated by assembling two PCR-amplified fragments between the
NdeI and EcoRI sites downstream of the expression
cassette of pJLA503. Primers D1/D2 were used to generate an
NdeI-to-BamHI fragment with the NdeI
site encompassing the ATG start codon and extending to the internal
BamHI site of MG1655 rpoD. Primers D3/D4 were
used to generate BamHI-to-EcoRI fragments from
wild-type or mutant rpoD derivatives to reconstitute
full-length rpoD from the internal BamHI to 12 bp
downstream of the termination codon.
lac harboring pVI687
(dmpR-Po-tet) were grown for 2 h with
shaking at 30 °C in 2 ml of LB supplemented with carbenicillin (50 µg/ml) and 0.5 mM 2-methylphenol (the most potent
aromatic effector of DmpR). Tetracycline was then added to 20 µg/ml
and the culture incubated under the same conditions for another 2 h prior to dilution and plating on equivalent solid media and growth
overnight at 30 °C. Equal numbers of large and small independent
isolates were re-streaked and screened for DmpR dependence of the
tetracycline resistance phenotype by testing for TcR in the
presence and absence of 0.5 mM 2-methylphenol. Of the original 102 isolates, all exhibited DmpR
effector-dependent tetracycline resistance. However, 33 were discarded as being unstable, P1 phage-resistant, and/or having
suppressor phenotypes too poor to map by P1 transduction as described below.
lac harboring the reporter plasmid pVI686. Plasmid pVI686 mediates DmpR-Po-dependent expression of the
dmpB-encoded catechol-2,3-dioxygenase, which converts
catechol to the bright yellow product 2-hydroxymuconic semialdehyde.
Co-transduction of thi-39::Tn10 and a
suppressor mutation was scored among TcR transductants
grown in the presence of 0.5 mM 2-methylphenol by
determination of the number of yellow versus white colonies after spraying with catechol (41).
relA251::Km of the host. Therefore, the detected mutant rpoD and rpoN alleles of this
strain were only analyzed independently in authentic
CF1693
lac.
PCR primers
N, IHF,
A2*-His-DmpR, and DmpR-His has previously
been described (12, 39). Core RNA polymerase was purchased from
Epicentre Technology. For purification of E. coli
D and its mutant derivatives, expression plasmids pVI690
to pVI694 were introduced into the cognate CF1693
lac
derivatives prior to purification as previously described (42).
-D-thiogalactopyranoside (IPTG) during the
last 1 h of growth, was essentially as described (43) except that
equimolar ratios of GTP and GDP were used as substrates. Synthesis
using a purified His-tagged RelA protein was essentially as described
in Ref. 27, with the minor modifications described in Ref. 22. Pure
ppGpp was lyophilized and stored at
80 °C until use. Purity of the preparations was monitored by thin layer chromatography on
polyethyleneimine cellulose plates (Merck), using 1.5 M
KH2PO4 (pH 3.4) as chromatographic buffer.
Concentrations of ppGpp were determined spectrophotometrically at
A260 using the molar extinction coefficient of
13,700.
D and/or
N for 5 min in
the presence of 4 mM ATP (required for DmpR activity) and
ppGpp, where added. Open complex formation was initiated by the
addition of 0.5 µg of supercoiled pVI695, IHF (10 nM),
DmpR-His (50 nM) or
A2*-His-DmpR (100 nM),
and the aromatic effector 2-methylphenol (0.5 mM). After 20 min of incubation to allow open complex formation, multiple-round
transcription was initiated by adding a mixture of ATP, GTP, and CTP
(final concentration, 0.4 mM each), UTP (final concentration, 0.06 mM), and [
-32P]UTP (5 µCi at >3000 Ci/mmol, Amersham Biosciences). Re-initiation was prevented after 5 min by the addition of heparin (0.1 mg/ml), and 5 min later the reactions were terminated by adding 5 µl of stop/load
buffer (150 mM EDTA, 1.05 M NaCl, 14 M urea, 3% glycerol, 0.075% xylene cyanol, and 0.075%
bromphenol blue). Samples were analyzed on a 7 M urea,
4.5% acrylamide sequencing gel and quantified using an Amersham
Biosciences PhosphorImager.
D and anti-E
D (gift from M. Jishage)
or mouse monoclonal anti-
N sera (Neoclone) were as
previously described (45). Antibody decorated bands were revealed using
chemiluminescence reagents (Amersham Biosciences) as directed by the
supplier. Differences in expression levels were assessed by comparison
of different exposures of dilution series of crude extracts.
Specificity of antisera was monitored using genetic control strains
proficient and deficient in expression of the gene product and/or
purified proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
In vivo transcription from
the N-dependent Po
promoter in ppGpp-proficient and -deficient E. coli.
Panel A, luciferase activity (- - -) and
growth (
) of ppGpp + MG1655
lac
(closed squares) and its otherwise isogenic
ppGpp0 counterpart CF1693
lac (open
circles) harboring the dmpR-Po-luxAB
reporter pVI466. The results are the average of triplicate independent
experiments with cells grown and assayed as described under
"Experimental Procedures." Minimal and maximal values are 3 ± 2 to 10694 ± 265 and 2 ± 0.5 to 1177 ± 330 LU/A600 for MG1655
lac and
CF1693
lac, respectively. LU, luciferase
units. Panel B, luciferase activity of
ppGpp0 CF1693
lac suppressor derivatives
harboring pVI466 grown and treated as in A. Results are the
average of two independent experiments and represent the peak output
values that occur at the same point as shown for
MG1655
lac pVI466. Upper and lower
dashed lines indicate the peak values observed
for the ppGpp-proficient and -deficient strains shown in
panel A.
'), 1 had a mutation in
rpoD (
D), 1 had multiple mutations in
rpoN (
N) in addition to a mutation in
rpoD, and 2 had mutations elsewhere in the genome. Thus, the
majority of isolates harbor mutations in components of the RNA
polymerase, and, of these, 97% had mutations in rpoBC and
3% had mutations in rpoD.
lac varied considerably from
barely detectable to >4-fold the level observed in the
(p)ppGpp+ MG1655
lac counterpart in the case
of one rpoB allele (data not shown). This suggests that the
selection procedure is very sensitive and allows isolation of
suppressors that differ by >40-fold in their ability to restore
transcription from Po. The two rpoD alleles, the single
rpoN allele, and six rpoBC alleles (Table I) were chosen for direct comparison with two previously isolated
prototrophy-restoring rpoD alleles (P504L and S506F; Ref.
40) that also restore transcription to Po in (p)ppGpp0
E. coli (25). The majority of the mutants shown in Fig.
1B restored Po
N-dependent
transcription to between 0.5- and 1.5-fold of the levels observed in
(p)ppGpp+ MG1655
lac. However, the
rpoD-35 (Y571H) and rpoN-35 (E150D/I165M) mutants
are markedly poorer in their ability to restore transcription.
D Subunits Are Defective in Competition
against
N in Vitro--
The data above clearly
demonstrate that mutations in
D can efficiently
compensate for the effect of (p)ppGpp in vivo to restore transcription to the
N-dependent Po
promoter. Given the distinct classes of promoters recognized by the two
-factors, it is unlikely that the
D derivatives
directly affect Po output. The most plausible interpretation of this
data is that the mutant
D subunits mediate their effects
through deficient competition for limiting core RNA polymerase. To
directly test this idea, we set up a multiple-round in vitro
transcription (IVT) assay to simultaneously monitor the output of the
N-dependent Po promoter and the
D-dependent RNA1 promoter present on the
template pVI695. This assay system, employing either a constitutively
active form of DmpR deleted of its regulatory A-domain or aromatic
effector-activated DmpR, results in clearly distinguishable
N- and
D-dependent
transcripts (Fig. 2A, compare
lanes 1 and 2), which can be
simultaneously monitored within the same reaction (Fig. 2A,
lane 4). Inclusion of 0.5 mM aromatic
effector 2-methylphenol used in assays with DmpR-His did not alter
specificity or the output from the IVT assay.
View larger version (19K):
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Fig. 2.
Multiple-round in vitro
transcription from the
N-dependent Po promoter and
the
D-dependent RNA1
promoter of pVI695. Panel A shows the
specificity of the standard multiple-round transcription assay with 100 nM amount of the constitutively active
A2-His-DmpR
derivative with 10 nM core in the presence of the indicated
combinations of 20 nM
N, 20 nM
D, and the supercoiled pVI695 template (0.5 µg).
Indistinguishable results were obtained with the effector-activated
form of the regulator (data not shown). Panels B
and C show the saturation curves of 10 nM core
on the same template in the presence of effector activated DmpR-His (50 nM) and increasing concentrations wild type
D (closed squares),
D-Y571H (open triangles),
D-P504L (open
upside-down triangles),
D-S506F
(open diamonds),
D-
DSA-(536-538) (open
circles), and
N (closed
circles). The results are the average of duplicate
determinations from two independent experiments.
D subunits (Fig. 2B) or
N (Fig. 2C) to a set concentration of core
(10 nM) and supercoiled template (0.5 µg). In addition,
all assays contained effector-activated DmpR-His (50 nM)
and ATP (4 mM) required for
N-dependent transcription, and IHF (10 nM) required for optimal Po output (39). Consistent with
previous data, comparison of the saturation curves for wild type
D and
N (closed
symbols in Fig. 2, B and C) indicate
that these two
-factors have a similar high affinity for core
(46-48). For the
D mutants, the plateau saturation
values are all lower than observed for wild type
D (Fig.
2B). Because formation of the holoenzyme is a prerequisite for promoter binding and transcriptional initiation by
E
D, transcription by E
D can be considered
as at least a second order co-operative binding event. Lower plateau
values would thus be predicted for a defect in the initial binding
step, which would lead to lower levels of the holoenzyme at
equilibrium. However, it is possible or even probable that the
mutations may also have some other defect in the complex series of
events of transcriptional initiation that may also contribute to the
lower net output.
D-factors, we first performed a direct comparison of the
ability of increasing concentrations of
N to compete for
10 nM core polymerase in the presence of 20 nM
D-wt or the most severely affected
D-derivative (
D-
DSA-(536-538)). The
results in Fig. 3A illustrate
the severe competitive defect of
D-
DSA-(536-538) and
show that, at this fixed
D concentration, greater than
5-fold more
N is required to achieve a 50% reduction of
RNA1 transcript levels with
D-wt than with
D-
DSA-(536-538).
View larger version (23K):
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Fig. 3.
Multiple-round in vitro
competition assays between D
derivatives and
N.
A, multiple-round transcription assay performed with 0.5 µg of pVI695, 50 nM effector activated DmpR-His, 10 nM core, and either 20 nM wild type
D (closed squares) or
D-
DSA-(536-538) (open circles)
challenged with increasing concentrations of
N.
Transcript levels are given as a percentage of those achieved in the
presence of 20 nM of each
D-derivative in
the absence of
N (
), or of 160 nM
N in the absence of
D (- - -).
Lower panels show the RNA1 (open
bars) and Po (shaded bars) transcript
levels under the same conditions with the indicated
D-derivative at 40 nM (panel
B) or 20 nM (panel C), and
challenged with 100 nM
N. Bar
1,
D-wt; bar 2,
D-Y571H; bar 3,
D-S506P; bar 4,
D-P504L; bar 5,
D-
DSA-(536-538). Results are the average of
duplicate determinations from two independent experiments. The same
hierarchy of the mutations was also obtained using single-round IVT
assays (data not shown).
D subunits by measuring the ability of
100 nM
N to compete for 10 nM
core polymerase in the presence of 40 nM (Fig.
3B) or 20 nM (Fig. 3C) of the
D mutants. The results show that the mutants differ
substantially in their ability to compete, ranging from minimally
effected (
D-Y571H) to severely affected
(
D-
DSA-(536-538)). Consistent with a critical role
in
-factor competition in vivo, the relative order of the
defects of the these mutants follows the order of their ability to
restore Po transcription in the absence of (p)ppGpp (Fig.
1B), namely
D-
DSA-(536-538) >
D-P504L >
D-S506P >
D-Y571H.
N Transcription
from Po in Vitro--
To assess the potential direct effects of ppGpp
on Po promoter output, we measured Po transcription in the presence of
200 nM to 800 µM ppGpp in a multiple-round
IVT assay. As shown in Fig.
4A, no major stimulatory
effect of ppGpp was observed, and the highest concentration resulted in
moderate inhibition. The minor stimulatory effect observed in the low
micromolar ppGpp range was also obtained when the assay was performed
with a constitutively active form of DmpR,
A2-His-DmpR, deleted of
its regulatory A-domain (data not shown). No further stimulatory
effects were observed by changing assay parameters likely to modulate
promoter kinetics, namely (i) reduction of DmpR concentrations from the
saturating 50 nM concentration to as low as 2.5 nM, (ii) reducing temperature from 37 °C to 30 or
20 °C, or (iii) shortening open complex formation time from 20 min
to 8 or 3 min (data not shown). Hence we conclude that ppGpp has no
major stimulatory effects on DmpR-mediated Po transcriptional output
under the in vitro conditions used.
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Fig. 4.
In vitro effects of ppGpp on
transcription from the
N-dependent Po
promoter. A, multiple-round IVT reactions were
performed with 0.5 µg of pVI695, 50 nM effector activated
DmpR-His, 10 nM core, and 40 nM
N in the presence of indicated concentration of ppGpp.
Transcript levels in the absence of ppGpp were set as 100 to allow
comparison from independent experiments. The results are the average of
two to four determinations made within four independent experiments
performed with overlapping concentrations of ppGpp. B,
competition assays were performed as under A but in the
presence of increasing concentrations of
D. Results are
the average of two independent experiments.
N/
D competition in vitro, we
added ppGpp to a competition assay for limiting core (10 nM). In these experiments
N was held at a
constant concentration of 40 nM and challenged with
increasing amounts of
D from 0 to 100 nM. As
shown in Fig. 4B, although
D effectively
competes with
N, leading to a decrease in
N-dependent Po transcription to ~20% at
the highest concentration tested, addition of either 20 or 180 µM ppGpp had no discernible effect. Using a similar
experimental set-up, addition of 180 µM ppGpp to an
in vitro competition assay has recently been shown to result
in an ~2-fold difference in inhibition of transcription from the
H-dependent dnaK promoter caused
by competing
D (22). Thus, in contrast to the case of
the low affinity
D-like
H, ppGpp does not
appear to have a detectable direct effect on competition between high
affinity
N and
D in vitro.
N and
D have similar high
affinity for core when assessed in isolation (Fig. 2), clear
differences between these two
-factors become apparent under
conditions of in vitro competition for limiting core. The
data show that increasing concentrations of
N are
significantly poorer at out-competing a fixed concentration of
D than the converse. This is exemplified by the finding
that a 2.5 M excess of
D over
N causes a ~4-fold decrease in
N-dependent transcription (Fig. 4), whereas
the equivalent molar excess of
N over
D
causes only a ~1.5-fold decrease in
D-dependent transcription (Fig. 3). These
considerations prompted the series of experiments described below,
designed to assess the effect of modulation of
-factor levels on the
transcriptional output from the Po promoter in vivo.
N and
S Levels
on in Vivo Transcription of Po--
The levels of
N in
E. coli are constant throughout the different growth phases
(36, 49), and the cellular levels of both
N and DmpR are
independent of (p)ppGpp (Fig.
5A (inset), and
Ref. 25). To test the effect of overexpression of
N on
Po transcription, the E. coli MG1655-derived rpoN
gene was cloned under the control of the IPTG-inducible
Ptac promoter of a plasmid expression vector and introduced
in the (p)ppGpp-deficient and proficient strains. IPTG induction
results in 10-12-fold higher
N levels in both strains
as compared with vector control derivatives (Fig. 5A
(inset) and data not shown). This high level of
overexpression only partially restored transcription to the Po promoter
in the (p)ppGpp-deficient strain, increasing transcription from
10-14% to ~30% of the levels observed in the parental
(p)ppGpp-proficient strain with native levels of
N (Fig.
5A).
View larger version (26K):
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Fig. 5.
In vivo effects of modulation
of N and
S levels on transcription from the Po
promoter. A, CF1693
lac (open
bars) and MG1655
lac (shaded
bars) harboring pVI466 and either the
Ptac-rpoN expression plasmid pVI688 (
) or a
vector control plasmid pEXT21 (
) were cultured in the presence of 0.5 mM IPTG. The results are represented as -fold increase over
transcription in CF1693
lac (pVI466, pEXT21).
Inset shows a Western blot of the
N levels in
25 µg of soluble proteins from the assayed cultures harvested 1 h into stationary phase. B, CF1693
lac and
MG1655
lac derivatives with the indicated mutant
rpoD alleles and harboring pVI466 and either pVI688 or a
vector control plasmid pEXT21 were cultured as described under
A. The results are represented as -fold increase of maximal
transcription in strains overexpressing
N from pVI688
versus vector control. Absolute values of luciferase
activity from MG1655
lac derivatives harboring
pVI466/pEXT21 were 25-45% lower than their CF1693
lac
counterparts. C, MC4100 (open bars)
and its isogenic rpoS-null mutant RH90 (hatched
bars) harboring the dmpR-Po-luxAB
reporter pVI466 and either the Ptac-rpoN
expression plasmid pVI688 (
) or a vector control plasmid
pEXT21 (
) were grown and assayed as described under
A. Results are the average of the maximal luciferase output
from the Po promoter obtained from two or three cultures in independent
experiments.
lac strain is further
enhanced 2-2.5-fold by overexpression of
N (Fig.
5A). We also tested the effect of overexpression of
N on transcription from Po in strains carrying the three
rpoD alleles that all restore transcription in the absence
of (p)ppGpp to above that observed in (p)ppGpp-proficient
MG1655
lac (see Fig. 1B). The data shown in
Fig. 5B demonstrate that, for all these three derivatives,
overexpression of
N results in a 2-2.5-fold enhancement
of Po transcription in both (p)ppGpp-deficient and (p)ppGpp-proficient
strains. However, the absolute output levels in the parental
(p)ppGpp+ MG1655
lac derivatives are 25-45%
lower than in the cognate (p)ppGpp0 CF1693
lac
derivatives. Because both the level and the competitive ability of
S for available core are dramatically increased in
(p)ppGpp-proficient strains (Ref. 22 and references therein),
additional competition by
S is likely to occur in the
(p)ppGpp+ MG1655
lac derivatives. As shown in
Fig. 5C, the presence or absence of
S has the
anticipated effects on the levels of Po transcription in
vivo. As previously observed (44), lack of
S in the
(p)ppGpp-proficient RH90 strain results in a 2-fold increase in Po
transcription, whereas overexpression of
N in the
absence of
S results in a 3.5-fold net increase over the
maximal levels of
N-dependent Po
transcription observed in the parental counterpart (Fig.
5C). Hence, we conclude that
N is under
significant competition with both
S and
D
in (p)ppGpp+ cells.
D Levels Restores Output from Po in the
Absence of (p)ppGpp--
Both
N and
D
levels in E. coli are constant throughout the different
growth phases (49). The finding that
D more readily
out-competes
N than the converse in vitro
prompted us to assess the effect of decreased levels of
D on transcription from Po in vivo. To
achieve underproduction of
D, we utilized a genetic
system in which expression of
D is under control of the
Ptrp promoter, transcription from which can be regulated by
varying levels of the IAA that counteracts the action of the Trp
repressor (50). Culturing of both (p)ppGpp+ and
(p)ppGpp0 strains carrying the
Ptrp-rpoD system in the presence of 0.2 mM IAA results in
D levels comparable with
those present in the wild-type (p)ppGpp+ counterpart (Fig.
6A, compare lanes
1-4). Reduced IAA (0.002 mM) causes a 2-3-fold
underproduction of
D in both
Ptrp-rpoD strains, as compared with those
cultured with 0.2 mM IAA (see Fig. 6A,
lanes 5-8). These culturing conditions did not
alter the levels of the RNA polymerase
subunit (Fig. 6A,
lower panels) or
' subunits (data not
shown).
View larger version (44K):
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Fig. 6.
Effect of modulation of in vivo
D levels on transcription from the Po
promoter. A, Western blot analysis of 25 µg of crude
extract from (p)ppGpp-proficient (+) and (p)ppGpp-deficient
(0) strains grown in rich media supplemented with 0.2 mM IAA (+) or 0.002 mM IAA
(
). Expression of rpoD from chromosome in its
wild type context (native-rpoD) or from the
IAA-dependent Ptrp promoter
(Ptrp-rpoD) is indicated at the top
of the figure. Panel B shows the transcriptional
output from Po of the luciferase reporter plasmid pVI684 in
(p)ppGpp0 CF1963
lac
Ptrp-rpoD (open bars) or
in (p)ppGpp+ MG1655
lac
Ptrp-rpoD (shaded bars)
that harbor either the plasmid pBR-Rsd (indicated as Rsd
) or the pBR322 vector control (indicated as Rsd
). Results are the average of data from two independent
experiments with cultures inoculated into LB supplemented with 0.2 mM IAA (indicated as
D
) or 0.002 mM IAA (indicated as
D
). Qualitative similar
results were obtained when strains were cultured in rich defined
minimal media (data not shown). C, immediate Po
transcriptional response (open symbols) upon
down-regulation of
D levels in CF1963
lac
Ptrp-rpoD (pVI684, pBR322) (circles)
and its (p)ppGpp proficient counterpart (squares)
MG1655
lac Ptrp-rpoD (pVI684,
pBR322). Growth as measured by A600 is indicated
with closed symbols.
D levels are observed in the LB-grown
(p)ppGpp0 strain than in the (p)ppGpp+ strain
(data not shown, and Ref. 40) and transcription from Po in
(p)ppGpp-deficient strains is ~10% of that in (p)ppGpp-proficient strains (see Fig. 1). Culturing of Ptrp-rpoD
strains in LB supplemented with 0.2 mM IAA to obtain
D levels in the (p)ppGpp0
CF1693
lac Ptrp-rpoD derivative
equivalent to those in the (p)ppGpp+ counterpart results in
a comparative increase in
N-dependent Po
transcription to ~75% of that in the presence of (p)ppGpp (Fig.
6B). Further 2-3-fold down-regulation of
D
levels by culturing with 0.002 mM IAA results in an
additional increase in Po transcription in both strains (4-5-fold),
with transcription in the (p)ppGpp0 strain now exceeding
that in the presence of native
D levels and (p)ppGpp by
~3-fold (Fig. 6B). Thus, consistent with a major role of
-factor competition on Po transcription, down-regulation of
D levels can fully restore
N-dependent Po transcription in the absence
of (p)ppGpp. Down-regulation of
D levels to below those
in wild type cells also allows transcription from Po during exponential
growth where (p)ppGpp levels are low and the Po promoter is normally
silent (compare Figs. 6C and 1A). The finding
that a 2-3-fold reduction in
D causes a ~5-fold
increase in
N transcription (Fig. 6B),
whereas a >10-fold increase in
N over native levels
causes only a ~2.5-fold increase in Po output (Fig. 5), mirrors the
in vitro finding that increased levels of
N
are poorer at out-competing
D than the converse.
D-factor during stationary phase by binding to free
D and thereby reducing access to core RNA polymerase
(51, 52). Given the large effect of reduced levels of
D
on
N-dependent Po transcription, we also
determined the effect of increased Rsd levels on Po transcription
in vivo. This was achieved by introducing this gene on a
high copy number plasmid into the Ptrp-rpoD
strains. As shown in Fig. 6B, Rsd overproduction alone increases Po transcription ~3-fold in both strains and is sufficient to restore Po transcription in the absence of (p)ppGpp to above that
observed in the presence of (p)ppGpp and native levels of Rsd. Similar
-fold increases in Po transcription by the presence of the Rsd plasmid
were also observed in (p)ppGpp-proficient and deficient strains with
rpoD in its native context (data not shown). Thus, like
underproduction of
D, overproduction of Rsd has a major
impact on
N-dependent Po transcription
in vivo. The effects of these manipulations are more than
additive, with simultaneous underproduction of
D and
overproduction of Rsd resulting in a 10-12-fold increase over the
maximum levels of Po transcription in cells with wild-type levels of
these proteins (Fig. 6B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N competition for limiting core RNA
polymerase. This role for (p)ppGpp is based on data using transcription
of the
N-dependent Po promoter as a
functional probe for E
N activity in both in
vivo and in vitro assays. Four
D
suppressor mutations that functionally mimic (p)ppGpp share the common
property of being defective in their ability to compete with
N for limiting core RNA polymerase in vitro
(Figs. 2 and 3). These mutations were isolated by different genetic
strategies, two (P504L and S506F) on the basis of their ability to
restore prototrophy (40) and two (
DSA-(536-538) and Y571H) on the
basis of restoration of Po transcription, in (p)ppGpp-deficient
E. coli (Fig. 1). Consistent with the common property of
defects in
-factor competition underlying the mechanism of
suppression in both cases, the magnitude of the defect of these four
D mutations follows the same hierarchy as their ability
to restore Po transcription in (p)ppGpp0 cells. This idea
is further supported by the finding that underproduction of
D and/or sequestering of
D by Rsd
in vivo can restore Po transcription in the absence of (p)ppGpp (Fig. 6). Moreover, underproduction of
D allows
E
N-dependent Po transcription in
(p)ppGpp+ cells during exponential growth where (p)ppGpp
levels are low and the Po promoter is normally silent. Thus, we
conclude that (p)ppGpp per se is not an absolute requirement
for
N-dependent Po transcription. Rather,
elevated synthesis of (p)ppGpp at the onset of stationary phase allows
successful competition of
N for the available core,
resulting in elevated levels of E
N sufficient to occupy
and initiate transcription from the Po promoter. These findings do not
preclude that sufficient E
N exists during rapid
exponential growth to allow transcription from certain high affinity,
easy to saturate
N promoters. Thus, akin to the model
put forward to explain (p)ppGpp stimulation of specific
D-promoters (33), the extent to which (p)ppGpp
modulation of E
N levels is manifested at different
promoters will depend on their innate ability to recruit limiting
E
N.
-factor competition
(22), and extend the model to include the structurally and functionally
distinct
N. Within this model (p)ppGpp directly
modulates interaction of alternative
-factors with the increased
pool of core RNA polymerase generated by (p)ppGpp-mediated
down-regulation of stringent promoters. Facilitation of association of
alternative
-factor by (p)ppGpp has been postulated to explain both
decreases in the levels of
D-holoenzyme and increases in
the levels of
S- and
H-holoenzymes in
extracts from (p)ppGpp+ as compared with
(p)ppGpp0 cells (22, 40). Most directly, addition of ppGpp
to an in vitro competition assay has been shown to have a
direct stimulatory effect on the outcome of competition between the low
affinity
H-factor and
D for core. It is
as yet unclear whether (p)ppGpp has an inhibitory effect on
D binding, a stimulatory effect on
S and
H binding to core, or both (22). We could not document
any effect of ppGpp on
N competition against
D (Fig. 4) under assay conditions that gave the
predicted 2-fold effect on
H competition (data not
shown). These results suggest that detection of direct effects of
(p)ppGpp on
-factor competition may be limited to low affinity
D-like proteins, rendering systems dependent on
S and
H more sensitive to
-factor
competition. This idea is supported by the data from the
D suppressor mutants (
DSA-(536-538) and Y571H),
where the effect of the poor suppressor
D-Y571H that
barely has detectable effects on
N-competition (Figs. 1
and 3), has markedly greater effects on in vivo and in
vitro competition assays involving
S and
H (22).
-factor competition as described above does
not exclude the possibility that transcription of some promoters are
directly controlled by (p)ppGpp. Both positive and negative in
vitro effects of adding (p)ppGpp to reconstituted transcription
systems have been found; however, they are frequently notably lower
than when assessed in vivo. More extensive positive stimulatory effects have been observed using coupled in
vitro transcription-translation systems (53, 54). We have only
observed minor direct stimulatory effects of low micromolar
concentrations of (p)ppGpp on E
N transcription from the
Po promoter in vitro (Fig. 4A). More substantial stimulation has been observed in a similar in vitro
transcription assay employing the Po promoter and a truncated
constitutively active form of XylR (27). We cannot as yet explain the
differences in the stimulation levels observed in the two assays;
however, it is possible that it is attributable to one of a number of
differing properties between the two regulators (13). Nevertheless, the role of (p)ppGpp in determining the level of the pools of alternative holoenzyme RNA polymerases provides an in vivo mechanism to
amplify even minor direct effects of (p)ppGpp at specific promoters.
-factor competition significantly
influences the levels of
N-dependent Po
transcription, with overexpression of
N, or lack of
S, causing a ~2.5-fold increase, and underproduction
and sequestering of
D causing a >10-fold increase over
the maximum level of Po transcription achieved in wild-type cells.
Given the great impact of
-factor competition on Po output, it may
appear surprising that only one rpoN suppressor allele was
isolated during the genetic selection. This mutant possesses two
conservative substitutions (E150D and I165M) that both lie within a
subportion of region II (amino acids 120-215) of
N that
is intimately associated with core (reviewed in Ref. 8). These
mutations only mediate a low level suppressor phenotype (Fig. 1);
however, we were unable to overproduce and purify
N-E150D/I165M using the
D-dependent temperature-sensitive
PRPL expression system that has been
successfully employed for overexpression and purification of native
N. As with two other overexpression systems that are
ultimately dependent on
D
(lacIQ/Ptac, and the phage T7 system
Ptac-T7 RNAP/PT7), induction conditions were
found to result in rapid growth arrest. Although anecdotal, these
findings suggest that
N-E150D/I165M possesses an
enhanced ability to compete with
D, and that more
pronounced increases in competitiveness might be lethal. In this
respect it is interesting to note that, although both
N
and
D exhibit similar affinities for core when assessed
in isolation (Fig. 2),
N is poorer at competing
D than the converse in both in vivo and
in vitro assays. The levels of these proteins within the
cell are also disproportionate as compared with the number of genes
requiring their activity. For an estimated ~30 potential
N-dependent promoters, ~110 copies/cell of
N are available, whereas only 600-700 copies/cell of
D are available for >1000 actively transcribed
D-dependent promoters (36, 55). The
properties of constant comparatively high levels and high affinity of
N, together with (p)ppGpp stimulation of its otherwise
poor competitive ability against
D, would provide a
system for rapid alteration in occupancy and transcription of
N-dependent promoters in response to
nutritional changes in the environment without de novo
synthesis of prerequisite
. Thus, for the Po promoter controlling
the enzymes for methylphenol metabolism, lack of (p)ppGpp and
concomitant high
-factor competition fulfill the same function as
catabolite repression, namely causing silencing of energetically less
favorable specialized catabolic functions until needed.
lies spread out across the upstream face of the
enzyme with each of the
2-4 domains and the linkers connecting them making extensive contacts with the core enzyme. The
P504L and S506F substitutions lie just within the N-terminal end of the
conserved 3.2-linker that joins the
1-associated
3 and
4 that interacts with the
-flap. The 3amino
acid insertion mutation
DSA-(536-538) is located just within the
first
-helix of the
4-4.1 region, whereas the Y571H
substitution is directly adjacent to the
4-4.2 conserved
region (6). Thus, consistent with their defects in competition against
N, all these mutants could directly or indirectly
decrease the overall affinity of the extensive
D-core interaction.
-factor competition without specifically affecting the kinetics of
E
N at
N-dependent promoters.
The least and most severely affected proteins (Y571H and
DSA-(536-538), respectively) do not restore prototrophy, whereas
the intermediately affected proteins (P504L and S506F) do (Table I).
These results suggest that restoration of
N-Po
transcription may provide a broader activity window than prototrophy for isolating suppressor mutations within
D. The genetic
selection strategy, as anticipated, also identified many mutations in
the
(rpoB) and
' (rpoC) subunits of the
transcriptional apparatus that restore transcription to Po in the
absence of (p)ppGpp. These mutations include some (e.g.
R454H; Fig. 1) that have previously been isolated on the basis of
restoration of prototrophy
(32).2 RpoB-R454H and some of
the other newly isolated rpoBC suppressors also restore the
ability to grow on minimal media, whereas others do not (Table I and
data not shown). Similarly, only 7 of 15 rpoBC alleles
isolated on the basis of restoration of prototrophy also exhibited the
phenotype of restoring activity to the Po promoter in
(p)ppGpp-deficient strains (25). Thus, although some mutations in
rpoBC mediate both phenotypes, others are specific to one
phenotype. In addition to the potential to alter binding of
D to core, mutations located within the
and
'
subunits have the potential to directly and differentially affect
transcription kinetics at different classes of promoters. Our future
analysis of these mutations is aimed at clarifying the degree to which such modulation and effects on
-factor competition contribute to
their different suppressor phenotypes.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to M. Carmona, V. de Lorenzo,
R. Wagner, and M. Cashel for communicating unpublished results and
advice and protocols for purification of ppGpp. We thank M. Jishage for
the gift of anti-D and E
D antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Swedish Research Councils for Natural Sciences and the Swedish Foundation for Strategic Research.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.
§ Current address: Landcare Research, P. O. Box 69, Lincoln 8152, New Zealand.
¶ Current address: Dept. of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
** To whom correspondence should be addressed. Tel.: 46-90-785-2534; Fax: 46-90-771420; E-mail victoria.shingler@molbiol.umu.se.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209268200
2 M. Cashel, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
E, enzyme;
IAA, indole-3-acrylic acid;
IPTG, isopropyl
-D-thiogalactopyranoside;
ppGpp, guanosine
tetraphosphate;
pppGpp, guanosine pentaphosphate;
(p)ppGpp, ppGpp and
pppGpp;
(p)ppGpp0, (p)ppGpp-deficient;
LB, Luria broth;
IVT, in vitro transcription;
NIRCA, non-isotopic RNA
cleavage assay;
IHF, integration host factor.
![]() |
REFERENCES |
---|
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---|
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Chacon, P.,
Polyakov, A.,
Richter, C.,
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and Wriggers, W.
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