U-Turns and Regulatory Rnas Thomas Franch and Kenn Gerdes*

159

U-turns and regulatory RNAs Thomas Franch and Kenn Gerdes*

Conventional antisense RNAs, such as those controlling Escherichia coli chromosome act as promiscuous regulators plasmid replication and maintenance, inhibit the function of that control or modulate the functions of multiple RNAs. their target RNAs rapidly and efficiently. Novel findings show Here we review the importance of the U-turn motif in anti- that a common U-turn loop structure mediates fast RNA sense RNA regulation and the multifaceted regulatory pairing in the majority of these RNA controlled systems. properties of several small RNAs. Usually, an antisense RNA regulates a single, cognate target RNA only. Recent reports, however, show that antisense RNAs Rapid RNA–RNA pairing mediated by U-turn can act as promiscuous regulators that control multiple genes loops in concert to integrate complex physiological responses in The effects of antisense RNAs on their target RNAs are Escherichia coli. proportional to the rate of complex formation. In vitro bi- molecular reactions yield maximum RNA pairing rate Addresses constants of 106–107 M–1s–1 [9–11]. Thus, the interaction Department of Biochemistry and Molecular Biology, University of between RNA molecules is inherently constrained and Southern Denmark, Campusvej 55, 5230 Odense M, Denmark not simply diffusion limited. From such studies, it was *e-mail: [email protected] inferred that antisense RNAs have evolved structural ele- Current Opinion in Microbiology 2000, 3:159–164 ments that reduce constraints and ambiguity of the pairing reactions. Recently, a comparative sequence analysis of 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. antisense RNA recognition loops revealed the presence of a ubiquitous YUNR (where Y = pyrimidine. U = uracil, Abbreviations N = any nucleoside, R = purine) sequence motif [8••] (see SD Shine & Dalgarno TIR translational initiation region 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 Introduction argues for a conserved function of the motif in the initial Antisense RNAs are small, non-translatable, regulatory recognition steps. The motif was proposed to configure a entities usually transcribed from eubacterial chromosomes, U-turn loop structure similar to that observed in tRNA bacteriophages and accessory genetic elements [1]. Anti- anticodon loops [8••]. The U-turn is characterised by a sense RNAs regulate their target RNAs by different sharp turn in the phosphodiester-backbone between the mechanisms including translational repression, target RNA invariant uracil and the N base stabilised by three non- degradation and RNA folding interference [1]. Usually, Watson–Crick interactions involving the invariant uracil antisense RNAs act as post-transcriptional regulators in ribonucleoside [12–17,18•]. The nucleobases on the processes where fast adaptive responses are appropriate. 3′ side of the turning phosphate is presented in an A-form The majority of known antisense RNAs are regulators that structure creating an unpaired Watson–Crick surface pre- form paired complexes with their complementary target disposed for binding to a complementary set of RNAs. In several cases, the extensive base pairing nucleotides. Furthermore, the U-turn architecture retracts between antisense and target RNA does not occur within a the phosphodiester backbone within the loop, thereby feasible time-frame. Consequently, many antisense RNA decreasing the local electronegative potential surrounding regulated systems have evolved structural features that the bases presented. This, in turn, promotes the pairing to mediate target RNA inactivation upon formation of only a the complementary bases by reducing backbone repulsion limited number of interstrand base pairs [2–7]. Despite in the initial recognition step [19,20]. clear diversities in binding pathways, the rate-limiting step in complex formation between an antisense RNA and its The activity of the hok/sok locus of plasmid R1, which target is always an initial base-pairing interaction between mediates plasmid maintenance by killing of plasmid-free two loops, or a loop and a single-stranded RNA tail. Com- cells, is regulated by Sok antisense RNA [21]. The target parative sequence analysis of the recognition loops in loop in hok mRNA contains a U-turn structure that is antisense RNAs and their targets revealed the presence of recognised by the single-stranded tail of the complemen- a common structural motif that specifies so-called U-turns tary Sok antisense RNA [8••]. Substitutions of loop [8••]. U-turns are structural elements present in the anti- nucleotides that disrupt the YUNR U-motif in hok mRNA codon loops of tRNAs, where they facilitate rapid adversely affect the kinetics of RNA pairing, whereas sub- codon–anticodon interaction. Thus, the U-turn loop struc- stitutions that maintain the motif are silent [8••]. ture is a general binding-rate-enhancer that promotes Structure probing analyses support the formation of a RNA–RNA pairing. Usually, an antisense RNA regulates U-turn loop structure in the antisense-binding loop of the only one cognate, complementary target RNA. However, mRNA [8••]. A U-turn structure was also proposed to be newly described regulatory RNAs encoded by the 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 inter- or 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/RNA- OUT 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 RNA- regulated 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 inde- pendently. Clearly, the U-turn loop structure is a unique element in RNA–RNA recognition.

OxyS RNA 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 single- stranded, 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 Alignment of YUNR U-turn loop motifs in prokaryotic antisense and target RNA and thus expands the repertoire of regulatory target RNAs. The conserved YUNR sequence is highlighted in grey. mechanisms used by antisense RNAs. The underlined nucleotides form the top-stem base-pairs closing the loops. chr., chromosome. 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 plas- of plasmid ColIb-P9 [22•]. Here, the UpU dinucleotide of mid negatively regulates FhlA production at the the rUUGGCG hexa-nucleotide loop sequence was post-transcriptional level [30•]. A short segment of seven shown to enhance the rate of intermolecular binding to nucleotides centred in the transcriptional terminator hair- the IncI antisense RNA and to promote formation of an pin loop of OxyS RNA is complementary to the Shine & intramolecular pseudoknot structure. The UpU dinu- Dalgarno (SD) region of fhlA mRNA (Figure 2a). Muta- cleotide sequence does not appear to interact with the tional 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) (c) (a) In OxyS the proposed regulatory region complementary to the target genes fhlA and exbB (i.e. negative regulation) are indicated by fhlA - a bold line. The stoichiometry and exact rpoS - location of Hfq binding in OxyS RNA is Hfq exbB - ? unknown. (b) In DsrA the proposed regulatory 3' region complementary to the target genes 5'- rpoS (i.e. positive regulation) and hns OxyS (i.e. negative regulation) are indicated by double lines and bold lines, respectively. (b) hns - (c) The degenerated repetitive CAGGAUG sequences in CsrB, proposed to bind the rpoS + CsrA regulator, are indicated by bold lines. 5' 3'

CsrB 5'- DsrA Current Opinion in Microbiology

model in which OxyS pairs to the complementary bases in DsrA RNA the leader of the fhlA transcript, thereby preventing ribo- The cps genes that encode factors involved in the synthe- some binding at the translational initiation region (TIR). sis of the capsular polysaccharide colanic acid are Intriguingly, OxyS regulates rpoS and fhlA expression by regulated by the RcsC–RcsB phosphorelay system and different mechanisms involving separate regions of OxyS the auxiliary positive regulator RcsA [33]. Multiple copies (Figure 2a). Secondary structure predictions of the fhlA of a region closely linked to the rcsA gene increases rcsA- mRNA leader suggests that the OxyS target forms a part of dependent capsule synthesis [34]. This region encodes a a six-nucleotide YUNR U-turn loop structure presented by small, stable 85 nucleotide RNA, known as DsrA, that a short unstable stem (T Franch, unpublished data). This folds into a compact secondary structure with three con- U-turn loop may enhance the kinetics of OxyS/fhlA pairing secutive stem-loops (Figure 2b). DsrA-mediated consistent with the observed high level of fhlA repression activation of capsule polysaccharide production largely by OxyS. mimics the effect observed in strains devoid of H-NS, a major histone-like protein responsible for silencing of Highly reactive hydroxyl radicals generated by Fe2+ ions numerous bacterial genes, including rcsA [34]. Since over- are hazardous to cell components, especially DNA. A production of DsrA induced the proU and papA, also region of 11 nucleotides in OxyS overlapping with the known to be silenced by H-NS, DsrA was proposed to region responsible for fhlA regulation is complementary to promote rcsA transcription by reducing H-NS activity. the single-stranded SD region of the exbB mRNA DsrA exhibits 13 base-pairs of complementarity to a (T Franch, unpublished data), which encodes a compo- region immediately downstream of the AUG start codon nent promoting ferric-siderophore uptake via TonB and of hns [35••]. Nucleotide substitutions in DsrA in the the iron/B12 subset of ABC membrane transporters [31]. region of complementarity abolished DsrA repression of Consequently, OxyS might reduce in vivo iron concentra- H-NS activity, whereas introduction of compensating tion by repression of exbB synthesis during oxidative stress. mutations in the hns gene restored DsrA regulation [35••]. Thus, OxyS could help further to reduce detrimental These data support a model in which DsrA represses DNA lesions and maintain the integrity of biomolecules, expression of hns by direct RNA–RNA interaction, either an effect consistent with the proposed role of OxyS as a by ribosome exclusion at the TIR or by DsrA facilitated protector of mutagenesis upon oxidative stress [26]. decay of the hns mRNA. Since H-NS represses transcription of rpoS, DsrA indirectly stimulates expression of σs Upon challenge with superoxide, the SoxRS transcription via its repression of hns translation. factors activate numerous genes involved in the defence against oxidative stress in E. coli. Interestingly, SoxS Positive regulation is unusual in antisense control circuits also activates transcription of another antisense RNA, and have thus far only been proposed for the stimulation of MicF, that is responsible for repression of the outer-mem- the hla alpha-toxin mRNA by the RNAIII regulator of the brane OmpF porin synthesis [32]. Thus, OxyS and MicF agr virulence locus in Staphylococcus aureus [36]. As act by distinct mechanisms to increase resistance to vari- described below, DsrA RNA may also active as a positive ous compounds. regulator of gene expression. 162 Cell regulation

The level of σS is inversely correlated with growth rate and CsrA/CsrB ribonucleoprotein complex that consists of virtually no σS is present at optimal growth conditions at approximately 18 monomers of CsrA [40]. The stoi- 37oC. In contrast, σS accumulates during exponential chiometry of the complex indicates that CsrA recognises growth at low temperatures (20°C) coinciding with optimal and binds the imperfect repeats of CsrB via its KH RNA- synthesis of DsrA [37]. binding motif. A model has been proposed in which CsrB acts as a sink or a decoy molecule for CsrA and Surprisingly, DsrA exerts both H-NS-dependent and - thereby controls the concentration of free CsrA [40,42•]. independent control of rpoS expression [37]. Two short Although a detailed knowledge on the regulation of CsrA regions of 8 and 13 nucleotides in DsrA are complemen- and CsrB synthesis is still lacking, it is plausible that the tary to an element in the rpoS mRNA that is responsible ratio between the CsrA protein and the CsrB RNA deter- for cis-inhibition of rpoS translation (Figure 2b). Muta- mines the expression of a wide array of CsrA regulated tional analyses of DsrA and the complementary element genes ([42•]; see also Noguiera and Springer, this issue, in rpoS mRNA verified that DsrA activates rpoS transla- pp 154–158). tion by direct RNA–RNA interaction [35••,38••]. The Hfq protein is required for translation of rpoS. Since Hfq is A similar two-component system encoded by rsmA (csrA believed to bind to the same region as DsrA in the rpoS homolog) and rsmB (csrB homolog) is present in the plant transcript [27], DsrA may act either separate from or in pathogenic enterobacteria, Erwinia carotovora [45,46]. combination with Hfq to stabilise a translationally active During exponential growth, RsmA negatively regulates configuration of the rpoS mRNA. The dual actions of production and secretion of extracellular enzymes, quo- DsrA that ultimately promote RpoS production show that rum-sensing signals and other components necessary for DsrA is a highly flexible regulator that may serve an virulence [45]. The RsmB RNA counteracts RsmA by for- important function as an activator of the stress-response mation of a ribonucleoprotein similar to that observed for network at reduced temperatures. CsrA/CsrB [46]. The well-documented control of virulence factors by RsmA/RsmB could indicate that the homologous The regulation of the cellular content of σs is highly com- system CsrA/CsrB might have a related, undisclosed func- plex and involves control of rpoS transcription and tion in pathogenesis in E. coli. This, in turn, could explain translation, and σs proteolysis. Moreover, translation of the altered adherence and surface properties observed for rpoS mRNA is regulated by at least three factors: DsrA and static csrA– cells [39]. Hfq stimulate rpoS translation, whereas OxyS inhibits rpoS translation. The effect of OxyS appears counterintuitive, Conclusions since σs stimulates the expression of several genes that Conventional antisense RNAs such as those controlling protect against oxidative stress. Since OxyS is expressed plasmid replication and maintenance, regulate the func- when OxyR is activated, OxyS may act to prevent redun- tion of a single, cognate target RNA. Such antisense RNAs dant and wasteful activation of genes that are under or their targets contain YUNR motifs that specify dynamic positive control of both OxyR and σs [28••]. Thus, OxyS U-turn loop structures required for fast bi-molecular inter- and DsrA are interesting examples of promiscuous anti- action. The evolution of similar U-turn loop structures in sense RNAs that integrate different global regulatory tRNAs and antisense RNAs argues that the intrinsic prop- networks into an appropriate physiological response. erties of the U-turn loop configuration facilitate rapid CsrB RNA bi-molecular RNA–RNA interaction. In contrast, the OxyS and DsrA RNAs are promiscuous regulators, each modu- Recently, a 61 amino acid protein, CsrA, and a ∼360 nucleotide RNA, CsrB, were identified as important com- lating the expression of several or even many target genes. ponents in the regulation of carbohydrate metabolism in The negative regulation by these RNAs, exerted by short- E. coli [39,40]. High levels of csrA expression promote the stretch base pairing to their targets, resembles the activity of several enzymes of the glycolytic pathway and regulation by conventional antisense RNAs. However, the repress genes responsible for gluconeogenesis and glyco- extraordinary complex negative and positive control of gen biosynthesis [39,41]. Furthermore, a csrA– strain rpoS translation shows that OxyS and DsrA are highly ver- exhibits altered motility, adherence and cell morphology satile regulators of multiple target genes. The promiscuous phenotypes [39,42•]. regulation by OxyS and DsrA is important for the adaptive responses to adverse growth conditions, such as oxidative Studies on the glgCAP operon, which encodes genes stress and low temperatures, and appears to be an integral required for glycogen biosynthesis, have shown that part of global stress response networks. We anticipate the CsrA facilitates the decay of the glgCAP mRNA by bind- discovery of additional regulatory RNAs in E. coli and ing to the TIR of glgC [43,44]. The CsrB RNA consists of other prokaryotic organisms. 18 imperfect repeats of seven nucleotides that are located in loops of predicted hairpin structures (see Acknowledgement – Work in the group of Kenn Gerdes was supported by the Center for Figure 2c). Overexpression of CsrB yields a csrA pheno- Interaction, Structure, Function and Engineering of Macromolecules (CIS- type and antagonises CsrA by the formation of a globular FEM) of the Danish Biotechnology program. U-turns and regulatory RNAs Franch and Gerdes 163

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