Terminator still moving forward: expanding roles for Rho factor

COMICR-1061; NO. OF PAGES 7

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 ([email protected])

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

Please cite this article in press as: Boudvillain M, et al.: Terminator still moving forward: expandin

www.sciencedirect.com

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


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http://www.sciencedirect.com/science/journal/13695274

2 Cell regulation


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



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




Terminator still moving forward: expanding roles for Rho factor Boudvillain, Figueroa-Bossi and Bossi 3


Figure 2

(a) (b)

(c)

50S

50S

50S

30S

30S

30S


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



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




4 Cell regulation


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



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






Figure 3

Termination at theend of genes

Rho

Recruitmentby riboswitch

sRNA-inducedpolarity

RNAPol / replisomecollisions

Antisensetranscription

Nonsensepolarity

R-loopformation


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



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).




6 Cell regulation


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Terminator still moving forward: expanding roles for Rho factor

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