159
U-turns and regulatory RNAs
Thomas Franch and Kenn Gerdes*
Conventional antisense RNAs, such as those controlling
plasmid replication and maintenance, inhibit the function of
their target RNAs rapidly and efficiently. Novel findings show
that a common U-turn loop structure mediates fast RNA
pairing in the majority of these RNA controlled systems.
Usually, an antisense RNA regulates a single, cognate target
RNA only. Recent reports, however, show that antisense RNAs
can act as promiscuous regulators that control multiple genes
in concert to integrate complex physiological responses in
Escherichia coli.
Addresses
Department of Biochemistry and Molecular Biology, University of
Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
*e-mail: [email protected]
Current Opinion in Microbiology 2000, 3:159–164
1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
SD
Shine & Dalgarno
TIR
translational initiation region
Introduction
Antisense RNAs are small, non-translatable, regulatory
entities usually transcribed from eubacterial chromosomes,
bacteriophages and accessory genetic elements [1]. Antisense RNAs regulate their target RNAs by different
mechanisms including translational repression, target RNA
degradation and RNA folding interference [1]. Usually,
antisense RNAs act as post-transcriptional regulators in
processes where fast adaptive responses are appropriate.
The majority of known antisense RNAs are regulators that
form paired complexes with their complementary target
RNAs. In several cases, the extensive base pairing
between antisense and target RNA does not occur within a
feasible time-frame. Consequently, many antisense RNA
regulated systems have evolved structural features that
mediate target RNA inactivation upon formation of only a
limited number of interstrand base pairs [2–7]. Despite
clear diversities in binding pathways, the rate-limiting step
in complex formation between an antisense RNA and its
target is always an initial base-pairing interaction between
two loops, or a loop and a single-stranded RNA tail. Comparative sequence analysis of the recognition loops in
antisense RNAs and their targets revealed the presence of
a common structural motif that specifies so-called U-turns
[8••]. U-turns are structural elements present in the anticodon loops of tRNAs, where they facilitate rapid
codon–anticodon interaction. Thus, the U-turn loop structure is a general binding-rate-enhancer that promotes
RNA–RNA pairing. Usually, an antisense RNA regulates
only one cognate, complementary target RNA. However,
newly described regulatory RNAs encoded by the
Escherichia coli chromosome act as promiscuous regulators
that control or modulate the functions of multiple RNAs.
Here we review the importance of the U-turn motif in antisense RNA regulation and the multifaceted regulatory
properties of several small RNAs.
Rapid RNA–RNA pairing mediated by U-turn
loops
The effects of antisense RNAs on their target RNAs are
proportional to the rate of complex formation. In vitro bimolecular reactions yield maximum RNA pairing rate
constants of 106–107 M–1s–1 [9–11]. Thus, the interaction
between RNA molecules is inherently constrained and
not simply diffusion limited. From such studies, it was
inferred that antisense RNAs have evolved structural elements that reduce constraints and ambiguity of the pairing
reactions. Recently, a comparative sequence analysis of
antisense RNA recognition loops revealed the presence
of a ubiquitous YUNR (where Y = pyrimidine. U = uracil,
N = any nucleoside, R = purine) sequence motif [8••] (see
Figure 1). Strikingly, the YUNR motif of different antisense and target RNAs are located at the same relative
positions within the recognition loops (Figure 1). This
argues for a conserved function of the motif in the initial
recognition steps. The motif was proposed to configure a
U-turn loop structure similar to that observed in tRNA
anticodon loops [8••]. The U-turn is characterised by a
sharp turn in the phosphodiester-backbone between the
invariant uracil and the N base stabilised by three nonWatson–Crick interactions involving the invariant uracil
ribonucleoside [12–17,18•]. The nucleobases on the
3′ side of the turning phosphate is presented in an A-form
structure creating an unpaired Watson–Crick surface predisposed for binding to a complementary set of
nucleotides. Furthermore, the U-turn architecture retracts
the phosphodiester backbone within the loop, thereby
decreasing the local electronegative potential surrounding
the bases presented. This, in turn, promotes the pairing to
the complementary bases by reducing backbone repulsion
in the initial recognition step [19,20].
The activity of the hok/sok locus of plasmid R1, which
mediates plasmid maintenance by killing of plasmid-free
cells, is regulated by Sok antisense RNA [21]. The target
loop in hok mRNA contains a U-turn structure that is
recognised by the single-stranded tail of the complementary Sok antisense RNA [8••]. Substitutions of loop
nucleotides that disrupt the YUNR U-motif in hok mRNA
adversely affect the kinetics of RNA pairing, whereas substitutions that maintain the motif are silent [8••].
Structure probing analyses support the formation of a
U-turn loop structure in the antisense-binding loop of the
mRNA [8••]. A U-turn structure was also proposed to be
present in the IncI-antisense-binding loop of repZ mRNA
160
Cell regulation
Figure 1
complementary nucleotides in IncI. Instead, this
sequence serves a pivotal structural purpose in presenting
the subsequent sequence, rGGCG, for high affinity interor intramolecular base pairing. Furthermore, kinetic
analyses of several antisense/target RNA pairs including
RNAI/RNAII of ColE1 [23], CopA/CopT of R1 (EGH
Wagner, personal communication) and RNA-IN/RNAOUT of IS10 [24] support a similar rate-enhancing effect
of the YUNR U-turn motif. This suggests that, although
the binding pathways and the mechanisms of target RNA
inhibition are different for different systems, YUNR
U-turn loop structures act as rate-of-binding enhancers in
the majority of the naturally occurring antisense RNAregulated gene systems (Figure 1). Since most control
units appear unrelated in structure and function, the ubiquitous presence of U-turns represents an interesting
example of convergent evolution at the mechanistic level.
Furthermore, two distinct processes involving RNA–RNA
recognition, mRNA decoding and antisense RNA gene
control, have evolved the U-turn loop configuration independently. Clearly, the U-turn loop structure is a unique
element in RNA–RNA recognition.
OxyS RNA
Alignment of YUNR U-turn loop motifs in prokaryotic antisense and
target RNAs. The conserved YUNR sequence is highlighted in grey.
The underlined nucleotides form the top-stem base-pairs closing the
loops. chr., chromosome.
of plasmid ColIb-P9 [22•]. Here, the UpU dinucleotide of
the rUUGGCG hexa-nucleotide loop sequence was
shown to enhance the rate of intermolecular binding to
the IncI antisense RNA and to promote formation of an
intramolecular pseudoknot structure. The UpU dinucleotide sequence does not appear to interact with the
Respiratory organisms have evolved mechanisms that
counteract the damaging production of reactive oxygen
species [25]. In E. coli, the transcriptional activator, OxyR,
induces the expression of a number of genes including a
small 109 nucleotide RNA, OxyS [26]. The transient synthesis of OxyS affects more than 40 genes including the
stress σ-factor of RNA polymerase, σs, encoded by the
rpoS gene. OxyS RNA represses translation of rpoS mRNA
despite the lack of obvious complementary sequence
between the two RNAs. Translation of rpoS requires the
small RNA-binding protein Hfq ([27]; see also Noguiera
and Springer, this issue, pp 154–158). To activate translation of rpoS, Hfq presumably counteracts a negatively
acting secondary structure in the rpoS mRNA [27]. OxyS
RNA was proposed to bind the Hfq protein via its singlestranded, A-rich linker region (see Figure 2a) and, thereby,
counteracts the Hfq effect on rpoS translation, perhaps via
formation of a translationally inactive rpoS/Hfq/OxyS
ternary complex [28••]. If true, OxyS converts Hfq from a
translational activator into a co-repressor. This observation
suggests that gene control by regulatory RNAs can be
exerted in the absence of sequence complementarity to a
target RNA and thus expands the repertoire of regulatory
mechanisms used by antisense RNAs.
The fhlA gene encodes a transcriptional activator of genes
necessary for the assembly of the formate-hydrogenlyase
complex [29]. Expression of OxyS from a multi-copy plasmid negatively regulates FhlA production at the
post-transcriptional level [30•]. A short segment of seven
nucleotides centred in the transcriptional terminator hairpin loop of OxyS RNA is complementary to the Shine &
Dalgarno (SD) region of fhlA mRNA (Figure 2a). Mutational analyses and toeprinting experiments support a
U-turns and regulatory RNAs Franch and Gerdes
161
Figure 2
Schematic secondary structures of the OxyS,
DsrA and CsrB regulatory RNAs of E. coli.
(a) In OxyS the proposed regulatory region
complementary to the target genes fhlA and
exbB (i.e. negative regulation) are indicated by
a bold line. The stoichiometry and exact
location of Hfq binding in OxyS RNA is
unknown. (b) In DsrA the proposed regulatory
region complementary to the target genes
rpoS (i.e. positive regulation) and hns
(i.e. negative regulation) are indicated by
double lines and bold lines, respectively.
(c) The degenerated repetitive CAGGAUG
sequences in CsrB, proposed to bind the
CsrA regulator, are indicated by bold lines.
(a)
(c)
fhlA rpoS Hfq
5'-
exbB - ?
3'
OxyS
(b)
hns rpoS +
5' 3'
CsrB
5'DsrA
Current Opinion in Microbiology
model in which OxyS pairs to the complementary bases in
the leader of the fhlA transcript, thereby preventing ribosome binding at the translational initiation region (TIR).
Intriguingly, OxyS regulates rpoS and fhlA expression by
different mechanisms involving separate regions of OxyS
(Figure 2a). Secondary structure predictions of the fhlA
mRNA leader suggests that the OxyS target forms a part of
a six-nucleotide YUNR U-turn loop structure presented by
a short unstable stem (T Franch, unpublished data). This
U-turn loop may enhance the kinetics of OxyS/fhlA pairing
consistent with the observed high level of fhlA repression
by OxyS.
Highly reactive hydroxyl radicals generated by Fe2+ ions
are hazardous to cell components, especially DNA. A
region of 11 nucleotides in OxyS overlapping with the
region responsible for fhlA regulation is complementary to
the single-stranded SD region of the exbB mRNA
(T Franch, unpublished data), which encodes a component promoting ferric-siderophore uptake via TonB and
the iron/B12 subset of ABC membrane transporters [31].
Consequently, OxyS might reduce in vivo iron concentration by repression of exbB synthesis during oxidative stress.
Thus, OxyS could help further to reduce detrimental
DNA lesions and maintain the integrity of biomolecules,
an effect consistent with the proposed role of OxyS as a
protector of mutagenesis upon oxidative stress [26].
Upon challenge with superoxide, the SoxRS transcription
factors activate numerous genes involved in the defence
against oxidative stress in E. coli. Interestingly, SoxS
also activates transcription of another antisense RNA,
MicF, that is responsible for repression of the outer-membrane OmpF porin synthesis [32]. Thus, OxyS and MicF
act by distinct mechanisms to increase resistance to various compounds.
DsrA RNA
The cps genes that encode factors involved in the synthesis of the capsular polysaccharide colanic acid are
regulated by the RcsC–RcsB phosphorelay system and
the auxiliary positive regulator RcsA [33]. Multiple copies
of a region closely linked to the rcsA gene increases rcsAdependent capsule synthesis [34]. This region encodes a
small, stable 85 nucleotide RNA, known as DsrA, that
folds into a compact secondary structure with three consecutive stem-loops (Figure 2b). DsrA-mediated
activation of capsule polysaccharide production largely
mimics the effect observed in strains devoid of H-NS, a
major histone-like protein responsible for silencing of
numerous bacterial genes, including rcsA [34]. Since overproduction of DsrA induced the proU and papA, also
known to be silenced by H-NS, DsrA was proposed to
promote rcsA transcription by reducing H-NS activity.
DsrA exhibits 13 base-pairs of complementarity to a
region immediately downstream of the AUG start codon
of hns [35••]. Nucleotide substitutions in DsrA in the
region of complementarity abolished DsrA repression of
H-NS activity, whereas introduction of compensating
mutations in the hns gene restored DsrA regulation [35••].
These data support a model in which DsrA represses
expression of hns by direct RNA–RNA interaction, either
by ribosome exclusion at the TIR or by DsrA facilitated
decay of the hns mRNA. Since H-NS represses transcription of rpoS, DsrA indirectly stimulates expression of σs
via its repression of hns translation.
Positive regulation is unusual in antisense control circuits
and have thus far only been proposed for the stimulation of
the hla alpha-toxin mRNA by the RNAIII regulator of the
agr virulence locus in Staphylococcus aureus [36]. As
described below, DsrA RNA may also active as a positive
regulator of gene expression.
162
Cell regulation
The level of σS is inversely correlated with growth rate and
virtually no σS is present at optimal growth conditions at
37oC. In contrast, σS accumulates during exponential
growth at low temperatures (20°C) coinciding with optimal
synthesis of DsrA [37].
Surprisingly, DsrA exerts both H-NS-dependent and independent control of rpoS expression [37]. Two short
regions of 8 and 13 nucleotides in DsrA are complementary to an element in the rpoS mRNA that is responsible
for cis-inhibition of rpoS translation (Figure 2b). Mutational analyses of DsrA and the complementary element
in rpoS mRNA verified that DsrA activates rpoS translation by direct RNA–RNA interaction [35••,38••]. The Hfq
protein is required for translation of rpoS. Since Hfq is
believed to bind to the same region as DsrA in the rpoS
transcript [27], DsrA may act either separate from or in
combination with Hfq to stabilise a translationally active
configuration of the rpoS mRNA. The dual actions of
DsrA that ultimately promote RpoS production show that
DsrA is a highly flexible regulator that may serve an
important function as an activator of the stress-response
network at reduced temperatures.
The regulation of the cellular content of σs is highly complex and involves control of rpoS transcription and
translation, and σs proteolysis. Moreover, translation of
rpoS mRNA is regulated by at least three factors: DsrA and
Hfq stimulate rpoS translation, whereas OxyS inhibits rpoS
translation. The effect of OxyS appears counterintuitive,
since σs stimulates the expression of several genes that
protect against oxidative stress. Since OxyS is expressed
when OxyR is activated, OxyS may act to prevent redundant and wasteful activation of genes that are under
positive control of both OxyR and σs [28••]. Thus, OxyS
and DsrA are interesting examples of promiscuous antisense RNAs that integrate different global regulatory
networks into an appropriate physiological response.
CsrB RNA
Recently, a 61 amino acid protein, CsrA, and a ∼360
nucleotide RNA, CsrB, were identified as important components in the regulation of carbohydrate metabolism in
E. coli [39,40]. High levels of csrA expression promote the
activity of several enzymes of the glycolytic pathway and
repress genes responsible for gluconeogenesis and glycogen biosynthesis [39,41]. Furthermore, a csrA– strain
exhibits altered motility, adherence and cell morphology
phenotypes [39,42•].
Studies on the glgCAP operon, which encodes genes
required for glycogen biosynthesis, have shown that
CsrA facilitates the decay of the glgCAP mRNA by binding to the TIR of glgC [43,44]. The CsrB RNA consists of
18 imperfect repeats of seven nucleotides that are located in loops of predicted hairpin structures (see
Figure 2c). Overexpression of CsrB yields a csrA– phenotype and antagonises CsrA by the formation of a globular
CsrA/CsrB ribonucleoprotein complex that consists of
approximately 18 monomers of CsrA [40]. The stoichiometry of the complex indicates that CsrA recognises
and binds the imperfect repeats of CsrB via its KH RNAbinding motif. A model has been proposed in which
CsrB acts as a sink or a decoy molecule for CsrA and
thereby controls the concentration of free CsrA [40,42•].
Although a detailed knowledge on the regulation of CsrA
and CsrB synthesis is still lacking, it is plausible that the
ratio between the CsrA protein and the CsrB RNA determines the expression of a wide array of CsrA regulated
genes ([42•]; see also Noguiera and Springer, this issue,
pp 154–158).
A similar two-component system encoded by rsmA (csrA
homolog) and rsmB (csrB homolog) is present in the plant
pathogenic enterobacteria, Erwinia carotovora [45,46].
During exponential growth, RsmA negatively regulates
production and secretion of extracellular enzymes, quorum-sensing signals and other components necessary for
virulence [45]. The RsmB RNA counteracts RsmA by formation of a ribonucleoprotein similar to that observed for
CsrA/CsrB [46]. The well-documented control of virulence
factors by RsmA/RsmB could indicate that the homologous
system CsrA/CsrB might have a related, undisclosed function in pathogenesis in E. coli. This, in turn, could explain
the altered adherence and surface properties observed for
static csrA– cells [39].
Conclusions
Conventional antisense RNAs such as those controlling
plasmid replication and maintenance, regulate the function of a single, cognate target RNA. Such antisense RNAs
or their targets contain YUNR motifs that specify dynamic
U-turn loop structures required for fast bi-molecular interaction. The evolution of similar U-turn loop structures in
tRNAs and antisense RNAs argues that the intrinsic properties of the U-turn loop configuration facilitate rapid
bi-molecular RNA–RNA interaction. In contrast, the OxyS
and DsrA RNAs are promiscuous regulators, each modulating the expression of several or even many target genes.
The negative regulation by these RNAs, exerted by shortstretch base pairing to their targets, resembles the
regulation by conventional antisense RNAs. However, the
extraordinary complex negative and positive control of
rpoS translation shows that OxyS and DsrA are highly versatile regulators of multiple target genes. The promiscuous
regulation by OxyS and DsrA is important for the adaptive
responses to adverse growth conditions, such as oxidative
stress and low temperatures, and appears to be an integral
part of global stress response networks. We anticipate the
discovery of additional regulatory RNAs in E. coli and
other prokaryotic organisms.
Acknowledgement
Work in the group of Kenn Gerdes was supported by the Center for
Interaction, Structure, Function and Engineering of Macromolecules (CISFEM) of the Danish Biotechnology program.
U-turns and regulatory RNAs Franch and Gerdes
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