TraR, a Homolog of a RNAP Secondary Channel
Interactor, Modulates Transcription
Matthew D. Blankschien1, Katarzyna Potrykus2, Elicia Grace1,3, Abha Choudhary1, Daniel Vinella2,
Michael Cashel2, Christophe Herman1,3*
1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 2Laboratory of Molecular Genetics, National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America, 3Department of Molecular Virology and
Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through
the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp,
along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in,
among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and
transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp.
Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for
dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR
known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third,
unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino
acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene
transfer and virulence are discussed.
Citation: Blankschien MD, Potrykus K, Grace E, Choudhary A, Vinella D, et al. (2009) TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates
Transcription. PLoS Genet 5(1): e1000345. doi:10.1371/journal.pgen.1000345
Editor: William F. Burkholder, Stanford University, United States of America
Received June 19, 2008; Accepted December 17, 2008; Published January 16, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work is funded in part by the NICHD intramural program of the NIH and HFSPO grant RGY0060/2006.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The ability to respond to changes in nutritional environment is
a universal need inherent in all cells and is characterized by rapid
global changes in gene expression. Regulation of transcription
initiation is a central way to control gene expression and is largely
achieved through the use of DNA-binding proteins (activators and
repressors) restricted to distinct promoters through recognition of
specific DNA elements. Study of the nutritional response in
Escherichia coli has detailed a novel mechanism of modulating
transcription initiation, both positively and negatively, through the
use of a single small nucleotide effector, guanosine tetraphosphate
(ppGpp), that interacts with RNA polymerase . In E. coli, the
accumulation of ppGpp causes rapid effects on transcription;
ppGpp binds to RNA polymerase, provoking an alteration in
transcription kinetics that is proposed to result from a reduction in
open complex stability . Such effects include, but are not limited
to, upregulation of amino acid biosynthesis and transport genes, as
well as genes involved in stasis/stress survival, and downregulation
of translational components such as rRNA and tRNA genes .
Recently, an additional factor, DksA, has been shown to
potentiate the action of ppGpp on RNAP both in vitro and in vivo
[4–6]. The loss of either ppGpp or DksA results in similar, though
not identical, phenotypes including the downregulation of several
amino acid biosynthetic pathways, and the inability to negatively
regulate ribosomal RNA transcription [7,8]. Separate from
mediating the stringent response, DksA has roles in other processes
including chromosome segregation, DNA repair, protein folding,
bacterial motility, virulence, and the expression of type 1 fimbriae
[7–14]. The crystal structure of DksA has been determined and
shows that the 151 amino acid-long protein folds into three distinct
structural domains: an N-terminal region containing two a-helices
(coiled coil), a globular domain with a C4 Zn+2finger motif, and a
short C-terminal helix . DksA is structurally analogous to
GreA and GreB, transcriptional anti-pausing/fidelity factors that
are homologs of the eukaryotic TFIIS [16–19]. The Gre factors
bind RNAP and protrude their coiled coils deep into the
secondary channel toward the active site [20,21]. DksA also binds
RNAP, and it has been suggested that, similar to the Gre factors,
DksA could also interact with the secondary channel. This is
supported by a growing body of evidence indicating that the DksA
and Gre proteins compete in vivo for the same substrate, the
secondary channel of RNAP [22,23]. A proposed mechanism of
action for DksA positions the coiled coil region deep within the
RNAP secondary channel near the active site. At the tip of the
coiled coil region, two invariant aspartic acid residues, Asp71 and
Asp74, are thought to coordinate the ppGpp bound Mg+2ion to
effectively position ppGpp near the active site, and allow it to exert
its transcriptional modulation effects . Mutation of these two
conserved aspartic acid residues abolishes DksA’s ability to
modulate transcription with ppGpp . The proposed DksA
mechanism remains highly speculative, and it is unknown exactly
how ppGpp and DksA influence each other or how their binding
alters RNAP activity. Furthermore, detailed mutational analysis of
PLoS Genetics | www.plosgenetics.org1January 2009 | Volume 5 | Issue 1 | e1000345
the defined RNAP binding site for ppGpp has cast doubt on the
biological relevance of the placement of ppGpp in the original
ppGpp/RNAP co-crystal structure .
Comparisons of DksA with sequence databases have previously
found similarities of DksA to several bacteriophage ORFs and
TraR, a protein found on conjugative plasmids that promote
horizontal gene transfer (for alignments, refer to [15,25]).
Horizontal transfer of DNA allows the acquisition of new traits
in the recipient bacterium, such as virulence or resistance to
antibacterial agents . The well-studied F plasmid of E. coli is
considered a model for bacterial conjugation and is facilitated by a
large protein complex, the F pilus, which bridges the donor and
recipient cell membranes, enabling F plasmid DNA transfer .
The components of the F pilus, along with regulatory, accessory,
and unknown factors, are encoded on the single 33-kb transfer (tra)
operon . traR encodes a gene in the downstream region of the
tra operon that is dispensable for F plasmid transfer, at least under
normal laboratory conditions [25,28,29]. The sequence homology
of TraR to DksA, while weak (30% identity), raises the possibility
that episomal TraR possesses some functional similarities to DksA.
In this study, we show that TraR modulates gene expression
similarly to ppGpp/DksA, but in the absence of any nucleotide
effector, like ppGpp. Expression of TraR compensates for dksA
transcriptional repression and activation activities in vivo.
Mutagenesis of a TraR amino acid corresponding to a critical
residue for DksA function abolishes activity, implying structural
similarity to DksA. Compensation by TraR is inhibited by
overexpression of GreB, a factor known to interact with the
RNAP secondary channel , suggesting, like ppGpp/DksA,
that TraR also interacts similarly with RNAP. Surprisingly, unlike
DksA, TraR does not require ppGpp for repression of the rrnB P1
promoter either in vivo or in vitro, or for activation of amino acid
biosynthesis/transport promoters in vivo at physiological levels.
The activity of TraR in the absence of ppGpp could provide clues
on the mechanistic role of DksA in modulating RNA polymerase
for at least several cellular processes. The implications of our
findings on current models of DksA/ppGpp action will be
discussed, as well as the implications for roles of episomal traR in
conjugation, pathogenicity, and the evolution of gene expression.
Endogenously Expressed TraR Functions in Amino Acid
Since TraR shares limited sequence homology to the transcrip-
tional modulator DksA, we hypothesized that the two proteins
could possess some functional similarities. Loss of DksA function
causes permanent downregulation of several amino acid biosyn-
thetic and transport pathways and results in an inability of E. coli
cells to grow on minimal media without supplementation of
required amino acids [4,6,11]. If TraR shares functions with
DksA, expression of TraR in a DdksA strain should compensate for
the multiple auxotrophies. Indeed, when a mini-F plasmid
(pOX38, see Supplemental Materials), which naturally contains
traR in the transfer operon , was conjugated into a dksA null
strain, prototrophic growth on minimal media was observed
(Table 1). The restoration to prototrophy by the F plasmid is
TraR-dependent since complete deletion of traR abolishes the
prototrophy observed in the DdksA F factor strain. The ability of
Control of gene expression is central for cell operation.
Transcription regulation is a first step to control gene
expression and is largely mediated by DNA-binding
factors. These recruit or prevent RNA polymerase binding
to promoters of specific genes. Recently, a novel way to
control transcription has emerged from studying nutri-
tional stress in bacteria. In this case, a small nucleotide
effector, ppGpp, with the help of a protein DksA, interacts
with the secondary channel of RNAP, affecting RNA
polymerase kinetics at promoters without binding to
specific DNA sequences. This interaction results in up-
regulation and down-regulation of genes involved in
responding to nutritional stress. This work describes TraR,
a factor found on conjugative plasmids that can regulate
gene expression similarly to DksA, but in the absence of
any nucleotide effector, like ppGpp. Thus, regulation of
transcription similarly to DksA/ppGpp may be a more
general mechanism. The presence of TraR on conjugative
plasmids suggests a role for TraR in pathogenicity,
virulence, and antibiotic resistance. These observations
should provide a basis for new studies designed to combat
antibiotic resistance and virulence in emerging pathogens.
Table 1. Expression of TraR Rescues DdksA Amino Acid Auxotrophies.
Genotype % cfu M9-glu / M9-glu-CAA% cfu M9-glu / M9-glu-CAA
(no IPTG)(0.1 mM IPTG)*
DdksA [F]9869.8 NA
DdksA [F DtraR] 3.46102462.961024
DdksA [F] [pGreB]10069 6.461.1
DdksA [pControl] 1.86102461.361024
DdksA [pDksA]10367.2 10166.2
DdksA [pTraR-D4N] 1.16102364.461024
Plating efficiencies of colony forming units (cfu) on M9-glucose (glu) vs. M9-glucose (glu)-casamino acids (CAA), 6IPTG. Percentages depict means6standard deviation
of the mean from 3 independent determinations. TraR, expressed either endogenously from the F plasmid or ectopically from multicopy plasmids (a pTrc99A
derivative), rescues DdksA amino acid auxotrophies. Overexpression of GreB, a factor known to interact with the RNAP secondary channel , inhibited the
compensation by TraR. (*) 1 mM IPTG was used for the GreB experiment.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org2 January 2009 | Volume 5 | Issue 1 | e1000345
TraR to compensate for the multiple auxotrophies of DdksA
suggests that TraR possesses functional similarities to DksA.
Furthermore, overexpression of GreB (from an inducible pTrc
plasmid, see Supplemental Materials), a factor known to interact
with the RNAP secondary channel , reduced the prototrophic
compensation by TraR expressed from the F episome (Table 1).
GreB was previously shown to compete with DksA and does not
rescue DdksA auxotrophies [22,23] (unpublished data). The
competition of GreB with TraR suggests that TraR interacts with
the secondary channel.
Predicted Structural Features of TraR
Given the sequence homology between TraR and DksA (for
alignments see Figure 1A, or refer to [15,25]) and our
demonstration that TraR rescues defects associated with loss of
DksA, it is likely that TraR and DksA share structural similarities.
Sequence analysis predicts that TraR possesses a globular domain
with the C4 Zn+2finger motif, characteristic of the DksA family
. The sequence of TraR also begins with the two conserved
aspartic acid residues that are important for DksA function .
Unlike DksA however, TraR is 73 amino acids long, making TraR
approximately half the size of DksA. Secondary structure
prediction suggests that TraR starts with a long helical structure,
which could correspond to half of the coiled coil domain of DksA
and the length of this predicted helix would be shorter than the
corresponding region of DksA (Figure 1A,B).
Ectopically Expressed TraR Upregulates Amino Acid
To address whether TraR is sufficient to compensate for dksA
defects, traR and dksA were separately cloned onto a multi-copy
plasmid under an inducible pTrc promoter (pBA169, see
Supplemental Materials). Ectopic expression of TraR rescued
the inability of DdksA cells to grow in the absence of required
amino acids. The plating efficiencies of DdksA cells containing
TraR or DksA plasmids grown on M9-glucose plates compared to
those grown on M9-glucose-casamino acid plates approached
100%, approximately 5 orders of magnitude higher than with the
control plasmid (Table 1). Interestingly, both uninduced and
induced pTraR and pDksA plasmids provided a complete rescue
of the DdksA auxotrophy, suggesting that only a few copies of TraR
or DksA in the cell are needed to restore appropriate regulation of
amino acid biosynthesis and transport. To address the functional
similarities between TraR and DksA, the second aspartic acid
residue of TraR, D6, which corresponds to the invariant Asp74 of
DksA (see above), was mutated to asparagine. As shown in Table 1,
TraR(D6N) was no longer able to fully compensate for DdksA
auxotrophy. This observation emphasizes the functional impor-
tance of this aspartic acid residue conserved in TraR and DksA.
TraR Activates the Stringently Induced livJ Promoter
To examine in more detail the restoration of prototrophy by
TraR in a DdksA strain, activation of the livJ promoter was
explored. livJ encodes a transporter for branched-chain amino
acids and is activated by ppGpp/DksA . b-galactosidase assays
with a wild-type PlivJ-lacZ fusion strain yielded strong activation of
the livJ promoter by induction of TraR in exponential growth
(Figure 2A). In contrast, we observed little effect with DksA
overexpression. The lack of seeing effects with DksA, which is
ppGpp-dependent with respect to the livJ promoter , is
probably due to the low levels of ppGpp present in rich media
during exponential growth. Identical results were obtained when
the experiments were repeated in a DdksA background, supporting
that TraR can work independently of DksA in the activation of livJ
(Figure 2B). Thus, TraR, unlike DksA, is able to activate the livJ
promoter in exponential phase.
TraR Inhibits Ribosomal RNA Transcription
DksA is a pleiotropic regulator of transcription with positive and
negative effects on a wide array of genes [4,6,7,13,15]. One of the
more extensively characterized effects of DksA (and ppGpp) is the
negative regulation of ribosomal RNA (rRNA) accumulation
[22,23]. Since TraR was shown to compensate for DksA in the
positive regulation of amino acid genes, we explored whether
TraR can also negatively affect the transcription of rRNA. Since
expression of both TraR and DksA from the uninduced plasmids
fully compensated for DdksA auxotrophies (above), uninduced
levels of TraR and DksA on the rrnB P1 promoter were examined
first. Uninduced levels of TraR expressed from the plasmid did not
cause a significant negative effect on rrnB P1-lacZ activity
compared to the control plasmid in a wild-type background
(Figure 3A). We next examined rrnB P1-lacZ activity during IPTG-
Figure 1. Sequence Alignment Between DksA and TraR. (A) Alignment of TraR sequence with DksA. TraR secondary structure prediction was
performed with the PSIPRED prediction method  and is labeled below the TraR sequence. H=helix, E=strand, and C=loose coil. PSIPRED predicts
a high probability of an initial helix present in TraR. (B) Model of DksA  highlighting (red shading) residues aligned between TraR and DksA.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org3 January 2009 | Volume 5 | Issue 1 | e1000345
induced TraR and DksA overexpression. TraR reduced rrnB P1
transcription to negligible levels immediately after induction in
early exponential growth (Figure 3B). This inhibition of transcrip-
tion by overexpression of TraR was specific to the rrnB P1
promoter since no significant effect was observed on the wild-type
lac promoter (Figure 3E). DksA, when overexpressed, also had
inhibitory effects on the rrnB P1-lacZ construct compared to the
control plasmid, though the effect was much less pronounced than
that of TraR (Figure 3B). The DksA-mediated repression became
greater as cells exited exponential growth, corresponding to the
accumulation of ppGpp as cells approach stationary phase [1,30].
The repression of rRNA transcription exerted by TraR on rrnB P1
activity in a wild-type strain strongly reinforces the suggestion that
TraR can act like DksA.
Since TraR expressed from the mini-F compensated for the
DdksA amino acid auxotrophies in minimal media, we asked
whether endogenous expression of traR from the mini-F plasmid
could also affect the rrnB P1 promoter under the same conditions.
Upon mating traR+and DtraR mini-F plasmids into a wild-type
rrnB P1-lacZ fusion strain, we observed that the presence of TraR
negatively affected activity from of rrnB P1 (Supplemental
Materials, Figure S1). Although this effect was modest, a large
effect of endogenously expressed traR on the rrnB P1 promoter was
not expected since the presence of the F episome does not
significantly diminish growth rate and the tra operon is only
partially derepressed on the F plasmid in E. coli [31,32]. That
endogenous traR expressed from the F plasmid rescues the DdksA
amino acid auxotrophies near 100%, but the F plasmid’s effects on
the rrnB P1 promoter are modest, suggests that activation of amino
acid synthesis is more sensitive to a lower concentration of TraR
than the inhibition of rRNA synthesis. While activation of several
amino acid genes could conceivably take only a few copies of
TraR, inhibition of even small portion of rRNA transcription,
which constitutes the majority of all transcription in E. coli [33,34]
would take many more copies of TraR.
The Absence of DksA Enhances TraR Inhibition of rRNA
Given that derepression of rRNA synthesis is observed in dksA
mutants [5,22] and that TraR compensates for the amino acid
can fully compensate for the derepression seen in a DdksA strain. As
expected, rrnB P1-lacZ activity was derepressed about 50% in DdksA
compared to its dksA+counterpart during exponential growth
(compare pControl curves from Figure 3A and Figure 3C).
Uninduced pDksA complemented the DdksA rrnB P1 derepression
to wild-type levels. Furthermore, uninduced pTraR not only fully
compensated for DdksA, but also again exerted stronger inhibition of
rrnB P1 transcription compared to DksA (Figure 3C). We then
studied the effects of overexpression of TraR and DksA on rrnB P1-
lacZ activity in a DdksA background. As in the uninduced
experiments, rrnB P1 was derepressed in DdksA cells, and
overexpression of DksA fully complemented the derepression to
wild-type levels during exponential growth (Figure 3D). Overex-
pression of TraR in the DdksA rrnB P1-lacZ background again
resulted in a strong repression of rrnB P1-lacZ activity immediately
for the uninduced plasmids, the magnitude of TraR-mediated
repression in early exponential growth was greater in the DdksA
background (40-fold) than wild-type (27-fold) (Figure 3D and
Figure 3B, respectively). This difference had a variation of about
10% and matches the 50% rrnB P1 derepression observed in DdksA.
The enhanced effects of TraR in the DdksA strain further reveal
shared functions between TraR and DksA. The combined rrnB P1
results not only extend the functional similarity of TraR and DksA
to negative regulatory effects, but also further demonstrate that
TraR may function as a more effective modulator of transcription
than DksA in exponential phase.
TraR Inhibits Growth
The strong downregulation of transcription from the rrnB P1
promoter by TraR is expected to decrease the growth rate due to
an inhibition of ribosome synthesis [1,35]. Hence, we asked
whether TraR could inhibit bacterial growth. Figure 4A shows
growth curves for WT rrnB P1-lacZ strains overexpressing TraR,
DksA, or control plasmids. When overexpressed, TraR is shown to
slow growth compared to DksA and control plasmids. Specifically,
the doubling time increased after the second generation from 31
minutes (control plasmid) to 49 minutes when TraR is expressed.
Figure 2. Activation of the livJ Promoter by Expression of TraR. b-galactosidase activity of the PlivJ-lacZ promoter fusion in LB media.
Differential activity of b-galactosidase plotted. (A) Wild-type background. Induced (0.1 mM IPTG) expression of the TraR (triangles) activates the livJ
promoter. WT DksA plasmid (squares) and control plasmid (circles). Rates of b-galactosidase synthesis in exponential phase (from OD6000.2 to 0.6) are
,520, 480, and 1010 b-gal activity/OD600for pControl, pDksA, and pTraR, respectively. R2values.0.95 in this linear range. (B) As in A, but a DdksA
background. Rates of b-galactosidase synthesis in exponential phase (from OD6000.2 to 0.6) are ,520, 670, and 1500 b-gal activity/OD600for
pControl, pDksA, and pTraR, respectively. R2values.0.95 in this linear range.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org4January 2009 | Volume 5 | Issue 1 | e1000345
Little growth difference resulted from DksA overexpression in
logarithmic growing cells (doubling time 35 minutes), highlighting
an important difference between the two homologs.
With near complete inhibition of ribosome synthesis and cell
reliance on pre-existing ribosomes, it was further predicted that
doubling time would be progressively reduced due to dilution of
ribosome pools during subsequent growth. To test this, we diluted
an overnight culture (pTraR) one hundred-fold in LB media
containing IPTG and measured growth over time. When the
logarithmic culture reached an OD600of 0.6, a six-fold dilution
Figure 3. Inhibition of the rrnB P1 Promoter by Ectopically Expressed TraR. Differential b-galactosidase activity of the rrnB P1-lacZ promoter
fusion in LB media. (A,B) Wild-type background. (A) Effects of uninduced ectopic expression of TraR (open triangles) or DksA (open squares) on rrnB
P1 activity. Plasmid control indicated by open circles. Rates of b-galactosidase synthesis in exponential phase (from OD6000.2 to 0.6) are ,100, 100,
and 84 b-gal activity/OD600for pControl, pDksA, and pTraR, respectively. R2values.0.95 in this linear range. (B) Induced (0.1 mM IPTG) ectopic
expression of TraR (triangles) or DksA (squares) represses rrnB P1 activity. Circles, plasmid control. Rates of b-galactosidase synthesis in exponential
phase (from OD6000.2 to 0.6) are ,95, 67, and 2.0 b-gal activity/OD600for pControl, pDksA, and pTraR, respectively. R2values.0.95 in this linear
range. (C,D) DdksA background. (C) Repression of b-galactosidase activity of rrnB P1-lacZ from uninduced ectopic expression of TraR (open triangles)
or DksA (open squares) in the DdksA cells. Plasmid control indicated by open circles. Rates of b-galactosidase synthesis in exponential phase (from
OD6000.2 to 0.6) are ,140, 84, and 88 b-gal activity/OD600for pControl, pDksA, and pTraR, respectively. R2values.0.95 in this linear range. (D)
Induced (0.1 mM IPTG) ectopic expression of TraR (triangles) or DksA (squares) represses rrnB P1 activity in DdksA. Circles, plasmid control. Rates of b-
galactosidase synthesis in exponential phase (from OD6000.2 to 0.6) are ,140, 80, and 3.3 b-gal activity/OD600for pControl, pDksA, and pTraR,
respectively. R2values.0.95 in this linear range. (E) TraR does not affect the Placpromoter. b-galactosidase activity assays of the wild-type lac operon.
TraR (triangles) induced (0.1 mM IPTG) from a multi-copy plasmid. Control plasmid indicated by circles.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org5January 2009 | Volume 5 | Issue 1 | e1000345
was performed in the same media and growth continued to be
monitored. A second six-fold dilution was performed when the
culture reached OD600 0.6 a third time. The doubling time
increased from 49 to 89 and 125 minutes, respectively, and
showed an overall 4-fold increase after being carried out for seven
doublings (Figure 4B). In agreement with the growth results above
(Figure 4A), neither the pDksA (doubling times of 36, 36, and 37
minutes) or control plasmid (doubling times of 35, 33, and 35
minutes) resulted in any significant growth defects when serially
diluted (see Supplemental Materials, Figure S2).
Despite lowered growth rates, cells never stopped growing and
appeared to undergo an adaptation to the strong rrnB P1
repression by TraR. Assaying for rrnB P1 activity in WT rrnB
P1-lacZ cells overexpressing TraR after overnight growth, we
observed that rrnB P1 activity was still repressed, although not as
strongly as before (data not shown). This new low level of rrnB P1
transcription may result from a feedback inhibition mechanism.
Complete inhibition of the protein synthesis machinery by TraR
would ultimately inhibit its own production, resulting in
upregulation of rrnB P1 transcription. Protein synthesis would
resume until TraR reaccumulates. This negative feedback loop
would produce slower balanced growth. Such a scenario is also
likely for the colonies forming on plates containing IPTG. The
data described above indicate that TraR, unlike DksA, has
pronounced negative effects on cells during logarithmic growth.
These negative effects on bacterial growth became more
pronounced as the cells continued to divide and were likely
caused by dilution of the pre-existing ribosomes after each cell
TraR Is a Potent Regulator of rRNA Transcription in
Western blots were performed to measure induced levels of
TraR and DksA in order to ascertain whether differences in
activation and repression activity were due to differences in
protein expression. We first constructed C-terminal epitope-tagged
TraR-His6and DksA-His6fusions and confirmed their wild-type
behavior in vivo (data not shown). Western blot analysis, as well as
Coomassie blue staining, showed the presence of a DksA band
upon induction (,8-fold higher than wild-type DksA levels, data
not shown), but no band corresponding to TraR was detected
(Figure 4C). To rule out complications inherent to the Western
blot, we performed pull-down experiments to concentrate and
measure the relative amount of TraR-His6and DksA-His6present
in extracts made after two hours of induction. As shown in
Figure 4C, the amount of TraR pulled-down is significantly less
than DksA. As a positive control for Western blotting and pull-
downs, TraR induced from our purification plasmid (pET24a-
TraR-His6, see below) was successfully detected in both assays.
Overall, these data suggest that low levels of TraR, compared to
Figure 4. TraR Inhibits Growth and Is Expressed Less than DksA. (A,B) TraR Inhibits Growth. (A) Semilog plot of OD600vs. growth time
resulting from induced (0.1 mM IPTG) expression of TraR (triangles) or DksA (squares) in the WT rrnB P1-lacZ background grown in LB-ampicillin at
32uC. Circles, plasmid control. (B) Figure depicting increasing doubling times (early logarithmic growth) resulting from successive dilutions in LB
containing IPTG of a logarithmic culture induced for TraR (triangle). pControl and pDksA do not inhibit growth when treated similarly (35, 33, 35
minutes and 36, 36, 37 minutes were the respective doubling times, see Figure S2, Supplemental Materials). (C) Steady state levels of TraR expressed
from pTraR-His6are lower than corresponding DksA levels. (Left) Protein extracts from indicated plasmids (6IPTG) were separated by SDS-PAGE (12%)
and detected by Coomassie staining (upper left) or Western blotting using an anti-His6antibody (lower left). Bands corresponding to TraR and DksA
are indicated by asterisks. (Right) TraR-His6and DksA-His6were pulled-down from identical amounts of protein extracts made after two hours of
induction, subjected to SDS-PAGE, and detected by Coomassie blue staining (upper right) or Western blotting (anti-His6antibody) (lower right).
Purification plasmid pET24a-TraR-His6was used as a positive control to visualize TraR-His6in both assays.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org6 January 2009 | Volume 5 | Issue 1 | e1000345
DksA, can achieve both strong activation of amino acid
biosynthesis and potent inhibition of rRNA accumulation in
TraR Functions Independently of ppGpp In Vivo
It is well established that DksA influences the regulatory
activities of ppGpp. Cells lacking DksA share a wide variety of
phenotypes with those deficient for ppGpp . Because of the
intimacy between DksA and ppGpp, we sought to examine
whether this characteristic extends to TraR. Our first hints that
TraR and DksA might differ with respect to ppGpp were the
observed potent livJ activation and rrnB P1 repression caused by
TraR in early exponential growth, when little ppGpp is present
[1,30]. To directly examine the effects of ppGpp on TraR
function, we examined livJ promoter activity in a DrelA DspoT
(ppGpp0) strain, which lacks both synthetases for ppGpp
production . As observed previously , ppGpp deficiency
caused decreased expression of the livJ promoter compared to
wild-type (compare plasmid controls in Figure 5A and 2A), and
DksA overexpression compensated for this defect (Figure 5A). It is
interesting and unknown why the livJ promoter has lower activity
in a ppGpp0background. It is also unknown how overexpression
of DksA restores the livJ activity to wild-type levels in this
background. Induced TraR expression in ppGpp0cells resulted in
strong activation of the livJ promoter identical to a ppGpp+
background (Figure 5A and 2A,B, respectively).
Previous studies have shown the functional importance of the
conserved, invariant aspartic acid residues (D71 and D74, tip of
coiled coil domain) of DksA. To address the functional importance
of these residues with respect to the TraR, the second aspartic acid
residue of TraR was changed to asparagine (D6N) and assayed for
PlivJ-lacZ activity in the absence of ppGpp0(Figure 5A). Altering
this second aspartic acid residue abolished the strong activation
seen with TraR expression, supporting the shared functional
importance of the invariant aspartic acid residues in both TraR
We next examined the effects of ppGpp on the negative
regulatory aspects of TraR by measuring TraR-mediated
inhibition of rrnB P1 activity in the DrelA DspoT (ppGpp0)
background. As expected, loss of ppGpp disrupted rrnB P1
repression mediated by DksA, resulting in activity identical to
the control plasmid (Figure 5B). However, induction of TraR in
the ppGpp0strain caused an immediate and striking downregu-
lation of the rrnB P1 promoter as observed in a ppGpp+strain
(Figure 5B and Figure 3B, respectively). Therefore, with respect to
the rrnB P1 promoter, TraR and DksA are not entirely functionally
interchangeable because TraR appears to be ppGpp-independent
while DksA is ppGpp-dependent. Interestingly, the D6N mutation
of TraR, which alters a conserved aspartic acid residue,
completely abolished the rrnB P1 repression in the ppGpp0strain
(Figure 5B), again emphasizing the importance of the invariant
aspartic acid residues for TraR function.
Figure 5. TraR Functions In Vivo Without ppGpp. (A) Strong activation of b-galactosidase activity from PlivJ-lacZ in LB media with induced (0.1
mM IPTG) expression of TraR (triangles) in DrelA DspoT (ppGpp0) cells. DksA and control plasmids indicated by squares and circles, respectively. The
ppGpp-independent activation is dependent on the 6thAsp residue of TraR mutated in pTraR-D6N (X). Rates of b-galactosidase synthesis in
exponential phase (from OD6000.2 to 0.6) are ,140, 400, 1900, and 150 b-gal activity/OD600for pControl, pDksA, pTraR, and pTraR-D6N, respectively.
R2values.0.95 in this linear range. (B) Inhibition of b-galactosidase activity from rrnB P1-lacZ in LB media with induced (0.1 mM IPTG) expression of
TraR (triangles) in a DrelA DspoT (ppGpp0) background. DksA and control plasmids indicated by squares and circles, respectively. Repression by TraR is
abolished by mutation of 6thAsp residue (pTraR-D6N, diamonds). Rates of b-galactosidase synthesis in exponential phase (from OD6000.2 to 0.6) are
,82, 120, 0.0, and 110 b-gal activity/OD600for pControl, pDksA, pTraR, and pTraR-D6N respectively. R2values.0.95 in this linear range. (C) Ectopic
uninduced expression of DksA or TraR suppress the cell motility defects of either DdksA or DrelA DspoT (ppGpp0) DdksA cells. Strains were inoculated
on low agar plates (0.375%) and grown for ,24 hours at room temperature, at which the diameters of resulting growth areas produced by motile
cells were measured (see Figure S3 in Supplemental Materials for representative picture). Means6standard deviation plotted.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org7 January 2009 | Volume 5 | Issue 1 | e1000345
We next asked whether TraR could also satisfy the multiple
amino acid requirements of a ppGpp0strain . Table 2 shows
the TraR-mediated rescue of DrelA DspoT (ppGpp0) auxotrophies.
The control plasmid in the ppGpp0strain exhibited the expected
very low plating efficiency (1023%) on M9-glucose vs. M9-glucose-
casamino acid plates. Also, no compensation of the ppGpp0
auxotrophies was observed when TraR was not overexpressed. In
contrast, IPTG-induced levels of TraR resulted in an 85% plating
efficiency on M9-glucose plates in the ppGpp0strain. DksA
overexpression in the ppGpp0strain exhibited only a weak rescue
for growth on M9-glucose. The resulting plating efficiency of 0.5%
was similar to results reported by Magnusson et al.  in a
MG1655 ppGpp0background. However, their observation was
noted to be strain specific (see Discussion).
The predicted structural homology and functional similarities
between TraR and DksA suggest that TraR interacts with the
RNAP secondary channel, further supported by the in vivo GreB
competition data above (Table 1). It is likely that TraR, because of
low expression levels, has to compete for the secondary channel
with endogenous DksA to exert its positive biosynthetic effects. If
this is correct, then deleting dksA might not only enhance the
ability of TraR to rescue the multiple ppGpp0amino acid
auxotrophies, but also provide evidence of similar binding to
RNAP. As predicted, the uninduced TraR plating efficiency on
M9-glucose increased from 1023% in the ppGpp0
background to 47% in the ppGpp0DdksA background (Table 2).
For the DksA and control plasmids, the loss of dksA to the ppGpp0
caused no noticeable effects compared to the ppGpp0dksA+strain.
This striking difference in compensation activity suggests compet-
itive binding between TraR and DksA to RNA polymerase,
supporting a modulatory role of TraR within the RNA polymerase
secondary channel. In addition, these data further support the
ppGpp-independent nature of gene regulation by TraR.
No suppression of ppGpp0DdksA amino acid auxotrophies was
observed with the F plasmid (Table 2), contrasting the full
suppression of DdksA amino acid auxotrophies by endogenous traR.
The reason for this discrepancy remains unknown. Considering
that low uninduced levels of cloned traR can rescue ppGpp0DdksA
amino acid auxotrophies, one possibility is TraR expression from
the F plasmid is reduced in a ppGpp0background.
It has been previously shown that bacterial motility is impaired
in ppGpp0cells, and that expression of DksA could suppress this
motility defect . DksA may activate motility by enhancing the
competitiveness of the sigma factor, sF, required for the
production of flagella and chemotaxis [7,37]. Uninduced pTraR,
like DksA, activated motility in both DdksA and ppGpp0DdksA
backgrounds (Figure 5C).
The above results demonstrate that TraR in the absence of
ppGpp activates livJ and rescues the multiple ppGpp0amino acid
auxotrophies. TraR also suppresses motility defects of ppGpp0and
DdksA cells. Furthermore, TraR alone causes rapid, near complete
repression of rRNA transcription. These observations clearly
identify important (and unexpected) differences between TraR and
TraR Acts Independently of ppGpp In Vitro
The above in vivo results clearly demonstrate that TraR can
both repress rRNA transcription and activate amino acid
biosynthesis independently of ppGpp. To determine whether the
ppGpp-independent action of TraR on the rrnB P1 promoter is
direct, we performed a single round in vitro transcription assay
with purified TraR (His6-tagged) or DksA (His6-tagged) as
described in . Addition of increasing amounts of TraR to
the transcription mixture showed a linear inhibition of rrnB P1
transcription with or without ppGpp (Figure 6A,B). Using identical
conditions for this range of concentrations, DksA only showed
inhibition of rrnB P1 promoter in the presence of ppGpp
(Figure 6A,B and  ). The in vitro data confirm that TraR
alone can directly inhibit the rrnB P1 promoter in a ppGpp-
Transcriptional control via interactions with the RNA poly-
merase secondary channel is an emerging field for both
prokaryotes and eukaryotes . Unlike classical regulators of
transcription that bind to specific DNA sites associated with their
target promoters, the transcription factor described in this work,
similar to its homolog DksA, most likely employs the secondary
channel of RNA polymerase to modulate gene expression. Here
we show that a small protein, like TraR, can repress and activate
gene expression similarly to DksA, but without any nucleotide
effector, such as ppGpp. TraR can act similarly to DksA in both
activation of amino acid biosynthetic and transport pathways and
repression of rRNA transcription, and that overexpression of a
known secondary channel interactor inhibits TraR’s compensation
ability. However, TraR differs functionally from DksA in several
important aspects. The effects of TraR on rrnB P1 activity occur
Table 2. TraR Rescues ppGpp0Amino Acid Auxotrophies.
Genotype% cfu M9-glu / M9-glu-CAA % cfu M9-glu / M9-glu-CAA
(no IPTG) (0.1 mM IPTG)
ppGpp0DdksA [pControl] 7.56102364.661023
ppGpp0DdksA [pTraR] 4763.59960.5
ppGpp0DdksA [F] 1.36102561.261025
ppGpp0DdksA [F DtraR]2.46102561.761025
Plating efficiencies (M9-glucose vs. M9-glucose-casamino acids, 6IPTG) for either ppGpp0dksA+cells or ppGpp0DdksA cells with uninduced and induced expression of
the plasmid control, DksA, or TraR or with the presence of a F plasmid (traR+or DtraR). Means of 3 independent determinations are plotted6standard deviation.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org8January 2009 | Volume 5 | Issue 1 | e1000345
much earlier in the growth cycle of E. coli and are noticeably
stronger than those of DksA. The pronounced effects of TraR are
not due to higher levels of TraR present in the cell, but result from
an increased potency of TraR on transcription; very low levels of
TraR exert stronger effect(s) in exponential growth compared to
higher levels of DksA. The most surprising difference between
TraR and DksA concerns ppGpp: DksA function is ppGpp-
dependent at physiological levels while TraR acts independently of
the nucleotide effector, in vitro, and this independence extends to
both the positive and negative effects of TraR in vivo.
TraR, a Putative Member of the RNAP Secondary Channel
TraR highlights a growing class of transcriptional regulators
that may interact directly with the secondary channel. This class
spans both prokaryotes and eukaryotes and includes DksA, GreA,
GreB, TFIIS, Microcin J25, Gfh1, and Rnk [11,15,19–21,39–43].
Other putative members are bacteriophage ORFs  and the
uncharacterized E. coli ORF ybiI, which clusters in an iron
metabolism operon. YbiI was identified through a BLAST search
for sequences sharing homology to TraR and is capable of
rescuing the amino acid auxotrophies of DdksA, but not of DrelA
DspoT, when expressed at high levels (data not shown). However,
YbiI is more likely involved in iron metabolism. Secondary
channel interactors can be sequence diverse, but structurally
similar, as is the case between the Gre proteins and DksA
[15,20,21]. Despite likely structural similarities throughout this
class of regulators and their interactions inside the secondary
channel, their transcriptional effects can vary with respect to
initiation and their relationships to ppGpp. TraR, like DksA, is
unique compared to the rest of the known secondary channel
interacting proteins in that it functions in both positive and
negative regulation of transcription initiation. GreB, for instance,
has been shown to function similarly to DksA with respect to
rRNA inhibition, but is unable to activate amino acid promoters
. GreA, on the other hand, activates rRNA transcription in
vitro at the level of open complex formation, but not by altering
the half-life of formed complexes . The molecular basis for the
differences in transcriptional initiation by these factors remains to
be determined. Identification of factors like TraR will provide a
basis for a better understanding of the molecular mechanism(s) of
the secondary channel regulators.
Mechanistic Implications Resulting From the Differences
Between TraR and DksA
DksA, together with ppGpp, activates amino acid biosynthetic
pathways and represses rRNA transcription [5,6]. Prevailing
thought presents ppGpp as the mechanistic effector of these
aspects of the stringent response because ppGpp levels correlate
with the transcriptional effects observed, whereas the levels of
DksA remain constant during the growth phases of E. coli [5,6,44].
DksA is therefore thought to act primarily as a cofactor to stabilize
binding of ppGpp to RNAP, enhancing ppGpp effects on
transcription initiation [5,15]. These ideas arose from the
ppGpp/RNAP co-crystal structure localizing ppGpp binding near
the active center of RNAP in the secondary channel . In
addition, in vitro experiments have shown that ppGpp directly
affects the rrnB P1 rRNA promoter by decreasing the stability and
half-lives of RNAP open complexes . We show that TraR can
substitute for DksA function in the absence of ppGpp, indicating
that ppGpp may not be required, either for positive or negative
transcriptional regulation. In addition, recent studies have also
suggested that DksA can work independently of ppGpp, fueling
the question of exactly how DksA modulates the activity of ppGpp
(or vice versa) [7,8]. Magnusson et al.  have shown that high
levels of DksA can partially rescue the multiple ppGpp0amino
acid auxotrophies observed in a MC4100 background, although
these effects were not seen in wild-type MG1655, the strain used in
this study. Both in vivo and in vitro, a large excess of DksA over
RNAP can repress the rrnB P1 promoter in the absence of ppGpp,
and in vitro, ppGpp alone has no effect on amino acid biosynthetic
promoters [5,6,46]. Furthermore, the biological significance of the
placement of ppGpp in the original ppGpp/RNAP co-crystal
structure is questionable . Based on these findings and our
results with TraR, we postulate that DksA may be more than a
passive coregulator for at least several promoters during the
Since TraR can mimic DksA function in vivo, the ppGpp-
independent nature of TraR may reveal several important
mechanistic implications for ppGpp and DksA. DksA has two
N-terminal a-helices (coiled coil) and is ppGpp-dependent for
many processes while TraR may possess only one a-helix and can
function independently of ppGpp. Two coils interacting inside the
secondary channel would be more spatially restrictive than one.
TraR, with only one putative protruding a-helix, would be more
Figure 6. TraR Inhibits rrnB P1 Transcription In Vitro Independent of ppGpp. Single round transcriptions were performed in the presence
(filled symbols) or absence (open symbols) of 250 mM ppGpp with increasing concentrations (0–600 nM) of TraR-His6(triangles) or DksA-His6
(squares). Amounts of RNA were measured by phosphorimaging (A) and are quantified in (B), normalized to the equivalent units observed at TraR=0
or DksA=0 for each set of reactions separately. Means of 3 independent determinations of TraR (6standard deviation) and a DksA control
experiment performed in parallel are plotted. In vitro DksA results agree with previously published data [5,22].
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org9 January 2009 | Volume 5 | Issue 1 | e1000345
dynamic within the secondary channel and more able to adopt a
conformation required to modulate transcription.
The structural/functional conservation of the invariant aspartic
acid residues of DksA in TraR is of particular interest. When the
second conserved aspartic acid residue of TraR was mutated
(D6N), the ability of TraR to function both in positively activating
amino acid biosynthesis and repressing the rrnB P1 promoter was
abolished, similar to results seen with DksA on the phage T7 A1
promoter . Surprisingly, the D6N mutation abolished TraR-
mediated activation of livJ and repression of rRNA transcription in
a ppGpp0strain. Since TraR can work without ppGpp, the
invariant aspartic acid residues are unlikely to function solely in
the coordination of ppGpp and are likely exerting the transcrip-
tional effects in some other manner. Although such speculation
remains to be verified, the results presented in this study suggest
that only one coil tipped by aspartic residues is sufficient to
substitute for DksA function in the absence of ppGpp.
Possible Roles of TraR in Horizontal DNA Transfer and
Homologs for traR are found in the transfer operons of many
naturally occurring transmissible plasmids, including those in-
volved in pathogenicity and multidrug resistance, highlighting
selective pressure for an important function(s). To date, neither we
or others  have identified a role for TraR in F plasmid transfer
in E. coli (data not shown). This lack of traR function might be due
to the laboratory domestication of the F factor to constitutively
promote conjugation in E. coli. TraR may be a broad range host
factor important for conjugation in other species or required for
transfer in a ‘‘wild-type’’ F (the F plasmid carries a mutation in
finO, a gene involved in the repression of the transfer operon ).
In addition, little is known about conjugation in the wild, and it
remains to be determined if TraR plays a role in conjugation
under more natural conditions. The presence of TraR could also
provide indirect fitness advantages for the host during conjugation.
Both DksA and ppGpp modulate the cellular responses of
autoaggregation and bacterial motility [7,8]. Both functions can
easily be imagined to be important for conjugation (e.g. the quest
for a recipient bacteria and maintaining physical contact during
DNA transfer). TraR, independently of nutritional stress, may also
control these processes. One of the consequences of the stringent
response is the reallocation or partitioning of RNAP among
promoters in the cell [48,49]. Here, we show that induction of
TraR decreases rRNA synthesis, which encompasses the majority
of transcription [33,34]. This inhibition of rRNA synthesis would
free a major portion of RNAP which could be re-assigned, in this
case, to the transcription of episomal genes or stress genes induced
by the act of conjugation (e.g. periplasmic stress due to pilus
formation). Indeed, we have observed a role of TraR in the
upregulation of several stress response pathways similar to DksA
and ppGpp together (manuscript in preparation) . Finally,
genes found in pathogenicity islands are preferentially activated by
DksA/ppGpp [12–14,42,51,52]. The presence of TraR on
congugative plasmids may allow the bacteria to control expression
of these genes independently of ppGpp accumulation. Thus, TraR
may play an important role in activation of virulence in presence
of conjugative plasmids.
TraR may represent a novel and unique member of the growing
family of RNAP secondary channel regulators. Its small size
compared to DksA and its regulatory differences with respect to
ppGpp provide the ability to dissect the functional similarities and
differences between the two homologs, providing not only a better
mechanistic understanding of TraR and DksA/ppGpp, but that of
the other secondary channel regulators as well. Based on the
functions of TraR and its presence on conjugative plasmids, we
propose that TraR and DksA may have a fitness role during
bacteria mating, promoting horizontal gene transfer, and conse-
quently, bacterial evolution. These observations may provide the
basis for new studies designed to combat antibiotic resistance and
virulence in emerging pathogens.
Materials and Methods
Media and Bacterial Growth
Standard methods of E. coli genetics were performed .
Unless otherwise stated, all work was done at 32uC with either LB
medium or M9 medium, supplemented, when required, with
sodium citrate (5–20 mM), ampicillin (50 mg/mL), kanamycin (30
mg/mL), chloramphenicol (12.5 mg/mL), tetracycline (3.33 mg/mL
with sodium citrate and 10 mg/mL without), glucose (0.1%),
casamino-acids (0.3%), and IPTG (0.1 mM). M9 media was
always supplemented with FeCl2(10 mM) and thiamine (vitamin
B1) (2 mg/mL).
Bacterial Strains, Plasmids, Mutant Alleles, and Primers
The backgrounds, genotypes, and sources of the strains of E. coli
and plasmids used in this study are listed in Table S1 (Supplemental
Material). Primers for construction of deletion alleles and plasmids
are listed in Table S2 (Supplemental Material). Unless otherwise
stated, all strains used are derivatives of MG1655. Mutant alleles
were moved into this background via standard P1 transduction 
or linear transformation techniques with subsequent elimination of
the drug-resistance marker by FLP recombinase if necessary .
Plasmids were constructed and transformed into strains by standard
cloning, mutagenesis, and transformation techniques . For traR
and dksA, the Shine-Dalgarno and ORF were amplified from
sequencing, and/or phenotypic assays were performed for verifica-
tion of alleles and plasmids.
Plating Efficiencies of Auxotrophic Strains
Serial dilutions of overnight cultures grown in LB were
performed in 10 mM MgSO4. Appropriate volumes of the
dilutions of interest were then plated on M9-glucose and M9-
glucose-casamino-acid plates, both supplemented with IPTG (0.1
mM) and antibiotics when appropriate. The plates were incubated
for 4 days at 32uC and colonies were counted. Percentages were
obtained from the ratio of colonies growing on M9-glucose vs. the
M9-glucose (glu)-casamino-acid (CAA) plates. Errors bars depict 1
standard deviation calculated from 3 independent experiments.
b-Galactosidase Activity Assays
Overnight cultures were diluted 1/100 and grown aerobically at
32uC in LB supplemented with ampicillin, and, when appropriate,
IPTG for plasmid induction. For b-galactosidase assays involving the
F plasmid, M9 media supplemented with glucose and tetracycline
was used. Samples were taken at appropriate OD600intervals and
assayed as previously described . For b-galactosidase activity (per
ml), OD42061000 / reaction time vs. OD600was plotted. All graphs
are representative of multiple independent experiments that had a
maximum variability of 12%. Polynomial (3rdorder) regression lines
were plotted using Microsoft Excel.
Bacterial cells were grown overnight in LB media containing
ampicillin, after which each strain was inoculated via toothpick
onto low agar (0.375%) LB plates. The plates were incubated at
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org 10January 2009 | Volume 5 | Issue 1 | e1000345
room temperature for ,24 hours at which the growth halos
formed were measured directly .
Measurements of Expression Levels of TraR-His6and
Assays were performed as previously described . Briefly, 26
mL cultures of MG1655 [pTraR-His6], MG1655 [pDksA-His6],
and BL21 [pET24-TraR-His6] were grown to an OD600of 0.4 at
which a 1 mL aliquot was spun down and resuspended in 100 mL of
SDS loading buffer. The remaining 25 mL cultures were induced
withIPTG(1mM)for2 hoursafterwhichanother1 mLaliquotwas
taken, spun down, and resuspended in 100 mL of SDS loading
buffer for every OD6000.4 of culture. Extracts were made of the
remaining 24 mL cultures by spinning down and resuspending the
pellets in 1 mL of 6 M guanidine, Tris-HCl, (pH 7.4) for every 24
mL of OD6001.0. TALON 50% slurry resin (100 mL) (Qiagen) was
added to 1 mL of the extracts and incubated for 2 hours at 4uC after
which the resin was washed twice with the above buffer and once
with 6 M Urea, 25 mM Tris-HCl, 500 mM NaCl (pH 7.9) and
resuspended in 100 mL of TBS buffer. Samples (15 mL) were then
run on 12% polyacrylamide gels and analyzed by Western blotting
and Coomassie blue staining. Western blots were performed with
antibodies against His6(primary) (1:2000 dilution of His-probe (H-
15)rabbitpolyclonalIgG, Santa Cruz Biotechnology) and goat anti-
rabbit IgG (secondary) (1:2000 dilution of Alex Fluor 647,
Invitrogen). The PVDF membrane was scanned with a Typhoon
Trio according to the manufacturer (GE).
TraR-His6 (encoded by pET24-TraR-His6 plasmid) was
purified with nickel-nitrilotriacetic acid-agarose columns basically
as described by Qiagen, except that the binding buffer (BB) was 50
mM NaPO4, (pH 8.0), 0.5 M NaCl, 20 mM imidazole, and 10%
glycerol. The resin with bound proteins was washed extensively
with BB containing 40 mM imidazole, followed by TraR-His6
elution with 300 mM imidazole in BB. Pure protein fractions were
then dialyzed against storage buffer (10 mM Tris-Cl, (pH 8.0), 0.1
mM EDTA, 0.1 mM DTT, 250 mM NaCl, 50% glycerol). DksA-
His6was purified as previously described .
In Vitro Transcription
In vitro transcription reactions were performed as previously
described . Briefly, 30 nM RNAP was pre-incubated (25uC)
with or without 250 mM ppGpp for 7 min prior to the addition of
potassium glutamate (90 mM), and this was followed by a 20 min
incubation at 30uC with rrnB P1 DNA (10 nM final) and the
indicated TraR or DksA concentrations (0–600 nM). The reactions
were initiated by adding NTP substrates (100 mM ATP, GTP, and
CTP, and 10 mM UTP (10 mCi/reaction [a32P]UTP, Amersham
Biosciences))withheparin (100 mg/mL final) and terminatedafter 8
min by the addition of an equal volume of stop solution (95%
formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05%
xylene cyanol). Samples were analyzed on 7 M urea, 6%
polyacrylamide sequencing gels andquantifiedbyphosphorimaging
on a GE Healthcare imaging system.
Supplemental material includes: three data figures and legends,
a table listing bacterial strains and plasmids used, and a table
listing the primers used in this study to construct new deletion
alleles and plasmids.
P1 Promoter. b-galactosidase activity of the rrnB P1-lacZ promoter
fusion performed in M9 glucose media. traR (open triangles), as
present on the F plasmid, represses rrnB P1 activity. DtraR indicated
by open circles. Graph is representative of 3 independent
experiments. At OD600 0.8, the data had up to 12% variation
between experiments and a 1.2160.01 fold difference in b-
galactosidase activity between DtraR and traR+strains. Lower rrnB
P1-lacZ activity reflects the use of minimal media (M9 glucose).
Found at: doi:10.1371/journal.pgen.1000345.s001 (2.61 MB TIF)
TraR, Expressed From the F Plasmid, Inhibits the rrnB
Inhibit Growth in Logarithmic Cultures. Figure depicting growth
curves (early logarithmic growth) of strains containing pDksA or
pControl resulting from successive dilutions in LB containing
IPTG (0.1 mM). Cultures with induced pControl or pDksA do not
inhibit growth (35, 33, 35 minutes and 36, 36, 37 minutes were the
respective doubling times) when treated similarly to a culture with
induced pTraR (see Figure 4B).
Found at: doi:10.1371/journal.pgen.1000345.s002 (2.62 MB TIF)
DksA and Control Plasmids, Unlike pTraR, do not
Motility and Compensates for ppGpp0Motility Defects. Repre-
sentative picture of cell motility from cultures inoculated on low
agar (0.375%) plates with strains of the indicated genotypes
harboring respective plasmids (uninduced). Growth was observed
after 24 hours of incubation at room temperature and resulting
diameters measured (see Figure 5C).
Found at: doi:10.1371/journal.pgen.1000345.s003 (5.66 MB TIF)
Simulation of ctrA401ts. TraR, Like DksA, Activates
K12 strains and plasmids used in this study. See references [57–
62] for the original sources of the plasmids and strains.
Found at: doi:10.1371/journal.pgen.1000345.s004 (0.07 MB
Escherichia coli K12 Strains and Plasmids. Escherichia coli
Plasmids. Primers for construction of new deletion alleles and
plasmids used in this study.
Found at: doi:10.1371/journal.pgen.1000345.s005 (0.02 MB
Primers for Construction of New Deletion Alleles and
We would like to thank J. Halliday, J. West, A. Gordon, R. D’Ari, G. Ira, J.
Wang, A. Srivatsan, R. Galhardo, and G. Weinstock for their helpful
comments; H. Murphy, P. Katsonis, and D. Satory for excellent technical
help; and L. Frost, T. Nystrom, R. Gourse, A. Farewell, and S. Rosenberg
Conceived and designed the experiments: MDB CH. Performed the
experiments: MDB KP EG. Analyzed the data: MDB DV MC CH.
Contributed reagents/materials/analysis tools: AC MC CH. Wrote the
paper: MDB CH.
1. Cashel M, Gentry D, Hernandez VJ, Vinella D (1996) The stringent response.
In: Neidhardt FC, Curtiss R 3rd, Ingraham EC, Lin ECC, Low KB, et al., eds.
Escherichia coli and Salmonella: cellular and molecular biology. 2nd ed.
Washington, D.C.: American Society for Microbiology. pp 1458–1496.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org11January 2009 | Volume 5 | Issue 1 | e1000345
2. Paul BJ, Ross W, Gaal T, Gourse RL (2004) rRNA transcription in Escherichia
coli. Annu Rev Genet 38: 749–770.
3. Magnusson LU, Farewell A, Nystrom T (2005) ppGpp: a global regulator in
Escherichia coli. Trends Microbiol 13: 236–242.
4. Brown L, Gentry D, Elliott T, Cashel M (2002) DksA affects ppGpp induction of
RpoS at a translational level. J Bacteriol 184: 4455–4465.
5. Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, et al. (2004) DksA: a
critical component of the transcription initiation machinery that potentiates the
regulation of rRNA promoters by ppGpp and the initiating NTP. Cell 118:
6. Paul BJ, Berkmen MB, Gourse RL (2005) DksA potentiates direct activation of
amino acid promoters by ppGpp. Proc Natl Acad Sci USA 102: 7823–7828.
7. Magnusson LU, Gummesson B, Joksimovic P, Farewell A, Nystrom T (2007)
Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli.
J Bacteriol 189: 5193–5202.
8. Aberg A, Shingler V, Balsalobre C (2008) Regulation of the fimB promoter: a
case of differential regulation by ppGpp and DksA in vivo. Mol Microbiol 67:
9. Yamanaka K, Mitani T, Ogura T, Niki H, Hiraga S (1994) Cloning, sequencing,
and characterization of multicopy suppressors of a mukB mutation in Escherichia
coli. Mol Microbiol 13: 301–312.
10. Meddows TR, Savory AP, Grove JI, Moore T, Lloyd RG (2005) RecN protein
and transcription factor DksA combine to promote faithful recombinational
repair of DNA double-strand breaks. Mol Microbiol 57: 97–110.
11. Kang PJ, Craig EA (1990) Identification and characterization of a new Escherichia
coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.
J Bacteriol 172: 2055–2064.
12. Song M, Kim HJ, Kim EY, Shin M, Lee HC, et al. (2004) ppGpp-dependent
stationary phase induction of genes on Salmonella pathogenicity island 1. J Biol
Chem 279: 34183–34190.
13. Nakanishi N, Abe H, Ogura Y, Hayashi T, Tashiro K, et al. (2006) ppGpp with
DksA controls gene expression in the locus of enterocyte effacement (LEE)
pathogenicity island of enterohaemorrhagic Escherichia coli through activation of
two virulence regulatory genes. Mol Microbiol 61: 194–205.
14. Thompson A, Rolfe MD, Lucchini S, Schwerk P, Hinton JC, et al. (2006) The
bacterial signal molecule, ppGpp, mediates the environmental regulation of both
the invasion and intracellular virulence gene programs of Salmonella. J Biol Chem
15. Perederina A, Svetlov V, Vassylyeva MN, Tahirov TH, Yokoyama S, et al.
(2004) Regulation through the secondary channel-structural framework for
ppGpp-DksA synergism during transcription. Cell 118: 297–309.
16. Orlova M, Newlands J, Das A, Goldfarb A, Borukhov S (1995) Intrinsic
transcript cleavage activity of RNA polymerase. Proc Natl Acad Sci USA 92:
17. Erie DA, Hajiseyedjavadi O, Young MC, von Hippel PH (1993) Multiple RNA
polymerase conformations and GreA: control of the fidelity of transcription.
Science 262: 867–873.
18. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM (2003) Backtracking by
single RNA polymerase molecules observed at near-base-pair resolution. Nature
19. Kettenberger H, Armache KJ, Cramer P (2003) Architecture of the RNA
polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114:
20. Stebbins CE, Borukhov S, Orlova M, Polyakov A, Goldfarb A, et al. (1995)
Crystal structure of the GreA transcript cleavage factor from Escherichia coli.
Nature 373: 636–640.
21. Opalka N, Chlenov M, Chacon P, Rice WJ, Wriggers W, et al. (2003) Structure
and function of the transcription elongation factor GreB bound to bacterial
RNA polymerase. Cell 114: 335–345.
22. Potrykus K, Vinella D, Murphy H, Szalewska-Palasz A, D’Ari R, et al. (2006)
Antagonistic regulation of Escherichia coli ribosomal RNA rrnB P1 promoter
activity by GreA and DksA. J Biol Chem 281: 15238–15248.
23. Rutherford ST, Lemke JJ, Vrentas CE, Gaal T, Ross W, et al. (2007) Effects of
DksA, GreA, and GreB on transcription initiation: insights into the mechanisms
of factors that bind in the secondary channel of RNA polymerase. J Mol Biol
24. Vrentas CE, Gaal T, Berkmen MB, Rutherford ST, Haugen SP, et al. (2008)
Still looking for the magic spot: the crystallographically defined binding site for
ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription
regulation. J Mol Biol 377: 551–564.
25. Doran TJ, Loh SM, Firth N, Skurray RA (1994) Molecular analysis of the F
plasmid traVR region: traV encodes a lipoprotein. J Bacteriol 176: 4182–4186.
26. Narra HP, Ochman H (2006) Of what use is sex to bacteria? Curr Biol 16:
27. Frost LS, Ippen-Ihler K, Skurray RA (1994) Analysis of the sequence and gene
products of the transfer region of the F sex factor. Microbiol Rev 58: 162–210.
28. Moore D, Wu JH, Kathir P, Hamilton CM, Ippen-Ihler K (1987) Analysis of
transfer genes and gene products within the traB-traC region of the Escherichia coli
fertility factor, F. J Bacteriol 169: 3994–4002.
29. Maneewannakul K, Ippen-Ihler K (1993) Construction and analysis of F plasmid
traR, trbJ, and trbH mutants. J Bacteriol 175: 1528–1531.
30. Kramer M, Kecskes E, Horvath I (1981) Guanosine polyphosphate production
of Escherichia coli stringent and relaxed strains in the stationary phase of growth.
Acta Microbiol Acad Sci Hung 28: 165–170.
31. Will WR, Frost LS (2006) Hfq is a regulator of F-plasmid TraJ and TraM
synthesis in Escherichia coli. J Bacteriol 188: 124–131.
32. Will WR, Frost LS (2006) Characterization of the opposing roles of H-NS and
TraJ in transcriptional regulation of the F-plasmid tra operon. J Bacteriol 188:
33. Nomura M, Gourse R, Baughman G (1984) Regulation of the synthesis of
ribosomes and ribosomal components. Annu Rev Biochem 53: 75–117.
34. Bremer H, Dennis P (1996) Modulation of chemical composition and other
parameters of the cell by growth rate. In: Neidhardt FC, Curtiss R 3rd,
Ingraham EC, Lin ECC, Low KB, et al., eds. Escherichia coli and Salmonella:
cellular and molecular biology. 2nd ed. Washington, D.C.: American Society for
Microbiology. pp 1553–1569.
35. Hernandez VJ, Bremer H (1990) Guanosine tetraphosphate (ppGpp) depen-
dence of the growth rate control of rrnB P1 promoter activity in Escherichia coli.
J Biol Chem 265: 11605–11614.
36. Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, et al. (1991) Residual
guanosine 3’,5’-bispyrophosphate synthetic activity of relA null mutants can be
eliminated by spoT null mutations. J Biol Chem 266: 5980–5990.
37. Kundu TK, Kusano S, Ishihama A (1997) Promoter selectivity of Escherichia coli
RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and
chemotaxis genes. J Bacteriol 179: 4264–4269.
38. Nickels BE, Hochschild A (2004) Regulation of RNA polymerase through the
secondary channel. Cell 118: 281–284.
39. Adelman K, Yuzenkova J, La Porta A, Zenkin N, Lee J, et al. (2004) Molecular
mechanism of transcription inhibition by peptide antibiotic Microcin J25. Mol
Cell 14: 753–762.
40. Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004)
Antibacterial peptide microcin J25 inhibits transcription by binding within
and obstructing the RNA polymerase secondary channel. Mol Cell 14: 739–751.
41. Symersky J, Perederina A, Vassylyeva MN, Svetlov V, Artsimovitch I, et al.
(2006) Regulation through the RNA polymerase secondary channel. Structural
and functional variability of the coiled-coil transcription factors. J Biol Chem
42. Lamour V, Hogan BP, Erie DA, Darst SA (2006) Crystal structure of Thermus
aquaticus Gfh1, a Gre-factor paralog that inhibits rather than stimulates transcript
cleavage. J Mol Biol 356: 179–188.
43. Lamour V, Rutherford ST, Kuznedelov K, Ramagopal UA, Gourse RL, et al.
(2008) Crystal Structure of Escherichia coli Rnk, a New RNA Polymerase-
Interacting Protein. J Mol Biol.
44. Gralla JD (2005) Escherichia coli ribosomal RNA transcription: regulatory roles for
ppGpp, NTPs, architectural proteins and a polymerase-binding protein. Mol
Microbiol 55: 973–977.
45. Artsimovitch I, Patlan V, Sekine S, Vassylyeva MN, Hosaka T, et al. (2004)
Structural basis for transcription regulation by alarmone ppGpp. Cell 117:
46. Barker MM, Gaal T, Josaitis CA, Gourse RL (2001) Mechanism of regulation of
transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation
in vivo and in vitro. J Mol Biol 305: 673–688.
47. Yoshioka Y, Ohtsubo H, Ohtsubo E (1987) Repressor gene finO in plasmids
R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F
finO. J Bacteriol 169: 619–623.
48. Barker MM, Gaal T, Gourse RL (2001) Mechanism of regulation of
transcription initiation by ppGpp. II. Models for positive control based on
properties of RNAP mutants and competition for RNAP. J Mol Biol 305:
49. Jishage M, Kvint K, Shingler V, Nystrom T (2002) Regulation of sigma factor
competition by the alarmone ppGpp. Genes Dev 16: 1260–1270.
50. Costanzo A, Nicoloff H, Barchinger SE, Banta AB, Gourse RL, et al. (2008)
ppGpp and DksA likely regulate the activity of the extracytoplasmic stress factor
sigmaE in Escherichia coli by both direct and indirect mechanisms. Mol Microbiol
51. Haack KR, Robinson CL, Miller KJ, Fowlkes JW, Mellies JL (2003) Interaction
of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect Immun
52. Olekhnovich IN, Kadner RJ (2007) Role of nucleoid-associated proteins Hha
and H-NS in expression of Salmonella enterica activators HilD, HilC, and RtsA
required for cell invasion. J Bacteriol 189: 6882–6890.
53. Miller JH (1992) A short course in bacterial genetics: a laboratory manual and
handbook for Escherichia coli and related bacteria. Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory Press.
54. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes
in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:
55. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
56. Jones DT (1999) Protein secondary structure prediction based on position-
specific scoring matrices. J Mol Biol 292: 195–202.
57. Klimke WA, Rypien CD, Klinger B, Kennedy RA, Rodriguez-Maillard JM, et
al. (2005) The mating pair stabilization protein, TraN, of the F plasmid is an
outer-membrane protein with two regions that are important for its function in
conjugation. Microbiology 151: 3527–3540.
58. Walsh NP, Alba BM, Bose B, Gross CA, Sauer RT (2003) OMP peptide signals
initiate the envelope-stress response by activating DegS protease via relief of
inhibition mediated by its PDZ domain. Cell 113: 61–71.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org12 January 2009 | Volume 5 | Issue 1 | e1000345
59. Feng GH, Lee DN, Wang D, Chan CL, Landick R (1994) GreA-induced Download full-text
transcript cleavage in transcription complexes containing Escherichia coli RNA
polymerase is controlled by multiple factors, including nascent transcript
location and structure. J Biol Chem 269: 22282–22294.
60. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, et al. (1997) The
complete genome sequence of Escherichia coli K-12. Science 277: 1453–1474.
61. Casadaban MJ (1976) Transposition and fusion of the lac genes to selected
promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:
62. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning
vectors and host strains: nucleotide sequences of the M13mp18 and pUC19
vectors. Gene 33: 103–119.
TraR, a Novel Modulator of Transcription
PLoS Genetics | www.plosgenetics.org13January 2009 | Volume 5 | Issue 1 | e1000345