Autoregulation allows Escherichia coli RNase E to adjustcontinuously its synthesis to thatof its substrates

Sandra Sousa,† Isabelle Marchand and Marc Dreyfus*

Laboratoire de Genetique Moleculaire, CNRS UMR 8541,

Ecole Normale Superieure, 46 rue d’Ulm, 75230 Paris,

France.

Summary

The Escherichia coli endonuclease RNase E plays a

key role in rRNA maturation and mRNA decay. In

particular, it controls the decay of its own mRNA by

cleaving it within the 50-untranslated region (UTR),

thereby autoregulating its synthesis. Here, we report

that, when the synthesis of an RNase E substrate is

artificially induced to high levels in vivo, both the rne

mRNA concentration and RNase E synthesis increase

abruptly and then decrease to a steady-state level that

remains higher than in the absence of induction.

Using rne–lacZ fusions that retain or lack the rne

50UTR, we show that these variations reflect a

transient mRNA stabilization mediated by the rne

50UTR. Finally, by putting RNase E synthesis under

the control of an IPTG-controlled promoter, we show

that a similar, rne 50UTR-mediated mRNA stabilization

can result from a shortage of RNase E. We conclude

that the burst in substrate synthesis has titrated

RNase E, stabilizing the rne mRNA by protecting its

50UTR. However, this stabilization is self-correcting,

because it allows the RNase E pool to expand until its

mRNA is destabilized again. Thus, autoregulation

allows RNase E to adjust its synthesis to that of its

substrates, a behaviour that may be common among

autoregulated proteins. Incidentally, this adjustment

cannot occur when translation is blocked, and we

argue that the global mRNA stabilization observed

under these conditions originates in part from this

defect.

Introduction

In Escherichia coli, proteins that play a general role in gene

expression, e.g. in the synthesis or translation of mRNAs

or in the folding, maturation or degradation of either RNAs

or proteins, often exploit their activity for autoregulating

their own expression. For instance, protein CRP, which

activates many promoters by binding upstream of them,

can also bind downstream of its own promoter, repressing

its activity (Hanamura and Aiba, 1991). Likewise, many

proteins that bind to specific sites on rRNA or tRNA, such

as ribosomal proteins (r-proteins) and certain aminoacyl

tRNA synthetases, can also bind to similar sites on their

own mRNAs, inhibiting their translation (Nomura et al.,

1980; Draper, 1987; Springer, 1996). Similarly, the heat

shock proteins specifically downregulate the activity of s32,

a factor required for the transcription of their genes; in

particular, the DnaK–DnaJ–GrpE chaperone team, which

normally associates with denatured proteins, can also

associate with s32, inactivating this factor and perhaps

promoting its degradation (Gross, 1996; Arsene et al.,

2000). Finally, RNase III and RNase E, two endonu-

cleases involved in rRNA maturation and mRNA decay,

also repress their own synthesis by initiating the decay of

their mRNAs (Bardwell et al., 1989; Jain and Belasco,

1995).

What is the benefit for the bacterial cell of these

autoregulation loops? It is generally believed that they act

as sensors of the metabolic demand for the corresponding

proteins. Thus, the overproduction of rRNA or tRNAThr

results in an increased synthesis of r-proteins or threonyl-

tRNA synthetase respectively. Presumably, these proteins

are titrated by the burst of their substrates so that the

autoregulation loop is derepressed and their synthesis

increases (Yamagishi and Nomura, 1988; Comer et al.,

1996). Similarly, the misfolded or denatured polypeptides

that accumulate during heat shock are thought to titrate

the DnaK–DnaJ–GrpE team, thereby releasing free s32

and contributing to the induction of heat shock protein

synthesis (Craig and Gross, 1991). Following workers in

the heat shock field, we use the term ‘homeostasy’ to

designate this adjustment between the synthesis of a

protein and that of its substrates.

RNase E is a key E. coli endonuclease that controls the

production of p5S and 16.3S rRNAs, the immediate

precursors of 5S and 16S rRNAs, as well as the decay of

many or most mRNAs in the cell (Ghora and Apirion, 1978;

Ono and Kuwano, 1979; Cohen and McDowall, 1997;

Coburn and Mackie, 1999; Li et al., 1999). In particular, it

Accepted 31 August, 2001. *For correspondence. E-mail mdreyfus@biologie.ens.fr; Tel. (133) 1 44 32 35 26; Fax (133) 1 44 32 39 41.†Present address: Unite des Interactions Bacteries-Cellules, InstitutPasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.

Molecular Microbiology (2001) 42(3), 867–878

Q 2001 Blackwell Science Ltd

initiates the decay of its own mRNA by cleaving it in the 50

untranslated region (UTR), thereby autoregulating its own

synthesis (Mudd and Higgins, 1993; Jain and Belasco,

1995; Diwa et al., 2000). Recently, we have observed that

situations that result in a translational block also cause an

apparent inhibition of RNase E; presumably, this inhibition

contributes to the well-known stabilization of bulk mRNA

observed under these conditions (Lopez et al., 1998). As

an explanation, we noted that the pool of free RNase E in

growing cells may be limited because of autoregulation

and that, once translation is blocked, it cannot expand

further. Under these conditions, the synthesis of rRNA is

boosted, and this newly synthesized rRNA is unstable,

presumably because it cannot assemble with r-proteins

(Shen and Bremer, 1977). We therefore proposed that

RNase E is then permanently titrated by excess substrates

(Lopez et al., 1998). This model yields the simple

prediction that, by inducing the synthesis of a highly

expressed RNase E substrate in normally growing cells, it

should be possible to titrate RNase E independently of any

translation block. However, titration should be transient

because the pool of RNase E is free to expand in response

to the stabilization of its cognate mRNA. Accordingly,

autoregulation would allow homeostasy of RNase E

expression as for heat shock or ribosomal proteins. It is

this critical point of the model that is tested here.

Results

A truncated rrnB transcript lacking most of the 16S and

23S sequence is a substrate for RNase E

Our first aim was to design a system that allows massive

and inducible synthesis of a well-defined RNase E

substrate in the cell. Ideally, this substrate should be

devoid of biological activity in order to avoid perturbing

growth; in particular, it should remain untranslated. On this

basis, we chose plasmid pNO2681 (Gourse et al., 1985)

as a source of substrate RNA. This pBR322 derivative

carries a defective version of the rrnB operon lacking most

of the 16S and 23S rRNA genes, but retaining the 50 and 30

ends of the operon, including the two RNase E cleavage

sites flanking the 5S rRNA and the RNase E site located

upstream of the 16S rRNA (Ghora and Apirion, 1978; Li

et al., 1999; Fig. 1A and B). Its putative 1.8 kb transcript

carries no known ribosome binding site, nor is it functional

in ribosome biosynthesis (Gourse et al., 1985). In

pNO2681, the defective rrnB operon lacks its own

promoter but is fused downstream of the strong PL

promoter from bacteriophage lambda. Whereas at 308C,

this promoter is tightly repressed in cells synthesizing the

thermolabile c I857 lambda repressor, it can be switched

on by shifting the cultures to 428C, which inactivates the

repressor (Gourse et al., 1985). Since the effect of

blocking translation upon RNase E activity (Lopez et al.,

1998) and upon rRNA synthesis and stability (Shen and

Bremer, 1977), as well as most information on E. coli

physiology (Bremer and Dennis, 1996), have been studied

in the E. coli B (or B/r ) background, the E. coli B strain

BL21(DE3) (Studier et al., 1990) was chosen as a host for

this study. In the derivatives used, the genuine lacZ gene

has been inactivated in order to allow the use of various

rne–lacZ fusions for testing RNase E activity (see below).

Plasmids pNO2681 or pBR322 were introduced into

cells carrying pc I857, a pBR322-compatible plasmid

encoding the repressor (Remaut et al., 1983; Fig. 1A).

The doubly transformed cells were grown at 308C and then

shifted to 428C to induce the PL promoter. Cells carrying

either pNO2681 or pBR322 grew at the same rate both

before and after the temperature shift, indicating that this

induction is not toxic. Total RNA was extracted from the

doubly transformed cells before or after the shift and

analysed on Northern blots, using oligonucleotide probes

complementary to the 5S rRNA or the immediately

Fig. 1. A. Outline of an experimental system designed for the inducibleexpression of a RNase E substrate in E. coli. Plasmid pNO2681(Gourse et al., 1985) carries a truncated version of the rrnB operon(grey box) under the control of the PL promoter (thick arrow), insertedbetween the HindIII (H) and Bam HI (B) sites of pBR322. At 308C (butnot at 428C), the PL promoter is repressed (curved line) by thethermolabile c I857 repressor encoded by the plasmid pc I857 (circle).B. Detail of the truncated rrnB operon. The corresponding transcript,which extends down to the natural rrnB terminators (rrnT), differs fromthe genuine rrnB transcript by a large internal deletion (symbolized bydotted lines), which removes most of the 16S and 23S sequences(open boxes). Note that, because of the deletion, the duplex regionsthat are cleaved by RNase III during rRNA maturation, (cleavage sitesnoted ‘III’) are no longer present so that the transcript is presumablynot a substrate for RNase III. The RNase E sites flanking the 5S rRNAand the RNase E site located upstream of the 16S rRNA are arrowed(‘E’). Starred bars noted ‘5S’ and ‘9S’ show the positions of theoligonucleotides used in Figs 2 and 7 to probe 5S rRNA or itsprecursors, respectively.

868 S. Sousa, I. Marchand and M. Dreyfus

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

upstream flanking sequence (‘5S’ and ‘9S’ probes

respectively; Fig. 1B). With pNO2681-carrying cells grow-

ing at 308C, a very faint signal corresponding to the 1.8 kb

species was detected with both probes (cf. Fig. 2A; only

the 9S probing is shown). As expected, this species was

absent from pBR322-carrying cells. After the 428C shift,

the 1.8 kb species from pNO2681-carrying cells became

much more prominent, reflecting the activation of the PL

promoter (Fig. 2A). To assess whether this species is a

substrate for RNase E, the same experiment was repeated

with cells carrying the rne-1 mutation. The corresponding

RNase E polypeptide is functional at 308C but is rapidly

inactivated at 428C (Ono and Kuwano, 1979; Mudd et al.,

1990). With these cells as with rne 1 cells, a very faint,

pNO2681-specific signal corresponding to the 1.8 kb

species was detected at 308C (Fig. 2A). After the 428C

shift, this species accumulated to much higher levels than

in rne 1 cells, suggesting that it is a substrate for RNase

E. The unprocessed precursors of 5S rRNA originating

from the genuine rrn operons (‘9S’ rRNA; see below) also

accumulated at 428C as a result of RNase E inactivation.

As expected, this accumulation was observed in both

pBR322- and pNO2681-carrying cells (Fig. 2A).

The 5S probing showed that, in rne 1 cells carrying

pNO2681, the abundance of 5S rRNA over other rRNA

species increased progressively after shifting to 428C

(compare the 5S and 23S probings in Fig. 2B). No

comparable increase was observed in pBR322-carrying

cells. This result shows that the 1.8 kb transcript can finally

be processed into 5S rRNA, i.e. that the RNase E cleavage

sites flanking the 5S rRNA sequence (Fig. 1B) are

functional. After 1 h (0.75 doubling times), the 5S rRNA

expression has increased by 70%. This figure is close to

the theoretical value that can be calculated using previous

estimates for the rate of transcription from plasmids similar

to pNO2681 (1.6- to 1.7-fold the rate of transcription

from all seven chomosomal rrn operons; Yamagishi and

Nomura, 1988), indicating that the processing of the 1.8 kb

transcript into 5S rRNA is nearly quantitative. Whereas

from the above figure the rate of synthesis for the 1.8 kb

species may seem very high indeed, this rate is

presumably actually less than that of mRNA molecules,

many of which are also RNase E targets (see Discussion ).

Altogether, these results indicate that, like the genuine

9S rRNA, the 1.8 kb transcript is a substrate for RNase

E. When induced, it presumably accounts for a significant

but not dominant fraction of total RNase E targets in the

cell (see Discussion ). In the following, it is referred to as

‘the substrate’.

The rne mRNA is stabilized transiently after induction of

substrate synthesis

We next recorded the effect of substrate induction upon

the steady-state concentration of the rne mRNA. To this

end, Northern blots prepared with total RNA from the same

cultures as above (rne 1 cells) were hybridized with an

rne-specific probe (Fig. 2B, rne probing). The major

species detected with this probe is 3.6 kb in length,

consistent with the known length of the rne transcript

(Casaregola et al., 1992; Jain and Belasco, 1995). With

pNO2681-carrying cells, the concentration of this species

Fig. 2. A. Induction of transcription from pNO2681 (cf. Fig. 1A). Cellsharbouring plasmids pc I857 and either pNO2681 (pNO lanes) orpBR322 (pBR lanes) have been harvested either before or at varioustimes after a shift from 308C to 428C, and total RNA has been probedon Northern blots with the 9S probe (cf. Fig. 1B). The cells usedencode an RNase E activity that is either thermostable (wt) orthermolabile (rne-1 ) at 428C. The position of the 1.8 kb transcript fromthe truncated rrnB operon is arrowed. The bracket marked ‘9S’pinpoints the polydisperse precursors of the 5S rRNA that aresynthesized from the seven chromosomal rrn operons andaccumulate when RNase E is inactive (rne-1 strain, 428C). Note that,beyond the 9S and 1.8 kb species, another species, presumably adegradation product from the 1.8 kb transcript, accumulates at 428C inrne-1 cells harbouring plasmid pNO2681 (starred).B. Effect of induction upon the accumulation of 5S rRNA and rnemRNA. Cells were grown and total RNA was extracted as in (A),except that only wild-type (rne 1) cells were used. Northern blots wereprobed with probes specific for the 5S and 23S rRNA and for the rnemRNA (see Experimental procedures ).

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Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

increased abruptly (by < sixfold) after 10 min at 428C, and

decreased progressively thereafter; however, even after

60 min at 428C, it remained < twofold higher than before

the temperature shift. In contrast, with cells carrying the

control plasmid pBR322, the concentration of the rne

mRNA changed more smoothly and over a more limited

range (Fig. 2B).

The transient accumulation of the rne mRNA after

substrate induction might reflect either an increased

promoter activity or a stabilization of the rne mRNA. In the

latter event, it would presumably reflect a transient

shortage in RNase E activity, because the stability of

the rne mRNA is controlled by the rate of cleavage of

its 50UTR, which itself responds to RNase E activity (Jain

and Belasco, 1995). To test this issue, we used two in-

phase rne– lacZ fusions designed by Jain and Belasco

(1995). These fusions consist of the rne promoter followed

by the same rne– lacZ hybrid coding sequences, but they

differ in the presence or absence of most of the 361-nt-

long rne 50UTR in between. Only with the former fusion

was mRNA stability and protein yield affected by RNase E

activity as for the rne gene (Jain and Belasco, 1995).

These fusions were then introduced onto the chromosome

of BL21(DE3). For simplicity, the corresponding deriva-

tives are qualified here as ‘150UTR’ and ‘250UTR’

respectively (Fig. 3A); by extension, the same qualifiers

are used to designate the rne–lacZ fusions themselves or

the mRNAs originating from them. The 150UTR and

250UTR cells carrying plasmids pc I857 and either

pNO2681 or pBR322 were then grown as before. Total

RNA was extracted before or after the 428C shift and

analysed on Northern blots using a lacZ probe. Whether

pNO2681 or pBR322 was present, most of the signal

detected before the shift consisted of an abundant smear

arising from incomplete molecules: the prevalence of these

species presumably reflects the fact that the time required for

synthesizing the long rne–lacZ mRNA exceeds its lifespan

(Yarchuk et al., 1992; Jain and Belasco, 1995). Only with the

250UTR cells was a weak signal corresponding in size

(3.7 kb) to the complete rne– lacZ fusion mRNA observed,

suggesting that, at 308C, this mRNA is slightly more stable

than its 150UTR counterpart. After the shift, the fusion mRNA

from 150UTR cells (4.0 kb in this case) became apparent

transiently in cells carrying pNO2681, whereas it remained

almost undetectable in the same cells carrying pBR322. In

contrast, with 250UTR cells, no difference in mRNA pattern

was detected whether pNO2681 or pBR322 was present

(Fig. 3B). Thus, even though the 150UTR and 250UTR

mRNAs are synthesized from the same rne promoter, only

the former behaves like the genuine rne mRNA in

accumulating after substrate induction. We conclude that

this accumulation reflects mRNA stabilization, probably

caused by a shortage in RNase E activity (see below for

further results on this point).

The accumulation of the rne or rne–lacZ mRNA after

substrate induction is paralleled by an increased synthesis

of the corresponding proteins

Next, we tested whether the stabilization of the rne or

150UTR mRNAs that follows substrate induction results in

an increased synthesis of the corresponding proteins

(Fig. 4). To this end, we used the same cultures as for

Fig. 3. A. The BL21(DE3) derivatives used here, ENS401 (‘150UTR’)and ENS406 (‘250UTR’), harbour constructs consisting of the rnepromoter followed by an in-phase fusion between the rne (dark greybox) and lacZ (open box) coding sequences. In between, the 50UTR ofthe rne gene (light grey box) is either present (150UTR cells) or absent(250UTR cells). These fusions are inserted in the malT–malPintergenic region of the chromosome. The lacZ gene is followed by thebeginning of the lacY gene (lacY 0 ) and by two tandemly arrangedterminators (Ter). Also represented is the rne gene under the controlof its genuine promoter (Prne) and 50UTR.B. 150UTR and 250UTR cells harbouring plasmids pc I857 and eitherpNO2681 or pBR322 (cf. Fig. 1A) were grown and used for RNAextraction as in Fig. 2B. The transcript from the rne– lacZ fusion wasprobed on Northern blots. Arrows indicate the position of the rne– lacZmRNA species from 150UTR and 250UTR cells (4.0 kb and 3.7 kbrespectively); given their size, these species presumably result from aprocessing of the initial transcript downstream of the lacZ gene(Murakawa et al., 1991). The position of the 23S and 16S rRNA isindicated by ticks. To check for uniform RNA loading, the 23S rRNAhas been visualized directly on the same membrane (bottom).

870 S. Sousa, I. Marchand and M. Dreyfus

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Fig. 3B (150UTR cells). At timed intervals after the

temperature shift, aliquots of the cultures were pulse labelled

with a [35S]-methionine–cysteine mixture. Extracts from the

labelled cells were then incubated with anti-RNase E and

anti-b-galactosidase antibodies, and immune complexes

were analysed by SDS–PAGE (see Experimental pro-

cedures ). Although several labelled proteins were apparent

on the gels, presumably reflecting the limited specificity of the

antibodies used, two polypeptides corresponding in size to

RNase E (apparent molecular weight < 180 kDa; see

Casaregola et al., 1992) and RNase E–b-galactosidase

hybrid protein (139 kDa) were detected. Their identity was

ascertained by running in parallel extracts of cells that either

overexpress or lack these proteins (see Experimental

procedures for details). Before the shift, the rate of RNase

E synthesis was essentially the same whether cells carried

pNO2681 or pBR322 (see Fig. 4A and B for quantification).

In contrast, after the shift, this synthesis was distinctly higher

in pNO2681- than in pBR322-carrying cells; the ratio

between the two went through a maximum at 20 min after

induction (fourfold) but, even after 60 min, it still amounted to

about twofold. The behaviour of the fusion protein was very

similar (see the legend to Fig. 4 for comment on the 60 min

point). Thus, the rate of synthesis of the two proteins

closely followed the changes in the accumulation of the

cognate mRNAs (compare Fig. 4B with Figs 2B and 3B).

The burst in the synthesis of RNase E–b-galactosidase

hybrid protein after substrate induction in 150UTR cells

could also be confirmed directly by b-galactosidase

activity measurements. Whereas before the temperature

shift, this activity was similar in pNO2681- or pBR322-car-

rying cells, after 90 min at 428C, it was 40% higher in

pNO2681-carrying cells. It is noteworthy that b-galactosi-

dase activity reflects the average rate of synthesis of the

hybrid protein over the whole duration of the culture, hence

the modesty of the difference observed.

The effect of varying RNase E expression on the stability

of the 15 0UTR and 25 0UTR mRNAs

Jain and Belasco (1995) used the rne-1 mutation to show

that the 150UTR and 250UTR mRNAs are stabilized

differently by lowering the RNase E activity in the cell. In

this section, we show that the same result can be achieved

by decreasing the expression of the wild-type enzyme.

This result supports the view that the differential

stabilization of these mRNAs after substrate induction is

caused by RNase E titration.

To gain experimental control over RNase E expression

in 150UTR or 250UTR cells, we replaced the promoter and

50UTR of the chromosomal rne gene with the promoter and

50UTR from the lac operon (Fig. 5A). This change allows

modulation of RNase E expression by the lac operon

inducer IPTG. The downstream lac–rne junction is so

tailored that the lacZ initiation codon is followed

immediately by the second codon of the rne gene, i.e.

the RNase E sequence is not modified (Fig. 5B). In the

following, this engineered rne gene is called ‘Plac–rne’;

cells harbouring it are referred to as ‘Plac’ cells. As

expected since RNase E is an essential enzyme, Plac cells

did not form colonies on LB plates lacking IPTG. Likewise,

growth in liquid medium was IPTG dependent: at high

IPTG concentrations ($ 160mM), it was equivalent to that

of cells carrying the wild-type rne gene but, for lower IPTG

concentrations, the growth rate declined progressively (at

40mM IPTG, it has dropped to one-half its natural value).

These variations correlated with the expression of RNase

E in the cell. The latter was quantified on Western blots

using an anti-RNase E antibody. Extracts of cells carrying

the wild-type rne gene, as well as samples of pure RNase

E, were run in parallel as controls (Fig. 5C). For high IPTG

concentrations ($ 320mM), the expression of RNase E in

Fig. 4. A. Aliquots of the same cultures used in Fig. 3 (150UTR cells)were pulse labelled with [35S]-methionine and cysteine, and the ratesof synthesis of labelled RNase E (E) and hybrid RNase E–b-galactosidase protein (E-Z) were detected by immunoprecipitation,using a mixture of antibodies directed against the two proteins. Lanesmarked ‘E overexp.’, ‘E-Z overexp.’ and ‘E truncation’ correspond toextracts in which each of the RNase E or hybrid polypeptides isoverexpressed or missing altogether. The star pinpoints a couple ofpolypeptides (presumably the b and b0 subunits of E. coli RNApolymerase) that cross-hybridize with these antibodies and are usedas internal standards.B. Histograms showing the relative rate of synthesis of RNase E(closed bars) or hybrid RNase E–b-galactosidase (hatched bars) incells carrying either pNO2681 or pBR322. This figure is aquantification of the experiment shown in (A). In this particularexperiment, the recovery of the E-Z protein in the (pBR, 600) samplewas low; hence, the high value in the corresponding histogram (dottedbox). This low recovery was not seen in another experiment and ispresumably artifactual.

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Plac cells reached a plateau, presumably reflecting full

induction of the Plac promoter; this plateau corresponds to

< 250% of the RNase E expression from cells carrying the

wild-type rne gene (hereafter, the latter expression is

referred to as ‘normal’). Below 320mM IPTG, RNase E

expression decreased progressively: for 160mM IPTG, it

matches closely the normal expression, whereas for

40mM IPTG, it has dropped to only 10% of this expression

(Fig. 5C). In summary, the Plac–rne construct allows

manipulation of RNase E expression over a broad range,

say from 1/10th to 2.5-fold the normal expression: the

lower limit is a practical one, being set by the slow growth

of the cultures.

The 150UTR and 250UTR cells carrying the Plac–rne

construct (Fig. 6A) were then grown in the presence of

variable IPTG levels; total RNA was prepared from these

cultures and probed on Northern blots with a lacZ internal

probe. With 150UTR cells, the full-length species

predominated when RNase E expression was below

normal, whereas for higher expression, only incomplete

species were detected, indicating mRNA destabilization

(Fig. 6B). In contrast, with 250UTR cells, the mRNA

pattern remained similar for all IPTG concentrations.

These changes were paralleled by variations in b-

galactosidase expression: with 150UTR cells, increasing

IPTG concentration from 40 to 320mM caused this

expression to decrease more than 10-fold, whereas in

250UTR cells, no such effect was seen (variations, if any,

were in the reverse direction; Fig. 6B). Altogether, these

results show that the 150UTR mRNA but not the 250UTR

mRNA is stabilized when RNase E expression falls below

normal, as with the rne-1 mutation.

It is noteworthy that, under conditions of low RNase E

Fig. 6. Effect of RNase E concentration upon the stability of the150UTR and 250-UTR mRNAs.A. The strains used here are identical to the 150UTR and 250UTRstrains (Fig. 3), except that the genuine rne gene is replaced by thePlac– rne fusion (Fig. 5A).B. The RNA from the above cells growing with different concentrationsof IPTG (as indicated below each lane) has been probed on Northernblots with a lacZ internal probe. The histograms below show the b-galactosidase activities in the same cultures used for mRNA analysis.All symbols are as in Fig. 3A and B.

Fig. 5. A. Control of RNase E expression in-wild type (rne 1) and Plac

cells. In wild-type (WT) cells, RNase E (scissors) autoregulates its ownexpression by cleaving the 50UTR of its mRNA, whereas in Plac cells,its expression is downregulated by the lac repressor (the lacI product).The lac and rne sequences are indicated by empty and grey boxesrespectively. Negative regulation loops are indicated by curved lines.Note that, in Plac and wt cells, these loops operate at the transcriptionaland post-transcriptional levels respectively.B. Sequence encompassing the downstream lac–rne junction on thechromosome of Plac cells. ‘11’ designates the lac transcription startpoint. The major lac operator site (nt 1–22) is underlined; the lacZShine–Dalgarno and initiation codon are in upper case, and the rnesequence is italicized.C. Total proteins from Plac cells grown at different IPTG concentrationswere analysed by Western blot using an antibody against RNase E. Tocheck for uniform protein loading, the blot was also treated with anantibody against an unrelated protein, threonyl-tRNA synthetase(ThrS). Extracts of WT cells as well as various amounts of pure RNaseE were also loaded as controls. Bound immunoglobulins wererevealed with [125I]-protein A and autoradiography.

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expression, the b-galactosidase level is higher in 150UTR

than in 250UTR cells. This difference, which has also been

observed in the presence of the rne-1 mutation (Jain and

Belasco, 1995), indicates that the 150UTR mRNA is

intrinsically more translatable and/or less susceptible to

RNase E-independent decay than the 250UTR mRNA.

Other effects of varying RNase E expression

We also assayed the effect of varying RNase E expression

upon the maturation or decay of other RNase E

substrates. A classical substrate is the so-called ‘9S’

rRNA. This RNase III-generated fragment encompasses

the 30 end of the rRNA precursor; it is further processed by

RNase E into p5S, the immediate precursor of the 5S

rRNA (Ghora and Apirion, 1978). 9S rRNA is in fact

polydisperse, owing to heterogeneities at the 30 end of the

seven rRNA operons (Condon et al., 1995; Fig. 7A). To

test the effect of RNase E expression upon 9S rRNA

processing, total RNA from Plac cells growing with various

concentrations of IPTG was probed on Northern blots with

the 5S and 9S probes. To achieve a better resolution,

acrylamide–urea electrophoresis was used here instead

of agarose electrophoresis (Fig. 7B). For RNase E

expression equal to or larger than normal, the signals

originating from 9S rRNA were quite faint compared with

that originating from the 5S rRNA, consistent with a rapid

processing of the 9S rRNA. Quantitatively, raising RNase

E expression from 100% to 250% of normal caused an

< twofold decrease in the 9S compared with the 5S rRNA

signals. Conversely, for lower RNase E expressions, the

9S rRNA signals rose progressively until, together, they

eventually became nearly as intense as the 5S rRNA

signal (Fig. 7B). Thus, lowering the RNase E concentration

below normal slows down 9S rRNA processing markedly.

Finally, we assessed the effect of varying RNase E

concentration upon the functional stability of bulk mRNA.

Plac cells growing exponentially in the presence of various

concentrations of IPTG were treated with the transcription

inhibitor rifampicin, and the residual protein-synthesizing

capacity of the cultures was subsequently recorded as a

function of time by following the ability of aliquots to

incorporate [35S]-methionine in proteins (Fig. 8A). This

capacity decayed nearly exponentially with time, allowing

the functional half-life of bulk mRNA to be determined

(Fig. 8B). For RNase E expressions equal to or higher than

normal (IPTG $ 160mM), this half-life was nearly constant

(1.7–1.9 min) and similar to that observed with cells

carrying the wild-type rne gene. However, for lower RNase

E expressions, the half-life increased significantly: when

RNase E expression dropped to 1/10th of the normal

value, the increase was twofold (Fig. 8A and B).

Discussion

Here, we have examined the effect of overexpressing an

RNase E substrate upon the decay of mRNAs that carry

the rne 50UTR, the target for RNase E autoregulation. To

facilitate the interpretation of results, we have also studied

the effect of varying RNase E expression upon the stability

of the same mRNAs and, more generally, of other RNase

E substrates. For clarity, the latter results are discussed

first.

Experimental control of RNase E expression

To gain experimental control over RNase E expression,

we have replaced the promoter and 50UTR from the rne

gene with the promoter and 50UTR from the lac operon. As

the lac fragment used in this replacement encompasses

not only the main operator site (cf. Fig. 5B), but also a

minor one (OIII) located upstream of the promoter, the

transcription of the engineered rne gene is expected to be

very tightly repressed by the lac repressor in the absence

of IPTG (by < 440-fold; Oehler et al., 1990). Under

conditions of maximal IPTG induction, RNase E

expression was 2.5-fold the normal level. Interestingly,

this modest overexpression has no effect on growth,

Fig. 7. The rate of 9S rRNA processing varies with RNase Eexpression.A. Schematic representation of the most promoter-distal RNase IIIfragment from the E. coli rrn transcript (‘9S rRNA’; cf. Fig. 1B). Thesequence heterogeneity among the seven rrn operons downstream ofthe 5S sequence is symbolized by the hatched box. All other symbolsare as in Fig. 1B.B. Total RNA from Plac cells growing with different IPTGconcentrations (as indicated below each lane) was separated byurea–PAGE, blotted and probed with the 5S (left) or 9S (right) probes.The arrow points to 5S rRNA. Stars or ticks on the left indicate speciesthat are detected with both probes. The starred species match in sizethe classical ‘9S’ RNA from the rrnB operon (243 nt; see Cormack andMackie, 1992).

Synthesis of RNase E fits that of its substrates 873

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

whereas overexpression of RNase E from high-copy-

number plasmids bearing the rne gene has been found to

be toxic (Claverie-Martin et al., 1991). In contrast, growth

was impaired when the RNase E expression fell below

normal and was completely abolished in the absence of

IPTG, confirming the essential nature of RNase

E. However, surprisingly, as little as 1/10th of the normal

expression is enough to sustain growth, albeit at a twofold

reduced rate. The same observation has been made by

Belasco and colleagues (cited in Jiang et al., 2000).

Besides these effects on growth, variations in RNase E

expression also affected the stability of RNase E

substrates. In particular, the functional decay of bulk

mRNA, the processing of 9S rRNA and the cleavage of

the rne 50UTR (as judged by the compared stabilities and

b-galactosidase yields of the ‘150UTR’ and ‘250UTR’

mRNAs; see Fig. 6) were all slowed when RNase E

expression dropped below normal. These effects mimic

those observed with the rne-1 mutation (Mudd et al., 1990;

Jain and Belasco, 1995), confirming that the phenotype

associated with this mutation results from a shortage of

RNase E activity and not from a property of the mutated

polypeptide per se. Conversely, increasing RNase E

expression from 100% to 250% of normal appears to affect

individual substrates differently. Thus, bulk mRNA is not

destabilized further, and the processing of 9S rRNA is only

modestly accelerated, as judged by the magnitude of the

9S signal on Northern blots (Fig. 7B). In contrast, the rne

50UTR is destabilized more significantly, as seen by the

fact that the b-galactosidase yield from the 150UTR mRNA

decreases fourfold in this range of RNase E expression

(Fig. 6B). Whereas the molecular basis for this differential

behaviour is unknown, the particular sensitivity of the rne

50UTR to changes in RNase E expression seems

reasonable given its role as a ‘sensor’ of RNase E

demand (see below).

Homeostasy of RNase E expression in wild-type cells

Turning back to cells carrying the wild-type rne gene, we

have observed that, when the synthesis of an artificial

RNase E substrate is induced to relatively high levels (see

below), the concentration of the rne mRNA and the

synthesis of the RNase E polypeptide first increase rapidly

and then decrease to a new steady-state level, which

nevertheless remains higher than in the absence of

induction. These changes are not the result of changes in

the activity of the rne promoter, but rather of variations in

the rate of cleavage of the rne 50UTR. Thus, whereas the

150UTR fusion mRNA accumulates after substrate

induction much as the rne mRNA itself, no accumulation

was seen with the 250UTR mRNA (Fig. 3B). Given the

inverse correlation between the stability of the 150UTR

mRNA and the availability of RNase E (Fig. 6B), these

observations suggest that RNase E is transiently titrated

after substrate induction. A straightforward interpretation

is that, because of autoregulation, the pool of free RNase

E in the cell is limited: a burst in substrate synthesis

causes its titration, stabilizing RNase E substrates

including the rne mRNA. However, this stabilization is

transient, because it allows the RNase E pool to expand

and correct the titration. Thereby, autoregulation allows

RNase E to adjust its expression to that of its substrates.

As noted in the Introduction, this behaviour is not unique

to RNase E, but is also observed with several other

Fig. 8. The functional stability of bulk mRNA varies with RNase Econcentration.A. Plac cells growing with 40 (top) or 160 (bottom) mM IPTG weretreated with rifampicin (zero time) and, at the indicated times, theresidual protein-synthesizing capacity of the culture was measured byincubating an aliquot with [35S]-methionine until incorporation hadceased. Labelled proteins were separated by SDS–PAGE andvisualized by autoradiography.B. Total radioactivity incorporated into proteins is plotted versus time,assuming exponential decay kinetics; bulk mRNA half-lives areestimated from the slopes. Open circles, 40mM IPTG (10% normalRNase E expression; cf. Fig. 5C). Closed diamonds, 80mM IPTG(50% normal). Open triangles, 320mM IPTG (250% normal).

874 S. Sousa, I. Marchand and M. Dreyfus

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

autoregulated proteins. Particularly significant in this

respect is the case of heat shock proteins. Upon heat

shock, their synthesis increases and then decreases to a

new steady state, which remains higher than before the

heat shock. These variations parallel the activity of the

sigma factor s32. Although the mechanisms modulating

s32 activity are multiple, the sequestration of s32 by the

chaperone team DnaK–DnaJ–GrpE clearly plays an

important role. According to current models, the misfolded

or denatured polypeptides that accumulate during heat

shock titrate the chaperones, releasing free s32 and

causing heat shock protein synthesis to increase.

However, this increase is only transient because, once

the pools of DnaK, DnaJ and GprE have expanded

enough, s32 will become sequestrated again (Craig and

Gross, 1991). The parallel between this ‘homeostatic’

model of heat shock response and our present interpret-

ation is striking. Accordingly, we regard the response of

RNase E expression to changes in the concentration of its

substrates as another example of homeostasy.

Quantitative considerations

The notion that RNase E can be titrated by excess

substrate leads to speculative but intriguing questions

about the turnover of this enzyme. We rely here on the

physiological data compiled by Bremer and Dennis (1996).

At 378C and in the medium used here (0.75

doublings h21), the rate of stable RNA synthesis is about

5.6� 105 nt min21 cell21 (extrapolated for the values listed

in the above reference for 0.6 and 1 doublings h21). About

85% of this synthesis corresponds to the 5.5 kb rRNAs

precursors; these precursors are therefore made at a rate

of (5.6� 105� 0.85)/5500, or < 90 copies min21 cell21.

After RNase III cleavage, each precursor will yield two

RNase E substrates (cf. Fig. 1B). Therefore, rRNA

synthesis will contribute a total of 180 (90� 2) new

RNase E substrates min21. As for mRNA synthesis, it

corresponds to < 6.1� 105 nt min21 cell21 (Bremer and

Dennis, 1996). Assuming, for the sake of this discussion,

that the average size of an mRNA is 2000 nt, then about

300 (6.1� 105/2000) new mRNA molecules are syn-

thesized per minute and per cell. Altogether, the total

number of RNase E substrates synthesized per minute will

range from 180 to 480, depending on how many mRNA

molecules are RNase E substrates. When induced, the

artificial substrate used here is synthesized at a rate

1.7-fold higher than the rRNA precursors, or < 150

(90� 1.7) molecules per min. According to the above

estimates, its synthesis represents an increase of 24%

[150/(150 1 480)] to 45% [150/(150 1 180)] of the total

RNase E substrates in the cell. That this relatively modest

increase is enough to titrate the enzyme suggests that

RNase E is already close to substrate saturation under

normal growth conditions.

RNase E is a relatively abundant protein, being present

at several hundred copies per cell (Kido et al., 1996). That

this rather large pool is nearly saturated by a flow of only

175–475 new substrate molecules per min suggests that

the turnover of this enzyme is rather low. Conceivably, the

cleavages themselves are slow or the enzyme remains

durably associated with substrate before or after cleavage;

alternatively, the degradation of most mRNA molecules

involves multiple cleavages, delaying enzyme release.

According to another interpretation, RNase E may be

compartmentalized in vivo so that only a fraction of its pool

is actually available for RNA decay/processing. Interest-

ingly, in this respect, whereas mRNA decay is usually

regarded as mostly cytoplasmic, most of the RNase E is

known to be localized in the vicinity of the inner membrane

(Liou et al., 2001).

The stabilization of mRNA after a translation block may

reflect RNase E titration

In E. coli B cells growing under the conditions used here

(minimal or amino acid-supplemented glycerol medium),

the total rate of RNA synthesis increases < twofold after a

block in translation. In particular, for rRNA, the increase is

two- to 2.5-fold. Moreover, the newly synthesized rRNA is

then unstable (Shen and Bremer, 1977), and RNase E

presumably participates in its decay, as suggested by the

presence of rRNA fragments within degradosome prep-

arations (Bessarab et al., 1998). We recently proposed

that this burst of substrate synthesis under conditions in

which the RNase E pool cannot expand causes permanent

RNase E titration, accounting for the stabilization of mRNAs

observed when translation is blocked. The artificial substrate

used here retains the three well-characterized RNase E

sites of the rRNA precursor, and its rate of synthesis is

similar to the extra rate of rRNA synthesis observed after a

translation block. Since RNase E can be titrated by

inducing the synthesis of this substrate, it is reasonable to

assume that it can also be titrated after a translational

block. Thus, RNase E titration is probably one of the

causes for the bulk mRNA stabilization under these

conditions; whether it is the only one remains to be seen.

Experimental procedures

Plasmids and strains

Plasmids pEZ201 and pEZ206, which carry in phase rne–lacZ fusions retaining or lacking most of the rne 50UTR,

respectively, were kindly donated by Drs Jain and Belasco, aswas plasmid pRNE101, a pACYC177 derivative carrying the

rne gene and flanking sequences (Jain and Belasco, 1995).

Plasmid pNO2681 was a gift from Dr R. Gourse (Gourse et al.,

Synthesis of RNase E fits that of its substrates 875

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

1985). Plasmid pc I857 (Remaut et al., 1983) was obtainedfrom Dr M. Springer.

Construction of strains carrying the 15 0UTR and

25 0UTR fusions

We started from plasmid pTlacZ-Arg5 (Lopez et al., 1994).Within its polylinker, this pUC18 derivative carries sequen-

tially: (i) a truncated lac operon encompassing the lacZ geneand the beginning of lacY; and (ii) two tandemly arranged

transcriptional terminators. The Kpn I –Dra III fragmentextending from the polylinker sequence down to within the

lacZ coding sequence was then replaced by the Kpn I–Dra IIIfragments from pEZ201 and pEZ206, which extend from

upstream of the rne promoter down to the same Dra III site

within lacZ. The whole Kpn I–Xba I inserts extending fromupstream of the rne promoter to downstream of the

terminators were then subcloned from the resulting plasmidsinto the shuttle plasmid pOM43 (Chevrier-Miller et al., 1990),

yielding plasmids pSS201 and pSS206 respectively. Theinserts were then transferred into the malT–malP intergenic

region of the chromosome of MO20 [a Lac– derivative ofBL21(DE3)] as described by Lopez et al. (1994), yielding

strains ENS401 and ENS406.

Construction of the Plac strains

Using the fusion polymerase chain reaction (PCR) technique

(Ho et al., 1989), we constructed a 1.8 kb chimeric Bam HI–Sal I fragment carrying sequentially: (i) the region extending

from nt 2853 to 239 with respect to the rne transcription start;(ii) the region extending from nt 2126 to 141 with respect to

the lac transcription start; and (iii) the region extending from nt1365 to 11194 with respect to the rne transcription start. The

resulting fragment carries the lac promoter and 50UTR,flanked by rne homology regions. The rne and lac fragments

were amplified from plasmids pRNE101 and pUC19

respectively. The chimeric fragment was then cloned betweenthe Bam HI and Sal I sites of the shuttle plasmid pKO3 (Link

et al., 1997). After sequencing, the fragment was transferredinto the rne region of the E. coli chromosome using

homologous recombination between the plasmid- andchromosome-borne rne sequences (Link et al., 1997), yielding

the Plac–rne construct (Fig. 5A and B). As a recipient strain,we used a derivative of MG1655 carrying a Tn10 transposon

near the rne gene. The latter was transferred from CH1828[ams (ts), zce-726::Tn10; Mudd et al., 1990] by transduction,

selecting for TetR and ability to grow at 428C. The Plac–rneconstruct was then transduced from the MG1655 background

into strains ENS401 and ENS406, selecting for TetR andIPTG-dependent growth. The presence of the Plac– rne

construct in the final strains was checked by PCR.

Growth of cells

Cells were grown in MOPS medium (Neidhardt et al., 1974)

containing glycerol (0.2% w/v) as the carbon source. Forsubstrate induction studies, the above medium was sup-

plemented with kanamycin (50mg ml21) and ampicillin

(100mg ml21). Cells were grown to an OD600 of 0.4 at 308C,

then shifted to 428C for the time indicated. For experimentswith Plac cells, this medium was supplemented with all amino

acids, nucleic acid bases and vitamins, together with variableconcentrations of IPTG, as indicated. Growth temperature

was 378C in this case. With Plac cells, the use of startercultures yielded inconsistent results: we suspect that the

expression of RNase E differs widely in exponential andsaturated cultures, generating ‘memory’ effects when the

latter are diluted. Therefore, cells were routinely reisolated onM63B1 plates (Miller, 1972) containing 0.5% casamino acids

and a low concentration (32mM) of IPTG. After 20 h at 378C,the very small colonies that appeared were used directly to

inoculate liquid cultures (one colony per 5 ml). Cells wereharvested at an OD600 of 0.3–0.4.

RNA analysis

RNA was extracted as described by Yarchuk et al. (1992).

Conditions for agarose or acrylamide–urea electrophoresis,RNA blotting and membrane hybridization with randomly32P-labelled DNA fragments or 50 32P-labelled oligonucleo-tides have been described before, as have the 1.8 kb lac

fragment used to probe the rne–lacZ mRNA and the ‘5S’ and‘9S’ oligonucleotides used to probe the 5S and 9S rRNA

(Yarchuk et al., 1992; Lopez et al., 1994; 1999). The

oligonucleotide probe used to detect 23S rRNA was 50-AAGGTTAAGCCTCACGGTTC-30; the probe used to detect

the rne mRNA was an 816 bp Bam HI fragment from plasmidpGM102 (Cormack et al., 1993), which encompasses the 30

region of the rne gene. Radioactive signals were quantifiedwith a BAS1000 imager (Fuji).

Protein analysis

Purification of RNase E. RNase E was overexpressedfrom BL21(DE3) cells harbouring plasmid pGM102 (Cormack

et al., 1993). The overexpressed protein was eluted from apreparative 7.5% SDS–polyacrylamide gel. Its concentration

was estimated by running aliquots on an analytical gel inparallel with known amounts of bovine serum albumin (BSA),

staining with Coomassie blue and comparing the intensities ofthe stained bands by densitometry.

Quantification of RNase E in Plac cells. Pellets of

exponentially growing cells (equivalent to 0.1 OD600) wereresuspended directly in Laemmli sample buffer and separated

by SDS–PAGE (7.5% acrylamide). The proteins were

transferred to a nitrocellulose membrane (Amersham),which was incubated for 1 h at 378C in buffer A (20 mM Tris-

HCl, pH 7.5, 0.9% NaCl, 0.1% Triton X-100) containing 5%BSA. The membrane was incubated overnight with anti-

RNase E and anti-ThrS antibodies [gifts from Dr A. J.Carpousis (Toulouse) and Drs H. Putzer and C. Condon

(Paris); antibodies were used at 1:5000 in buffer A with 0.5%(BSA)], then washed (3� 15 min) with buffer A and, finally,

incubated for 2 h with buffer A containing 0.1mCi ml21 [125I]-protein A (Amersham). After washing again, the membrane

was exposed to a Fuji imager screen.

Functional half-life of bulk mRNA (Plac cells). Rifampicin

(500mg ml21 final) was added to exponentially growing

876 S. Sousa, I. Marchand and M. Dreyfus

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 867–878

cultures (OD600 < 0.4) of Plac cells (or control wild-type cells).Aliquots (500ml) were subsequently collected at timed

intervals and incubated with 10mCi of [35S]-methionine–[35S]-cysteine mix (Amersham) until 30 min after rifampicin

addition. As methionine and cysteine are present in excess inthe growth medium (see above), only a small fraction of the

label is incorporated in these experiments, so that theincorporated radioactivity reflects the protein-synthesizing

activity of the culture. The labelled samples were thenseparated by SDS–PAGE (12% acrylamide); after drying the

gels, the total incorporated radioactivity was quantified withthe Fuji imager.

Rate of synthesis of RNase E and hybrid RNase E–b-

galactosidase polypeptides after substrate induction. Cells

growing exponentially at 308C were shifted to 428C asdescribed above. At timed intervals after the shift, aliquots of

the cultures (0.5 ml) were removed, incubated for 1 min at428C with 10mCi of [35S]-methionine–cysteine mix and then

for 5 min with 10 mM cold methionine20.5 mM cold cysteine(final concentration). Cells were collected by centrifugation,

resuspended in SDS–PAGE loading buffer without thiols andheated (1008C for 4 min). Samples were diluted 20-fold in the

immunoprecipitation buffer described by Carpousis et al.(1994), except that Triton X-100 was used in place of Genapol

and BSA (0.2 mg ml21) was added. Extracts were incubatedwith anti-RNase E and anti-b-galactosidase antibodies (2.5ml

each for 0.5 ml of culture) for 1 h at 48C and treated with 100mlof a 20% (v/v) suspension of protein A–sepharose beads

(Pharmacia). After centrifugation, the immune complexeswere washed with the immunoprecipitation buffer (without

BSA), analysed by SDS–PAGE (7.5% acrylamide) and,finally, quantified with the Fuji imager. The anti-b-galactosi-

dase antibody was from Rockland. As controls, cultures

overexpressing RNase E or RNase E–b-galactosidase fusionpolypeptides or lacking them altogether were grown and

processed as above. For this purpose, we used BL21(DE3)rne-1 cells that had been incubated at 428C (at this

temperature, the inactive RNase E polypeptide encoded bythe rne-1 allele accumulates), BL21(DE3) cells carrying

pSS206, a multicopy plasmid encoding the fusion protein (seeabove) and BL21(DE3)rne131 cells, which synthesize a

truncated RNase E (Lopez et al., 1999).

Acknowledgements

We are much indebted to Drs C. Jain, J. G. Belasco andR. Gourse for plasmids, and to Dr A. J. Carpousis for

antibodies. We thank Dr P. J. Lopez for his participation inearly experiments, and Drs I. Iost and M. Springer for fruitful

discussions. This work was supported by CNRS, ENS and bygrants from ARC (no. 5474) and MENRT (programme

‘Microbiologie’) to M.D. S.S. was supported by the Fundacaopara a Ciencia e Tecnologia (fellowship PRAXIS

XXI/BM/19113/99), and I.M. by the Fondation pour laRecherche Medicale.

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