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Terminator still moving forward: expanding roles for Rho factorMarc Boudvillain1,2, Nara Figueroa-Bossi3,4 and Lionello Bossi3,4
Available online at www.sciencedirect.com
Rho factor is a molecular motor that translocates along nascent
RNA and acts on the transcription elongation complex to
promote termination. Besides contributing to transcriptional
punctuation of the bacterial genome, Rho can act intragenically
under conditions that perturb coupling of translation and
transcription. Recent advances have shed new light onto
several aspects of Rho function, including the translocation
mechanism, the avoidance of potential conflicts between DNA
replication and transcription, suppression of pervasive
antisense transcription and recruitment in riboswitch and small
RNA-dependent regulation. Altogether, these findings further
highlight the relevance of Rho factor, both as a multi-task
housekeeper and gene regulator.
Addresses1 Centre de Biophysique Moleculaire, CNRS UPR4301, Orleans 45071,
France2 affilie a l’Universite d’Orleans, Orleans 45100, France3 Centre de Genetique Moleculaire, CNRS UPR3404, Gif-sur-Yvette
91198, France4 affilie a l’Universite Paris Sud XI, Orsay 91400, France
Corresponding author: Bossi, Lionello (bossi@cgm.cnrs-gif.fr)
Current Opinion in Microbiology 2013, 16:xx–yy
This review comes from a themed issue on Cell regulation
Edited by Bonnie Bassler and Jorg Vogel
1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.mib.2012.12.003
IntroductionTranscription involves the elongation of an RNA chain
between two sites on a DNA template. While choice of
the start site is solely dictated by protein–DNA inter-
actions, the choice of the stop site depends on features of
the nascent RNA sequence, making termination some-
thing of a negative feedback step. Two types of mech-
anisms can cause the elongating RNA polymerase
(RNAP) to halt and dissociate from the template. They
are generally referred to as intrinsic (or Rho-independent)
termination and Rho-mediated (or factor-mediated)
termination. Intrinsic termination occurs in the absence
of auxiliary factors at sites where the nascent RNA can
form a stable hairpin-like structure immediately preced-
ing a run of uridine residues. Rho-mediated termination
results from the action of the Rho protein, which requires
its binding to specific sequences called rut (rho utiliz-
ation) sites in the nascent RNA. Discovered as a factor
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promoting termination of phage lambda transcripts in an
in vitro assay [1], Rho is linked to the seminal work on
lambda development (reviewed in [2]). Excellent review
articles with emphasis on Rho’s biochemical/enzymatic
properties [3], mechanism of action [4,5�], and roles in cell
physiology and gene regulation [6�] have been published
in recent years. We refer the reader to these publications
and the references therein for a full coverage of Rho
research. In the present article, we focus on selected
aspects that relate to recent developments in the field.
How Rho worksRho is a complex enzyme with interdependent RNA
binding, ATP hydrolysis, translocase, and roadblock-dis-
placement (duplex unwinding or protein dissociation)
activities. The active form is a ring-shaped, homo-hex-
amer with multiple sites for interaction with RNA and
ATP cofactors. Rho contacts RNA in an open, ‘lock
washer’ configuration wherein the ring is both distorted
and open so as to permit entry of RNA into its central
channel [7–9] (Figure 1). Crystallographic data suggest a
circular, crown-like path for RNA around the top of the
ring. Contact surfaces on each monomer compose the
primary binding site (PBS). Contacts take place within
clefts only large enough to accommodate pyrimidine
dimers with a preference for cytosines [10]. As a result,
rut sites are typically C-rich, although not traceable to a
consensus sequence [11]. Upon anchoring the RNA in the
open configuration, the ring closes around the RNA with
the residues in the central cavity of the ring — the sec-
ondary binding site (SBS) — interacting with the sugar-
phosphate backbone [12��]. RNA binding to the SBS is
required to activate Rho’s ATPase [13], which in turn
promotes translocation along the RNA [14,15]. Data from
single-molecule force/extension experiments with a
reconstructed transcription termination complex support
a ‘tethered tracking’ mode of translocation whereby the
RNA remains anchored to the PBS while Rho threads
the downstream RNA sequence in 50–30 direction through
the SBS [16�,17] (Figure 1). Upon reaching RNAP, Rho
promotes its dissociation from the DNA template and the
release of the RNA transcript. This can occur as close as
less than 10 nucleotides (nts) from the 30 edge of the rutsite and usually no more than 80–100 nts farther [18–21].
As with intrinsic termination, key details of these final
events are still missing. Two types of scenarios can be
envisaged: Rho causes termination either ‘by force’ —
pulling the RNA until the RNA:DNA hybrid is sheared
[22] or pushing the RNAP like a molecular bulldozer
[23] — or ‘by persuasion’, inducing an allosteric change in
the elongation complex that results in dissociation [24�].The latter model originates from in vitro experiments that
g roles for Rho factor, Curr Opin Microbiol (2013), http://dx.doi.org/10.1016/j.mib.2012.12.003
Current Opinion in Microbiology 2013, 16:1–7
2 Cell regulation
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Figure 1
RNA loading(open state)
Rho activation(closed state)
RNA translocation(tethered tracking)
RNAlooping
RNAtranslocation
RNAbindingto SBS3′
3′
5′ 5′5′
Current Opinion in Microbiology
RNA binding and translocation by Rho. The open configuration of the Rho ring (PDB# 1PVO) captures the RNA chain on the crown-like primary binding
site (PBS, in yellow) while allowing entry of 30-RNA into the central cavity (RNA loading). The direction of RNA circling on the PBS is debated [9]. Upon
ring closure, RNA binds the secondary binding site (SBS) side-chains (in cyan) in the central channel (Rho activation). The exact mechanism driving this
rearrangement — whether random (thermally-driven) or stimulated by RNA circling on the PBS combined to allosteric communication between the
PBS and SBS — remains uncertain [9,63,64]. The maintenance of PBS contacts promotes formation of a loop upon RNA translocation in the SBS
(tethered tracking model; [16�,17]). RNA ligands resolved in the crystal structures are shown as magenta sticks. Note that the low affinity rU12 ligand is
not bound to the PBS in the closed Rho structure (PDB# 3ICE). The putative RNA path is delineated by magenta ribbons.
suggest that Rho can enter RNAP’s main channel,
unwind part of the DNA–RNA hybrid, and cause a
conformational perturbation that hampers RNAP activity.
Inactivation of the transcription elongation complex
(TEC) then leads to its dissociation from the template.
Another relevant finding from this study is the apparent
ability of Rho to associate with RNAP in the absence of
RNA. This led the authors to challenge the conventional
view of Rho chasing RNAP and to propose that Rho binds
RNAP before, or concomitant with, transcription
initiation, remaining associated with the TEC throughout
the elongation cycle [24�]. While consistent with data
from in vivo chromatin immunoprecipitation (ChIP)
experiments [25��], this new model is at odds with the
results from other studies and remains somewhat con-
troversial [16�,26].
Rho and the ribosome: polarity effectsEarly on in the analysis of operon function, it became
apparent that mutations causing translation to stop in
promoter-proximal cistrons strongly depressed expres-
sion of downstream cistrons [27,28]. Genetic analyses
revealed that these polarity effects, initially ascribed to a
ribonuclease activity, were in fact due to intragenic
transcription termination mediated by Rho [29]. This
led to a model for polarity that has stood the test of time.
According to the model, rut sites occur frequently in
messenger RNA (mRNA) sequences but are inactive
because translating ribosomes obstruct access to Rho.
If translation is halted or slowed down upstream of a rutsite, then Rho can bind and promote termination. The
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model rests on the assumption that under optimal trans-
lation conditions the leading ribosome covers the rut site
before Rho can anchor to it. Some recent data provide
support for this assumption. One study showed that the
rate of displacement of the TEC in vivo is tightly de-
pendent on the rate of translation, implying that the
leading ribosome closely follows RNAP and may even
‘mechanically’ push it through roadblocks [30��]. Further
evidence for the existence of a direct physical link
between the leading ribosome and the TEC was
obtained in an NMR study which showed that ribosomal
protein S10 contacts residues in the carboxyl terminal
domain of NusG [31��]. NusG is a conserved protein that
associates with RNAP, modulating its processivity and
termination properties ([32,33]; for a more detailed
description of NusG and Nus factors, see article by
Shipper and Nudler, in this issue). Significantly, NusG’s
C-terminal domain also binds Rho and this interaction
stimulates termination [34,35]. It thus appears that Rho
must compete with the ribosome at two sequential levels,
initially for the binding to the RNA and subsequently for
the interaction with NusG [5�]. The second competition
step may serve as an additional checkpoint to block Rho
molecules that could occasionally succeed in capturing
the rut site during short lapses in ribosome progression
(Figure 2a,b).
Coupling Rho activity to regulation by smallRNA (sRNA)Polarity can be easily rationalized in terms of cell
economy, as it would not be advantageous for the cell
g roles for Rho factor, Curr Opin Microbiol (2013), http://dx.doi.org/10.1016/j.mib.2012.12.003
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Figure 2
(a) (b)
(c)
50S
50S
50S
30S
30S
30S
Current Opinion in Microbiology
Players modulating intragenic Rho-dependent transcription termination. (a) Under optimal translation conditions, the leading ribosome contacts the
transcription elongation complex (green) through the interaction between proteins S10 (cyan) and NusG (magenta) [31��]. The ribosome obstructs the
access to Rho (orange), which cannot bind to a rut site (cluster of yellow dots) in the nascent RNA. (b) By sequestering NusG, the ribosome hampers
Rho action even if the latter succeeds in capturing the rut sequence during a transient translational pause [5�]. (c) Pairing of a small RNA (red) to the
translation initiation region of the target mRNA prevents docking of ribosomal 30S subunit [18]. Rho can then anchor to the rut site, and upon reaching
the polymerase, cause dissociation of the elongation complex and release of the nascent mRNA. This step is stimulated by a Rho:NusG interaction
[34,35].
to waste energy and resources to continue making an
mRNA that is not going to be translated [36]. This applies
not only to polarity effects associated with mutations, but
also to more common conditions, like starvation or stress
that limit aminoacyl-tRNA availability and cause protein
synthesis to slow down or stall. Although the cell may be
able to respond by adjusting transcription rates [30��], the
ability to abort the process under severe conditions might
be crucial for viability, as such conditions could compro-
mise chromosomal integrity (see below). Besides this
general physiological role, Rho-dependent polarity can
be an integral component of specific regulatory mechan-
isms, as illustrated by recent work on an sRNA-regulated
locus in Salmonella.
The chiPQ operon encodes two proteins involved in the
uptake of chitin-derived oligosaccharides. Under unin-
duced conditions, synthesis of both proteins is repressed
by a constitutively made sRNA, ChiX, which blocks chiPmRNA translation through base pairing with the Shine-
Dalgarno region [37]. Investigating the mechanism by
which ChiX concomitantly downregulates the distal chiQ
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gene, we discovered that the action of the sRNA induces
Rho-dependent transcription termination within the pro-
moter-proximal portion of the chiP gene [18]. Apparently,
by hampering ribosome binding to the nascent chiPmRNA, ChiX allows Rho to bind instead (Figure 2c).
In vitro, termination occurs near a rut site approximately
200 nucleotides downstream from the ChiX pairing
region and requires the combined presence of Rho and
NusG in the transcription reaction. Mutations affecting
Rho or NusG activity partially relieve ChiX-mediated
chiQ repression in vivo. It seems reasonable to anticipate
that other sRNA-regulated systems may likewise incorp-
orate a Rho-dependent step. In fact, one may predict Rho
to play a role in a wide range of mechanisms targeting
translation initiation, as already proposed for a class of
riboswitches [38].
Modulating Rho binding with riboswitchesA novel transcription attenuation mechanism, based on
the conditional recruitment of Rho factor, was uncovered
during the study of the regulation of Salmonella Mg2+
transporter gene, mgtA [39�]. Briefly, the leader region of
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Current Opinion in Microbiology 2013, 16:1–7
4 Cell regulation
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mgtA mRNA can adopt two mutually exclusive confor-
mations as a function of intracellular Mg2+ levels. This
Mg2+-sensing riboswitch includes a rut sequence that is
either exposed (high Mg2+ conformer) or sequestered in a
secondary structure (low Mg2+ conformer). Thus, when
Mg2+ levels are high, Rho can bind to the nascent RNA
and induce termination, whereas no binding can take
place at low Mg2+, allowing transcription to proceed into
the structural gene. The study showed that a similar
mechanism operates at the flavin mononucleotide-
sensing riboswitch of the Escherichia coli ribB gene,
suggesting that modulation of Rho activity by ribos-
witches is a common regulatory mechanism in Gram-
negative bacteria [39�]. Interestingly, the terminated
form of the ribB riboswitch was previously known as a
small RNA, SroG [40] and the role of Rho in its pro-
duction was first described in the framework of a genome-
wide analysis of Rho-directed termination in E. coli. This
work identified other sRNAs whose 30 ends are generated
by Rho and suggested that for some of them termination
could play a role in the regulation of the downstream
genes [41�].
An intriguing twist in the mgtA system is the presence of a
coding sequence in the leader RNA and the finding that
translation of this region is required to observe premature
transcription termination in vivo. The coding segment
specifies a small proline-rich peptide, MgtL, whose rate
of translation was proposed to determine whether or not
transcription stops ahead of mgtA, thus linking mgtA expres-
sion to proline availability [42]. This conclusion has been
challenged by an independent study that presented evi-
dence for a direct effect of Mg2+ on mgtL translation [43].
Nonetheless, both groups agree that mgtL translation must
reach the end of the coding sequence for transcription
termination to occur. Since the mgtL stop codon is virtually
adjacent to the 50 boundary of the rut site, Rho must be able
to anchor to this sequence (possibly starting at its 30 end) as
soon as the ribosome is released.
Rho and the chromosome: recruitmentpatterns and protectionTranscriptomic and RNAP distribution analyses have
provided genome-wide snapshots of Rho activity in E.coli cells [25��,41�,44�,45�]. One of the main findings from
this work is the clustering of Rho activity in prophages
and other horizontally acquired material [41�,44�]. This
has led to the idea that Rho plays the physiological role of
silencing potentially harmful foreign genes [44�]. Less
finalistic interpretations consider that Rho-mediated
termination is a recurrent feature in phage regulation
and/or a predictable consequence of DNA insertion into
transcription units [41�]. Another explanation for the
preferential association of Rho activity with foreign genes
is that the latter tend to accumulate alterations — for
example, deletions — that disrupt translation–transcrip-
tion coupling, leading to increased Rho recruitment. The
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same line of reasoning can explain another major finding
from the genome-wide analyses, namely the role of Rho
in suppressing antisense transcription [41�,45�]. Most
antisense transcripts are likely to be non-coding and thus
especially susceptible to Rho-mediated polarity. Inter-
estingly, Rho performs this function in concert with
NusG and histone-like protein H-NS in a way reminis-
cent of mechanisms suppressing antisense transcription in
eukaryotes [45�].
By removing stalled TECs from the DNA, Rho activity
may help clear the way for the incoming replisome. While
co-directional collisions between the replication fork and
elongating RNAPs — inevitable, because of the speed
differences between the two complexes — are not
thought to affect chromosomal integrity, this may not
be the case for co-directional collisions involving a stalled
TEC [46]. According to a recent model, the propensity of
stalled RNAP to undergo backtracking will cause a per-
sistent RNA:DNA hybrid (R-loop) to form when RNAP is
dislodged by a colliding replisome. Although the 30 end of
the RNA in the R-loop can serve as primer to reinitiate
leading strand synthesis, the primer switch will leave a
gap in the leading strand that will be converted into a
double strand break in the next round of replication if the
R-loop is not processed before [46]. Several lines of
evidence indicate a role of Rho in preventing this chain
of events. Most significant is the finding that treatment of
E. coli with bicyclomycin (Bcm), a Rho inhibitor, causes
chromosomal DNA breaks that are dependent on DNA
replication [47] and whose frequency is increased if
translation is inhibited [48�]. The latter study reported
that exposing E. coli to sublethal amount of chloramphe-
nicol (Cam) induces the SOS response and that Bcm,
itself a non-inducer, enhances the effect. These findings
are puzzling as Cam was not previously known to induce
SOS in E. coli and in fact was shown to alleviate, as
opposed to exacerbate, conditions leading to SOS induc-
tion ([49] and references therein). It would be interesting
to see if Bcm amplifies the chronic SOS induction in
mutants of R-loop-processing RNase H1 ribonuclease
[50], as predicted by the above model.
Direct evidence for Rho involvement in preventing R-
loop formation was recently obtained from genome-wide
surveys of reactivity to sodium bisulfite (which targets
unpaired C-residues in DNA) [51�]. This work found
bisulfite-induced conversions to occur nearly three times
more frequently in a nusG mutant defective in Rho-
dependent termination than in wild-type, and biased
towards non-coding strands in both genomes. Signifi-
cantly, ectopic expression of the R-loop helicase UwsW
suppressed the lethality of deletions of nusG or rho genes,
suggesting that excessive R-loop accumulation is the
primary cause of such lethality. According to this study,
R-loops may form as a result of 50-end invasion and
reannealing of untranslated transcripts to template
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Figure 3
Termination at theend of genes
Rho
Recruitmentby riboswitch
sRNA-inducedpolarity
RNAPol / replisomecollisions
Antisensetranscription
Nonsensepolarity
R-loopformation
Current Opinion in Microbiology
Multiple roles of Rho factor. Orange boxes denote long known functions;
green boxes indicate functions uncovered more recently. Pointed and
blunt-end arrows denote active involvement and inhibitory action,
respectively.
DNA (the larger size of the DNA–RNA hybrid fraction in
termination-defective mutants reflecting increased read-
through) [51�]. It is worth noting that in the work
described above [48�], RNAP backtracking, not read-
through, converts the transcription bubble into an R-loop
extending in a 30 to 50 direction. The latter interpretation
may better explain why R-loops were not reported to
accumulate within ribosomal RNA genes whose tran-
scription is notoriously termination-resistant and presum-
ably refractory to RNAP backtracking.
Unusual suspects: RNase E and HfqIn Gram-negative bacteria, RNase E is an essential
endoribonuclease that processes most, if not all, mRNAs
as well as rRNA and tRNA precursors [52]. The enzyme is
particularly active on untranslated mRNA, reminiscent of
the conditions that lead to Rho recruitment. Intriguingly,
RNase E and Rho co-purify from E. coli in certain
instances [53] and are both components of the degrado-
some in Rhodobacter [54]. A recent study showed that rhoand nusG mutations suppress the lethal phenotype result-
ing from certain RNase E defects in E. coli [55]. Suppres-
sion required ribonuclease H1, which cleaves the RNA
component of R-loops. These findings were interpreted
to suggest that R-loop accumulation, consequent to the
termination defect [51�,56], activates an alternative,
RNase H1-dependent RNA processing pathway [55].
One might also envisage that the overall decrease in
transcription rates, due to widespread RNAP stalling in
rho/nusG mutants, lowers the demand for RNase E
activity. The RNase H1 requirement could be simply
ascribed to the fact that RNase H1 improves the viability
of rho/nusG mutants [56].
Among potential Rho partners in the E. coli ‘interactome’
[57] three proteins share topological similarities: NusG,
YaeO, and Hfq. While NusG and YaeO have long been
known to modulate Rho function [58,59], the effect of
RNA chaperone protein Hfq on Rho activity was only
recently described [60]. In vitro, Rho and Hfq associate to
form a moderately stable binary complex. This inter-
action does not prevent Rho from anchoring to RNA,
but inhibits ATP hydrolysis, duplex unwinding and most
importantly, transcription termination activities [60]. At
the prototypical ltR1 terminator, Hfq inhibits termin-
ation by binding both Rho and a transcript segment
immediately upstream from the rut site. In vivo, however,
Hfq’s antitermination activity has only been observed
under conditions where the activity of Rho is comprom-
ised by mutation or Bcm [18,60]. Perhaps the terminators
examined thus far do not fully exhibit the features
required for Hfq-mediated antitermination. Identifi-
cation of these ‘true’ targets would help in evaluating
the role and relevance of the functional link between Rho
and Hfq. The place of Hfq in sRNA biology [61] makes
sRNAs and sRNA-regulated genes to be the primary
choices in the search for such targets. Comparing the
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available co-immunoprecipitation data sets of Hfq targets
[62] to the Rho ChIP data [45�] could help with this
analysis.
ConclusionsRecent years have seen a renewed interest in Rho
research (Figure 3). State-of-the-art crystallography and
single-molecule techniques have provided fresh insights
on how Rho translocates along the RNA [12��,16�], while
transcriptomic and ‘ChIP-on-chip’ analyses have offered
snapshots of Rho activity at the genome-wide scale
[25��,41�,44�]. Other studies have revived the notion that
besides being a ‘housekeeper’, Rho can function as a
regulator of gene expression. The regulatory role can be
totally independent of the housekeeping function, as in
Rho recruitment by riboswitch [39�] or intimately con-
nected with it, as in the coupling of sRNA-mediated
regulation with Rho-induced polarity [18]. Despite recent
progress, various aspects of Rho functions remain elusive.
Key details of the mechanism by which Rho dissociates
the TEC are still missing and will require examination by
new approaches, possibly through single molecule exper-
imentation. Also, further work is needed to assess the
extent of Rho’s participation in sRNA-mediated-regula-
tion and to better characterize the Hfq and RNase E
connections. These analyses should, not only improve our
knowledge of how Rho works, but also provide a better
understanding of the transcription process per se.
AcknowledgmentsWe apologize to all of our colleagues whose work was omitted, or notadequately presented, due to space constraints. We are grateful to SofiaCiampi, Glenn Herrick, Pepe Casadesus and David Friedman for criticalreading of the manuscript and to reviewers for constructive criticism. Thework from our laboratories was supported by the French CNRS and bygrants 2010-BLAN-152502 (MB) and ANR-BLAN07-1_187785 (LB) fromthe French National Research Agency (ANR).
g roles for Rho factor, Curr Opin Microbiol (2013), http://dx.doi.org/10.1016/j.mib.2012.12.003
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