RNAs reguladores.docx

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NIHPublic AccessAuthorManuscriptCell. Authormanuscript; available in PMC 2011 July 10.

Published in final edited form as:Cell. 2009 February 20; 136(4): 615628. doi:10.1016/j.cell.2009.01.043.

RegulatoryRNAsinBacteria

LaurenS.WatersandGiselaStorz*CellBiology andMetabolismProgram, EuniceKennedy ShriverNationalInstituteof ChildHealth

andHumanDevelopment, Bethesda, MD20892-5430

Abstract

RNAregulatorsin bacteria are a heterogenousgroup ofmoleculesthat act by variousmechanisms

to modulate a wide range ofphysiological responses. One classcomprisesriboswitches, which are

partsofthe mRNAsthey regulate. These leadersequencesfold into structuresamenable to

conformational changesupon the binding ofsmall molecules. Riboswitchesthussense and

respond to the availability ofvariousnutrientsin the cell. Othersmall transcriptsbind to proteins,

among them global regulators, and antagonize theirfunctions. The largest and most extensively

studied set ofsmall RNAregulatorsact through base pairing with RNAs, usually modulating thetranslation and stability ofmRNAs. The majority ofthese small RNAsregulate responsesto

changesin environmental conditions.Finally, a recently discovered group ofRNAregulators,

known asthe CRISPR RNAs, containshort regionsofhomology to bacteriophage and plasmid

sequences. CRISPR RNAsinterfere with bacteriophage infection and plasmid conjugation by

targeting the homologousforeign DNAthrough an unknown mechanism. Here we discusswhat is

known about these RNAregulators, aswell asthe many intriguing questionsthat remain to be

addressed.

RNAmoleculesthat act asregulatorswere known in bacteria foryearsbefore the first

microRNAs(miRNAs)andshort interfering RNAs(siRNAs)were discovered in eukaryotes.

In 1981, the ~108 nucleotide RNAIwasfound to block ColE1 plasmid replication by base

pairing with the RNAthat iscleaved to produce the replication primer(Stougaard et al.,

1981; Tomizawa et al., 1981). Thiswork wasfollowed by the 1983 discovery ofa ~70

nucleotide RNAwhich istranscribed from the pOUT promoterofthe Tn10 transposon and

repressestransposition by preventing translation ofthe transposase mRNA(Simonsand

Kleckner, 1983). The first chromosomally-encoded small RNAregulator, reported in 1984,

wasthe 174 nucleotide EscherichiacoliMicFRNA, which inhibitstranslation ofthe mRNA

encoding the majoroutermembrane porin OmpF(Mizuno et al., 1984). These first small

RNAregulators, and a handful ofothers, were identified by gel analysisdue to their

abundance, by multicopy phenotypes, orby serendipity (reviewed in (Wassarman et al.,

1999)).

While a fewbacterial RNAregulatorswere identified early on, theirprevalence and their

contributionsto numerousphysiological responseswere not initially appreciated. In 2001

2002, fourgroupsreported the identification ofmany newsmall RNAsthrough systematic

computational searchesforconservation and orphan promoterand terminatorsequencesin

the intergenic regionsofE.coli(reviewed in (Livny and Waldor, 2007)). Additional RNAs

were discovered by direct detection using cloning-based techniquesormicroarrayswith

probesin intergenic regions(reviewed in (Altuvia, 2007)). Variationsofthese approaches,

aided by the availability ofmany newbacterial genome sequences, have led to the

identification ofregulatory RNAsin an ever-increasing numberofbacteria. Enabled by

*Correspondence: [email protected].


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Watersand Storz Page 3

gene product. An ever-increasing numberand variety ofriboswitchesare being identified in

bacteria, aswell asin some eukaryotes. Forexample, asmany as2%ofall Bacillussubtilis

genesare regulated by riboswitcheswhich bind metabolitesranging from flavin

mononucleotide (FMN)and thiamin pyrophosphate to S-adenosylmethionine, lysine and

guanine.

Riboswitchesgenerally consist oftwo parts: the aptamerregion, which bindsthe ligand, and

theso-called exp

ress

ion platform,

which

regulate

sgene exp

ress

ion through alte

rnative R

NA

structuresthat affect transcription ortranslation (reviewed in (Mandal and Breaker, 2004;

Montange and Batey, 2008; Nudlerand Mironov, 2004))(Figure 1A). Upon binding ofthe

ligand, the riboswitch changesconformation. These changesusually involve alternative

hairpin structureswhich form ordisrupt transcriptional terminatorsorantiterminators, or

which occlude orexpose ribosome binding sites(Figure 1A). In general, most riboswitches

represstranscription ortranslation in the presence ofthe metabolite ligand; only a few

riboswitchesthat activate gene expression have been characterized.

Due to the modularnature ofriboswitches, the same aptamerdomain can mediate different

regulatory outcomesoroperate through distinct mechanismsin different contexts(reviewed

in (Nudlerand Mironov, 2004)). Forexample, the cobalamin riboswitch, which bindsthe

coenzyme form ofvitaminB12, operatesby transcription termination forthe btuBgenesin

Gram-positive bacteria but modulatestranslation initiation forthe coboperonsofGram-negative bacteria. Some transcriptscarry tandem riboswitches, which can integrate distinct

physiological signals, and one notable riboswitch, the glmSleadersequence, even actsasa

ribozyme to catalyze self-cleavage. Upon binding ofitscofactorglucosamine-6-phosphate,

the glmSriboswitch cleavesitselfand inactivatesthe mRNAencoding the enzyme that

generatesglucosamine-6-phosphate, thuseffecting a negative feedback loop formetabolite

levels(Collinset al., 2007). In principle, riboswitchescould be used in conjunction with any

reaction associated with RNA, not just transcription, translation and RNAprocessing, but

also RNAmodification, localization orsplicing.

Generally, the riboswitchesin Gram-positive bacteria affect transcriptional attenuation,

while the riboswitchesin Gram-negative bacteria more frequently inhibit translation

(reviewed in (Nudlerand Mironov, 2004)). Possibly the preferential use oftranscriptional

termination in Gram-positive organismsislinked to the fact that genesare clustered togetherin largerbiosynthetic operonswhere more resourceswould be wasted ifthe full-length

transcript issynthesized. Gram-positive organismsalso appearto rely more on cis-acting

riboswitchesthan Gram-negative organisms, forwhich more trans-acting sRNAregulators

are known. Research directionspursued in studiesofthe different organisms, however, may

biasthese generalizations.

sRNAsThatModulateProteinActivity

Three protein-binding sRNAshave intrinsic activity (RNase P)orcontribute essential

functionsto a ribonucleoprotein particle (4.5Sand tmRNA). In contrast, three otherprotein-

binding sRNAs(CsrB, 6S, and GlmY)act in a regulatory fashion to antagonize the activities

oftheircognate proteinsby mimicking the structuresofothernucleic acids(Figure 1B).

The CsrB and CsrC RNAsofE.colimodulate the activity ofCsrA, an RNA-binding proteinthat regulatescarbon usage and bacterial motility upon entry into stationary phase and other

nutrient-poorconditions(reviewed in (Babitzke and Romeo, 2007)). CsrAdimersbind to

GGAmotifsin the 5UTR oftarget mRNAs, thereby affecting the stability and/or

translation ofthe mRNA. The CsrB and CsrC RNAseach contain multiple GGAbinding

sites, 22 and 13 respectively, forCsrA. Thus, when CsrB and CsrC levelsincrease, the

sRNAseffectively sequesterthe CsrAprotein away from mRNAleaders. Transcription of

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the csrBand csrCgenesisinduced by the BarA-UvrB two-component regulatorswhen cells

encounternutrient poorgrowth conditions, though the signal forthisinduction isnot known.

The CsrB and CsrC RNAsalso are regulated at the level ofstability through the CsrD

protein, a cyclic di-GMPbinding protein, which recruitsRNase E to degrade the sRNAs

(Suzuki et al., 2006). CsrB and CsrC homologs(such asRsmYand RsmZ)have been found

to antagonize the activitiesofCsrAhomologsin a range ofbacteria including Salmonella,

Erwinia, Pseudomonas,and Vibriowhere they impact secondary metabolism, quorum

sensing and epithelial cell invasion (reviewed in (Lapouge et al., 2008; Lucchetti-Miganehet al., 2008)).

The E.coli6SRNAmimicsan open promoterto bind to and sequesterthe 70-containing

RNApolymerase (reviewed in (Wassarman, 2007)). When 6Sisabundant, especially in

stationary phase, it isable to complex with much ofthe 70-bound, housekeeping form of

RNApolymerase, but isnot associated with the S-bound, stationary phase form ofRNA

polymerase (Trotochaud and Wassarman, 2005). The interaction between 6Sand 70-

holoenzyme inhibitstranscription from certain 70promotersand increasestranscription

from some Sregulated promoters, in part by altering the competition between 70-and S-

holoenzyme binding to promoters. Interestingly, the 6SRNAcan serve asa template forthe

transcription of1420 nucleotide product RNAs(pRNAs)by RNApolymerase, especially

during outgrowth from stationary phase (Gildehauset al., 2007; Wassarman and Saecker,

2006). In fact, it isthought that transcription from 6Swhen NTPconcentrationsincreasemay be a way to release 70-RNApolymerase (Wassarman and Saecker, 2006). It isnot

known whetherthe pRNAsthemselveshave a function. The 6SRNAisprocessed out ofa

longertranscript and accumulatesduring stationary phase, but the detailsofthisregulation

have not been elucidated (reviewed in (Wassarman, 2007)). There are multiple 6Shomologs

in a numberoforganisms, including two in B.subtilis(Trotochaud and Wassarman, 2005).

The rolesofthese homologsagain are not known, but it istempting to speculate that they

inhibit the activitiesofalternative factorformsofRNApolymerase.

One additional sRNA, GlmY, hasrecently been proposed to have a protein-binding mode of

action and isthought to function by titrating an RNAprocessing factoraway from a

homologoussRNA, GlmZ (reviewed in (Grke and Vogel, 2008)). Both GlmZ and GlmY

promote accumulation ofthe GlmSglucosamine-6-phosphate synthase, howeverthey do so

by distinct mechanisms. The full-length GlmZ RNAbase pairswith and activatestranslationofthe glmSmRNA. Although the GlmYRNAishighly homologousto GlmZ in sequence

and predicted secondary structure, GlmYlacksthe region that iscomplementary to the glmS

mRNAtarget and doesnot directly activate glmStranslation. Instead, GlmYexpression

inhibitsa GlmZ processingevent that rendersGlmZ unable to activate glmStranslation.

Although not yet conclusively shown, GlmYmost likely stabilizesthe full-length GlmZ by

competing with GlmZ forbinding to the YhbJprotein that targetsGlmZ forprocessing. The

GlmYRNAisalso processed and itslevelsare negatively regulated by poly-adenylation

(Reichenbach et al., 2008; Urban and Vogel, 2008).

CsrB RNAsimulatesan mRNAelement, 6Simitatesa DNAstructure, and GlmYmimics

anothersRNA, raising the question asto what othermolecules, nucleic acid orotherwise,

might yet uncharacterized sRNAsmimic?

Cis-encodedBasePairingsRNAs

In contrast to the fewknown protein-binding sRNAs, most characterized sRNAsregulate

gene expression by base pairing with mRNAsand fall into two broad classes: those having

extensive potential forbase pairing with theirtarget RNA(Figure 2A)and those with more

limited complementarity (Figure 2B). We will first focuson sRNAsthat are encoded in cis

on the DNAstrand opposite the target RNAand share extended regionsofcomplete


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complementarity with theirtarget, often 75 nucleotidesormore (Figure 2A)(reviewed in

(Brantl, 2007; Wagneret al., 2002)). While the two transcriptsare encoded in the same

region ofDNA, they are transcribed from opposite strandsasdiscrete RNAspeciesand

function in transasdiffusible molecules. Forthe fewcaseswhere it hasbeen examined, the

initial interaction between the sRNAand target RNAinvolvesonly limited pairing, though

the duplex can subsequently be extended. The most well-studied examplesofcis-encoded

antisense sRNAsreside onplasmidsorothermobile genetic elements, however

chromosomal versionsofthese sRNAsincreasingly are being found.Most ofthe cis-encoded antisense sRNAsexpressed from bacteriophage, plasmidsand

transposonsfunction to maintain the appropriate copy numberofthe mobile element

(reviewed in (Brantl, 2007; Wagneret al., 2002)). They achieve thisthrough a variety of

mechanisms, including inhibition ofreplication primerformation and transposase

translation, asmentioned forplasmid ColE1 RNAIand Tn10 pOUT RNA, respectively.

Anothercommon group act asantitoxinsto repressthe translation oftoxic proteinsthat kill

cellsfrom which the mobile element hasbeen lost.

In general, the physiological rolesofthe cis-encoded antisense sRNAsexpressed from

bacterial chromosomesare lesswell understood. Asubset promote degradation and/or

represstranslation ofmRNAsencoding proteinsthat are toxic at high levels(reviewed in

(Fozo et al., 2008a; Gerdesand Wagner, 2007)). In E.coli,there are also two sRNAs, IstRand OhsC, that are encodeddirectly adjacent to genesencoding potentially toxic proteins.

Although these sRNAsare not true antisense RNAs, they do contain extended regionsof

perfect complementarity (19 and 23 nucleotides)with the toxin mRNAs. Interestingly, most

ofthese sRNAsappearto be expressed constitutively. Some ofthe chromosomal antitoxin

sRNAsare homologousto plasmid antitoxin sRNAs(forexample, the Hok/Sok loci present

in the E.colichromosome)orare located in regionsacquired from mobile elements(for

example, the RatARNAofB.subtilisfound in a remnant ofa cryptic prophage). These

observationsindicate that the antitoxin sRNAand corresponding toxin genesmight have

been acquired by horizontal transfer. The chromosomal versionsmay simply be non-

functional remnants. However, some cis-encoded antisense antitoxin sRNAsdo not have

known homologson mobile elements. In addition, given that bacteria have multiple copies

ofseveral loci, all ofwhich are expressed in the casesexamined, it istempting to speculate

that the antitoxin sRNAs-toxin proteinsencoded on the chromosome provide beneficialfunctions(Fozo et al., 2008b). Although high levelsofthe toxinskill cells, more moderate

levelsproduced from single-copy loci underinducing conditionsmay only slowgrowth.

Thusone model proposesthat chromosomal toxin-antitoxin modulesinduce slowgrowth or

stasisunderconditionsofstressto allowcellstime to repairdamage orotherwise adjust to

theirenvironment (Kawano et al., 2007; Unoson and Wagner, 2008). Anotherpossibility is

that certain modulesmay be retained in bacterial chromosomesasa defense against

plasmidsbearing homologousmodules, assuming that the chromosomal antisense sRNAcan

repressthe expression ofthe plasmid-encoded toxin.

Anothergroup ofcis-encoded antisense sRNAsmodulatesthe expression ofgenesin an

operon. Some ofthese sRNAsare encoded in regionscomplementary to intervening

sequence between ORFs(Figure 2A). Forexample, in E.coli, base pairing between the

stationary phase-induced GadYantisense sRNAand the gadXWmRNAleadsto cleavage ofthe duplex between the gadXand gadWgenesand increased levelsofa gadXtranscript

(Opdyke et al., 2004; Tramonti et al., 2008). Forthe virulence plasmid pJM1 ofVibrio

anguillarum, the interaction between the RNAantisense sRNAand the fatDCBAangRT

mRNAleadsto transcription termination afterthe fatAgene, thusreducing expression ofthe

downstream angRTgenes(Stork et al., 2007). In Synechocystis, the iron-stressrepressed

IsrR antisense sRNAbase pairswith sequenceswithin isiAcoding region ofthe isiAB


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increasing the local concentrationsofsRNAsand mRNAs. It should be noted that when the

E.coliSgrSRNAispre-annealed with the ptsGmRNAin vitro, the Hfq protein isno longer

required (Maki et al., 2008). However, in vivo in E.coli, sRNAsno longerregulate their

target mRNAsin hfqmutant strains, and all trans-encoded base pairing sRNAsexamined to

date co-immunoprecipitate with Hfq. In fact, enrichment ofsRNAsby co-

immunoprecipitation with Hfq proved to be a fruitful approach to identify and validate novel

sRNAsin E.coli(Zhang et al., 2003)and hasbeen extended to otherbacteria, such asS.

typhim

ur

ium

(Sittka et al., 2008).Beyond facilitating base pairing, Hfq contributesto sRNAregulation through modulating

sRNAlevels(reviewed in (Aiba, 2007; Brennan and Link, 2007; Valentin-Hansen et al.,

2004)). Somewhat counterintuitively, most E.colisRNAsare lessstable in the absence of

Hfq, presumably because Hfq protectssRNAsfrom degradation in the absence ofbase

pairing with mRNAs. Once base paired with target mRNAs, many ofthe known sRNA-

mRNApairsare subject to degradation by RNase E, and Hfq may also serve to recruit RNA

degradation machinery through itsinteractionswith RNase E and othercomponentsofthe

degradosome. In addition, competition between sRNAsforbinding to Hfq may be a factor

controlling sRNAactivity in vivo.

Although all characterized E.colitrans-encoded sRNAsrequire Hfq forregulation oftheir

targets, the need foran RNAchaperone may not be universal. Forexample, VrrARNArepression ofOmpAprotein expression in V.choleraeisnot eliminated in hfqmutant cells,

though the extent ofrepression ishigherin cellsexpressing Hfq (Song et al., 2008). In

general, longerstretchesofbase pairing, asisthe case forthe cis-encoded antisense sRNAs

that usually do not require Hfq forfunction, and/orhigh concentrationsofthe sRNAmay

obviate a chaperone requirement.

In contrast to cis-encoded sRNAs, several ofwhich are expressed constitutively, most ofthe

trans-encoded sRNAsare synthesized undervery specific growth conditions. In E.colifor

example, these regulatory RNAsare induced by lowiron (Fur-repressed RyhB), oxidative

stress(OxyR-activated OxyS), outermembrane stress(E-induced MicAand RybB),

elevated glycine (GcvA-induced GcvB), changesin glucose concentration (CRP-repressed

Spot42 and CRP-activatedCyaR), and elevated glucose-phosphate levels(SgrR-activated

SgrS)((De Lay and Gottesman, 2008; Johansen et al., 2008; Urbanowski et al., 2000)andreviewed in (Grke and Vogel, 2008; Gottesman, 2005)). In fact, it ispossible that every

majortranscription factorin E.colimay control the expression ofone ormore sRNA

regulators. It isalso noteworthy that a numberofthe sRNAsare encoded adjacent to the

gene encoding theirtranscription regulator, including E.coliOxyR-OxyS, GcvA-GcvB, and

SgrR-SgrS.

The fact that a given base pairing sRNAoften regulatesmultiple targetsmeansthat a single

sRNAcan globally modulate a particularphysiological response, in much the same manner

asa transcription factor, but at the post-transcriptional level (reviewed in (Bejerano-Sagie

and Xavier, 2007; Mass et al., 2007; Valentin-Hansen et al., 2007)). Well-characterized

regulatory effectsofthese sRNAsinclude the down regulation ofiron-sulfurcluster

containing enzymesunderconditionsoflowiron (E.coliRyhB), repression ofouter

membrane porin proteinsunderconditionsofmembrane stress(E.coliMicAand RybB),and repression ofquorum sensing at lowcell density (VibrioQrr). The fact that direct or

indirect negative feedback regulation isobserved fora numberofsRNAsemphasizesthat

sRNAsare integrated into regulatory circuits. In E.coliforexample, ryhBisrepressed when

iron isreleased afterRyhB down-regulatesiron-sulfurenzymes(Mass et al., 2005), and

micAand rybBare repressed when membrane stressisrelieved upon theirdown-regulation

ofoutermembrane porins(Johansen et al., 2006; Thompson et al., 2007). Asanother


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example, the QrrsRNAsin Vibriobase pairwith and inhibit expression ofthe mRNAs

encoding the transcription factorsresponsible forthe activation ofthe qrrgenes

(Svenningsen et al., 2008; Tu et al., 2008).

CRISPRRNAs

Aunique classofrecently discovered regulatory RNAsisthe CRISPR RNAs, which

provide resistance to bacteriophage (reviewed in (Sorek et al., 2008))and prevent plasmid

conjugation (Marraffini and Sontheimer, 2008). CRISPR systemsshare certain similaritieswith eukaryotic siRNA-driven gene silencing, although they exhibit distinct featuresaswell,

and present an exciting newarena ofRNAresearch. The CRISPR sequenceshave been

found in ~40%ofbacteria and ~90%ofarchaea sequenced to date (Sorek et al., 2008),

emphasizing theirwide-ranging importance.

CRISPR sequences(Clustered Regularly Interspaced Short Palindromic Repeats)are highly

variable DNAregionswhich consist ofa ~550 bp leadersequence followed by a seriesof

repeat-spacerunits(Figure 3)(reviewed in (Sorek et al., 2008)). The repeated DNAcan vary

from 24 to 47 base pairs, but the same repeat sequence usually appearsin each unit in a

given CRISPR array, and isrepeated two to 249 times. The repeat sequencesdiverge

significantly between bacteria, but can be grouped into 12 majortypesand often contain a

short 57 base pairpalindrome. Unlike otherrepeated sequencesin bacterial chromosomes,

the CRISPR repeatsare regularly interspersed with unique spacersof26 to 72 base pairs;these spacersare not typically repeated in a given CRISPR array. Although the repeatscan

be similarbetween species, the spacersbetween the repeatsare not conserved at all, often

varying even between strains, and are most often found to be homologousto DNAfrom

phagesand plasmids, an observation that wasinitially perplexing.

Adjacent to the CRISPR DNAarray are several CRISPR-associated (CAS)genes(reviewed

in (Sorek et al., 2008)). Two to six core CASgenesseem to be associated with most

CRISPR systems, but different CRISPR subtypesalso have specific CASgenesencoded in

the flanking region. OtherCASgenes, that are neverpresent in strainslacking the repeats,

may be found in genomic locationsdistant from the CRISPR region(s). The molecular

functionsofthe CASproteinsare still mostly obscure, but they often contain RNA-or

DNA-binding domains, helicase motifs, and endo-orexonuclease domains.

Afterthe initial report ofCRISPR sequencesin 1989, several different hypotheseswere

advanced asto possible functionsofthese repeats(reviewed in (Sorek et al., 2008)). The

proposal that CRISPRsconferresistance to phagescame in 2005 with findingsthat the

spacersoften contain homology to phage orplasmids. Anothermajoradvance wasthe

discovery that the CRISPR DNAarraysare transcribed in bacteria (Brounset al., 2008)and

archaea (Tang et al., 2002; Tang et al., 2005). The full-length CRISPR RNAinitially

extendsthe length ofthe entire array, but issubsequently processed into shorterfragments

the size ofa single repeat-spacerunit. Recently, it wasshown that the E.coliK12 CasA-E

proteinsassociate to form a complex termed Cascade, forCRISPR-Associated Complex for

Antiviral Defense (Brounset al., 2008). The CasE protein within the Cascade complex is

responsible forprocessing ofthe full-length CRISPR RNAtranscript.

Importantly, it wasdemonstrated that newspacerscorresponding to phage sequencesareintegrated into existing CRISPR arraysduring phage infection and that these newspacers

conferresistance to subsequent infectionswith the cognate phage, orotherphage bearing the

same sequence (Barrangou et al., 2007). The newspacersare inserted at the beginning ofthe

array, such that the 5end ofthe CRISPR region ishypervariable between strainsand

conveysinformation about the most recent phage infections, while the 3end spacersare

consequencesofmore ancient infections. Single nucleotide point mutationsin the bacterial


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spacersorthe phage genome abolish phage resistance and, further, introduction ofnovel

phage sequencesasspacersin engineered CRISPR arraysprovidesde novo immunity to

bacteria that have neverencountered thisphage. Similarobservationswere recently made

forspacersfound to correspond to sequencespresent on conjugative plasmids(Marraffini

and Sontheimer, 2008).

These findings, togetherwith the observation that some CASgenesencode proteinswith

function

spotentially analagou

sto euka

ryotic R

NAi enzyme

s(M

akarova et al., 2006

), have

led to a model forCRISPR RNAfunction (Figure 3). The CRISPR DNAarray istranscribed

into a long RNA, which isprocessed by the Cascade complex ofCASproteinsinto a single

repeat-spacerunit known asa crRNA(Brounset al., 2008). The crRNAs, which are single-

stranded unlike double-stranded siRNAs, are retained in the Cascade complex (Brounset al.,

2008). By analogy with eukaryotic RNAi systems, Cascade orotherCASeffectorproteins

may then direct base pairing ofthe crRNAspacersequence with phage orplasmid nucleic

acid targets. Until recently, it wasnot known whetherthe crRNAswould target DNAor

RNA, but CRISPR spacersgenerated from both strandsofphage genescan effectively

conferphage resistance (Barrangou et al., 2007; Brounset al., 2008). In addition, the

insertion ofan intron into the target gene DNAin a conjugative plasmid abolishes

interference by crRNAs, even though the uninterrupted target sequence isregenerated in the

spliced mRNA(Marraffini and Sontheimer, 2008). These resultsall point to DNAasthe

direct target, but howthe crRNAsinteract with the DNAand what occurssubsequently arestill unknown. Furtherstudiesaddressing the detailsofthe molecularmechanism behind

CRISPR RNA-mediated silencing offoreign DNAand hownewspacersare selected and

then acquired are eagerly anticipated and will provide furtherinsight into the similaritiesand

differenceswith the eukaryotic RNAi machinery.

The CRISPR system hasbroad evolutionary implications. The extreme variability of

CRISPR arraysbetween organismsand even strainsofthe same speciesprovidesuseful

toolsforresearchersto genotype strainsand to study horizontal gene transferand micro-

evolution (reviewed in (Sorek et al., 2008)). The CRISPR loci record the history ofrecent

phage infection and allowdifferentiation between strainsofthe same species. Thisproperty

can be used to identify pathogenic bacterial strainsand track disease progression world-

wide, aswell asto monitorthe population dynamicsofnon-pathogenic bacteria (Horvath et

al., 2008). Additionally, the presence ofphage sequenceswithin the CRISPR arraysthatconferresistance against infection provide a strong selective pressure forthe mutation of

phage genomesand may partially underlie the rapid phage mutation rate (Andersson and

Banfield, 2008).

DualFunctionRNAs

The distinctionsbetween some ofthe categoriesofRNAregulatorsdiscussed above aswell

asbetween the RNAregulatorsand otherRNAscan be blurry. Forexample, a fewofthe

trans-encoded base pairingsRNAsencode proteinsin addition to base pairing with target

mRNAs. The S.aureusRNAIIIhasbeen shown to base pairwith mRNAsencoding

virulence factorsand a transcription factor(Boisset et al., 2007), but also encodesa 26

amino acid -hemolysin peptide. Similarly, the E.coliSgrSRNA, which blockstranslation

ofthe ptsGmRNAencoding a sugar-phosphate transporter, istranslated to produce the 43

amino acid SgrT protein (Wadlerand Vanderpool, 2007). In thiscase, the SgrT protein is

thought to reinforce the regulation exerted by SgrSby independently down-regulating

glucose uptake through direct orindirect inhibition ofthe PtsGprotein. We predict that other

regulatory sRNAswill be found to encode small proteinsand that conversely some mRNAs

encoding small proteinswill be found to have additional rolesassRNAregulators. It also

deservesmention that some ofthe cis-encoded antisense sRNAs, in addition to regulating

theircognate sense mRNA, may base pairwith othermRNAsvia limited complementarity


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or, in independent roles, bind proteinsto affect otherfunctions. Similarly, while

riboswitchesare synthesized aspart ofan mRNA, the small transcriptsthat are generated by

transcription attenuation orautocleavage potentially could go on to perform otherfunctions

astheirown entities.

FactorsInfluencingRegulationbyRNAs

While there hasbeen a great explosion in the discovery and characterization ofRNA

regulatorsin the past ten years, a numberofcritical questionsabout theirregulatory

mechanismsremain to be answered.

RNAStructures,Levels,andLocalization

What are the structuresofthe RNAsand howdo they impact ligand, protein and mRNA

binding? Three-dimensional structuresforseveral riboswitches, both in the presence and

absence oftheirrespective ligands, have been solved in recent years(Montange and Batey,

2008). These studieshave shown that some riboswitcheshave a single, localized ligand-

binding pocket. In these cases, the conformational changesinduced by ligand binding are

confined to a small region.In otherriboswitches, the ligand-binding site iscomprised ofat

least two distinct sites, such that ligand binding resultsin more substantial changesin the

global tertiary structure. In contrast, no three-dimensional structureshave been solved for

bacterial sRNAs. In fact, the secondary structuresforonly a limited numberofsRNAshavebeen probed experimentally. Anothergenerally unknown quantity, which hasimportant

implicationsforhowan RNAinteractswith othermolecules, isthe concentration ofthe

RNA. Afterinduction, the OxySRNAhasbeen estimated to be present at 4,500 molecules

percell (Altuvia et al., 1997), but it isnot known whetherthisistypical forothersRNAsand

whetherall ofthe sRNAmoleculesare active. Do nucleotide modificationsormetabolite

binding alterthe abundance oractivitiesofany ofthe sRNAs? It isalso intriguing to ask

whetherany ofthe regulatory RNAsshowspecific subcellularlocalization orare even

secreted. In eukaryotes, localization ofregulatory RNAsto specific subcellularstructures,

such asPbodiesand Cajal bodies, isconnected to theirfunctions(reviewed in (Pontesand

Pikaard, 2008)). It isplausible that subcellularlocalization similarly impactsregulatory

RNAfunction in bacteria. In support ofthisidea, RNase E hasbeen found to bind

membranesin vitro (Khemici et al., 2008), and membrane targeting ofthe ptsGmRNA-

encoded protein isrequired forefficient SgrSsRNArepression ofthistranscript (Kawamotoet al., 2005). Anotherattractive, but untested, hypothesisisthat bacterial RNAsmight be

secreted into a host cell where they could modulate eukaryotic cell functions.

ProteinsInvolved

What proteinsare associated with regulatory RNAsand howdo the proteinsimpact the

actionsofthe RNAs? So farmuch ofthe attention hasbeen focused on the RNAchaperone

Hfq. Even so, the detailsofhowthisprotein bindsto sRNAsand impactstheirfunctionsare

murky. Forexample, structural and mutational studiesindicate that both facesofthe donut-

like Hfq hexamercan make contactswith RNA(reviewed in (Aiba, 2007; Brennan and

Link, 2007)), but it isnot clearwhetherthe sRNAand mRNAbind both faces

simultaneously, whetherthe sRNAand mRNAbind particularfaces, and whetherbase

pairing isfacilitated by changesin RNAstructure orproximity between the two RNAsorboth. The Hfq protein hasbeen shown to copurify with the ribosomal protein S1,

componentsofthe RNase E degradosome, and polynucleotide phosphorylase (Mohanty et

al., 2004; Morita et al., 2005; Sukhodoletsand Garges, 2003), among others, but these are

all abundant RNA-binding proteinsand the in vivo relevance ofthese interactionsispoorly

understood. In addition, only halfofall sequenced Gram-negative and Gram-positive

speciesand one archaeon have Hfq homologs(reviewed in (Valentin-Hansen et al., 2004)).


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Do otherproteinssubstitute forHfq in the organismsthat do not have homologs, ordoes

base pairing between sRNAsand theirtarget mRNAsnot require an RNAchaperone in

these cases?

It islikely still otherproteinsthat act on orin conjuction with the regulatory RNAsremain

to be discovered. The RNase E and RNase IIIendonucleasesare known to cleave base

pairing sRNAsand theirtargets(Viegaset al., 2007), but these may not be the only

ribonuclea

sesto deg

rade the R

NAs.P

ull-do

wn expe

riment

sw

ith taggedsRNAs

indicate that

otherproteins, such asRNApolymerase (Windbichleret al., 2008), also bind the RNA

regulators, but again the physiological relevance ofthisinteraction isnot known. In

addition, genetic studieshint at the involvement ofproteinssuch asYhbJ, which antagonizes

GlmYand GlmZ activity, though the activity ofthisprotein isstill mysterious(Kalamorz et

al., 2007; Urban and Vogel, 2008).

RequirementsforProductiveBasePairing

What are the rulesforproductive base pairing? Trans-encoded sRNAsbind to theirtarget

mRNAsusing discontiguousand imperfect base pairing, ofwhich often only a core set of

interactionsisessential, stimulating questionsasto howspecificity between sRNAsand

mRNAsisimparted and howsuch limited pairing can cause translation inhibition orRNA

degradation. Several algorithmsforthe predictionsofbase pairing targetsfortrans-encoded

sRNAshave been developed ((Tjaden, 2008)and reviewed in (Pichon and Felden, 2008;Vogel and Wagner, 2007)). However, the accuracy ofthese predictionshasbeen varied. For

some sRNAs, such asRyhB and GcvB, there are distinct conserved single-stranded regions,

which appearto be required forbase pairing with most targetsand are associated with more

accurate predictions(Sharma et al., 2007; Tjaden et al., 2006). ForothersRNAssuch as

OmrAand OmrB, fewknown targetswere predicted in initial searches(Tjaden et al., 2006).

Mutational studiesto define the base pairing interactionswith known OmrAand OmrB

targets(Guillierand Gottesman, 2008)highlight possible impedimentsto computational

predictions. These can include the lack ofknowledge about the sRNAdomainsrequired for

base pairing, limited base pairing interactions, and base pairing to mRNAregionsoutside

the immediate vicinity ofthe ribosome binding site. Recent systematic analysisindicates

sRNAscan block translation by pairing with sequencesin the coding region, asfar

downstream asthe fifth codon (Bouvieret al., 2008). Otherfactorssuch asthe position of

Hfq binding and the secondary structuresofboth the mRNAand sRNAare also likely to

impact base pairing in waysthat have not been formalized. In vitro studiesexploring the role

ofHfq in facilitating the pairing between the RprAand DsrARNAsand the rpoSmRNA

showthat binding betweenHfq, the mRNAand the sRNAsisclearly influenced by what

portion ofthe rpoS5leaderisassayed (Soperand Woodson, 2008; Updegrove et al., 2008).

With an increasing numberofvalidated targetsthat can serve astraining sets, the ability to

accurately predict targetsshould significantly improve.

Aswith eukaryotic miRNAsand siRNAs, there may be mechanistic differencesbetween the

trans-and cis-encoded base pairing sRNAsbased on theirdifferent properties. Trans-

encoded sRNAs, which have imperfect base pairing with theirtargetslike miRNAs, often

interact with Hfq. In contrast, cis-encoded sRNAs, which have complete complementarity

with targetslike siRNAs, do not appearto require Hfq, but tend to be more structured and

may use otherfactorsto aidin base pairing. These differencesmay have broader

implicationsforthe typesoftargetsregulated, the nature ofthe proteinsrequired, aswell as

the mechanistic detailsofbase pairing.


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NewMechanismsofAction

What novel mechanismsofaction remain to be uncovered? Most sRNAscharacterized to

date base pairin the 5UTR oftarget mRNAsnearthe ribosome binding site, howeverother

locationsforbase pairing and consequent mechanismsofregulation are possible. Only a few

bacterial ribozymeshave been described. Will othersRNAsorriboswitchesbe found to

have enzymatic activity? Asalready alluded to, the mechanism ofcrRNAaction in targeting

and interfering with DNAisnot understood. Completely novel mechanismsmay be revealed

by furtherstudiesofthe CRISPR sequences. Finally, nearly a third ofthe E.colisRNAsidentified to date, and the vast majority ofthose in otherorganisms, have yet to be

characterized in significant detail. These too may have unanticipated rolesand modesof

action.

PhysiologicalRolesofRegulatoryRNAs

In addition to furtherexploring the mechanismsby which riboswitches, sRNAsand crRNAs

act, it isworth reflecting on what isknown, aswell aswhat isnot understood, about the

physiological rolesofthese regulators.

AssociationwithSpecificResponses

Anumberofthemesare emerging with respect to the physiological rolesofriboswitches

and sRNAs. In general terms, riboswitches, protein binding sRNAs, trans-encoded base

pairing sRNAsand some cis-encoding base pairing sRNAsmediate responsesto changing

environmental conditionsby modulating metabolic pathwaysorstressresponses.

Riboswitchesand T-boxestend to regulate biosynthetic genes, asthese elementsdirectly

sense the concentrationsofvariousmetabolites, while some RNAthermometers, such asthe

5-UTR ofthe mRNAencoding the heat shock sigma factor32 (Morita et al., 1999),

control transcriptional regulators. The CsrB and 6SfamiliesofsRNAsalso control the

expression oflarge numbersofgenesin response to decreasesin nutrient availability by

repressing the activitiesofglobal regulators. The trans-encoded base pairing sRNAsmostly

contribute to the ability to survive variousenvironmental insultsby modulating the

translation ofregulatorsorrepressing the synthesisofunneeded proteins. In particular, it is

intriguing that a disproportionate numberoftrans-encoded sRNAsregulate outermembrane

proteins(MicA, MicC, MicF, RybB, CyaR, OmrAand OmrB)ortransporters(SgrS, RydC,

GcvB). Otherpervasive themesinclude RNA-mediated regulation ofiron metabolism, not

only in bacteria but also in eukaryotes, aswell asRNAregulatorsofquorum sensing.

Pathogenesispresentsa set ofbehaviorsone might expect to be regulated by sRNAssince

bacterial infectionsinvolve multiple roundsofrapid and coordinated responsesto changing

conditions. The central role ofsRNAsin modulating the levelsofoutermembrane proteins,

which are key targetsforthe immune system, aswell asotherresponsesimportant for

survival underconditionsfound in host cells, such asaltered iron levels, also implicates

these RNAregulatorsin bacterial survival in host cells. Indeed, although these studiesare

still at the early stages, several sRNAshave been shown to alterinfection. These include

membersofthe CsrB family ofsRNAsin Salmonella, Erwinia, Yersinia, Vibrioand

Pseudomonadswhich bind to and antagonize CsrAfamily proteinsthat are global regulators

ofvirulence genes; RyhB ofShigellawhich repressesa transcriptional activatorofvirulencegenes; RNAIIIofStaphylococcuswhich both base pairswith mRNAsencoding virulence

factorsand encodesthe -hemolysin peptide; and the QrrsRNAsofVibriowhich regulate

quorum sensing ((Heroven et al., 2008; Murphy and Payne, 2007)and reviewed in (Romby

et al., 2006; Toledo-Arana et al., 2007)). hfqmutantsofa wide range ofbacteria also show

reduced virulence (reviewed in (Romby et al., 2006; Toledo-Arana et al., 2007)). Some

sRNAs, such asa numberofsRNAsencoded in Salmonellaand Staphylococcus


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pathogenicity islands, showdifferential expression underpathogenic conditions(Padalon-

Brauch et al., 2008; Pfeifferet al., 2007; Pichon and Felden, 2005). OthersRNAs, such as

five in Listeriamonocytogenes, are specific to pathogenic strains(Mandin et al., 2007).

Finally, thermosensorsandriboswitchescan have rolesin asregulatorsofpathogenesis,

upregulating virulence genesupon increased temperature encountered in host cellsorupon

binding signalssuch asthe second messenger cyclic di-GMP(Johansson et al., 2002;

Sudarsan et al., 2008). Furtherstudiesofthese and otherpathogenesis-associated regulatory

RNAscould lead to opportunitiesforinterfering with disease.Asubset ofthe cis-encoded antisense sRNAsexpressed from bacterial chromosomesact as

antitoxinsbut theirphysiological rolesare not clear. They may also be involved in altering

cell metabolism in response to variousstressesenabling survival. Alternatively, they may

play a role in protecting against foreign DNA. Thisisclearly the function ofCRISPR RNAs,

which have been demonstrated to repressbacteriophage and plasmid entry into the cell, and

in principle could be used to silence genesfrom othermobile elements.

PhysiologicalRolesofMultipleCopies

Some sRNAsincluding OmrA/OmrB, Prr1/Prr2, Qrr15, 6Shomologs, CsrB homologs,

GlmY/GlmZ, and several toxin-antitoxin modulesare present in multiple copiesin a given

bacterium. Although the physiological advantagesofthe repeated sRNAgenesare only

understood in a subset ofcases, multiple copiescan have several different roles(Figure 4).

Firstly, homologousRNAscan act redundantly, serving asback upsin critical pathwaysor

to increase the sensitivity ofa response. In V.cholerae, any single QrrRNAissufficient to

repressquorum sensing bydown regulating the HapR transcription factor, and the deletion

ofall fourqrrgenesisrequired to constitutively activate the quorum sensing behaviors

(Lenz et al., 2004). Since the effectivenessofsRNAregulation isdirectly related to their

abundance relative to mRNAtargets, thisredundancy hasbeen proposed to permit an

ultrasensitive, switch-like response forquorum sensing in V.choleraeand may help amplify

a small input signal to achieve a large output.

Secondly, repeated RNAscan act additively, asin the case ofthe V.harveyiQrrsRNAs(Tu

and Bassler, 2007). In thiscase, the five qrrgeneshave divergent promoterregionsand are

differentially expressed, suggesting each QrrsRNAmay respond to different metabolicindicatorsto integrate variousenvironmental signals. Deletion ofindividual Qrrgenes

affectsthe extent ofquorum sensing behaviors, indicating they do not act redundantly.

Rather, the total amount ofQrrsRNAsin V.harveyiproducesdistinct levelsofregulated

genes, such that altering the abundance ofany given QrrsRNAchangesthe extent ofthe

response. Thisadditive regulation isthought to allowfine-tuning ofluxRlevelsacrossa

gradient ofexpression, leading to precise, tailored amountsofgene expression. It is

surprising that within the same quorum sensing system in two related speciesofVibrio, the

multiple QrrsRNAsoperate according to two distinct mechanisms. While the reason forthis

isnot clear, the difference illustratesthe evolvability ofRNAregulatorsand the regulatory

nuancesthat can be provided by having multiple copies.

Athird possibility isthat the duplicated RNAscan act independently ofeach other. This

could occurin several ways. Forbase pairing sRNAs, each sRNAcould regulate a differentset ofgenes, most likely in a somewhat overlapping manner. Forprotein-binding sRNAs,

different homologscould interact with distinct proteins, giving rise to variationsin the core

complexes. Asmentioned above, variousB.subtilis6Sisoformscould repressRNA

polymerase bound to different factors. HomologousRNAspeciesalso can employ very

different mechanismsofaction, asobserved forthe E.coliGlmYand GlmZ RNAs(Urban


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and Vogel, 2008). GlmZ functionsby base pairing, while GlmYlikely actsasa mimic to

titrate away YhbJand otherfactorsthat inactive GlmZ.

In some casesit isstill perplexing why multiple copiesare maintained. One example isthe

toxin-antitoxin modules, which are not only encoded by multiple genesin E.coli

chromosomes, but which can vary in gene numbereven within the same species(reviewed

in (Fozo et al., 2008a)). Redundant RNAsmay simply indicate a recent evolutionary event,

which ha

snot yet unde

rgone va

riation to

select ne

wfunction

s.A

lternatively, additional

genesmay be selected by the pressure to maintain at least one copy acrossa population.

Complete answersto the question ofwhy variousregulatory RNAgenesare duplicated

await more characterization ofeach set ofRNAs.

AdvantagesofRegulatoryRNAs

RNAregulatorshave several advantagesoverprotein regulators. They are lesscostly to the

cell and can be fasterto produce, since they are shorterthan most mRNAs(~100200

nucleotidescompared to 1,000 nucleotidesforthe average ~350 amino acid E.coliprotein)

and do not require the extra step oftranslation.

The effectsofthe RNAregulatorsthemselvesalso can be very fast. Forcis-acting

riboswitches, the coupling ofa sensordirectly to an mRNAallowsa cell to respond to the

signal in an extremely rapid and sensitive manner. Similarly, since sRNAsare fastertoproduce than proteinsand act post-transcriptionally, it wasanticipated that, in the short term,

they could shut offorturn on expression more rapidly than protein-based transcription

factors. Indeed thisexpectation issupported by some dynamic simulations(Mehta et al.,

2008; Shimoni et al., 2007). Otherunique aspectsofsRNAregulation revealed by recent

modeling studiesare related to the threshold linearresponse provided by sRNAs, in contrast

to the straight linearresponse provided by transcription factors(Legewie et al., 2008; Levine

et al., 2007; Mehta et al., 2008). Most sRNAscharacterized thusfaract stoichiometrically

through the noncatalytic mechanismsofmRNAdegradation orcompetitive inhibition of

translation, reactionsin which the relative concentrationsofthe sRNAand mRNAare

critical. Thusfornegatively-acting sRNAs, when [sRNA][mRNA], gene expression is

tightly shut off, but when [mRNA][sRNA], the sRNAhaslittle effect on expression.

Thisthreshold property ofsRNArepression suggeststhat sRNAsare not generally as

effective asproteinsat transducing small ortransient input signals. In contrast, when input

signalsare large and persistent, sRNAsare hypothesized to be betterthan transcription

factorsat strongly and reliably repressing proteinslevels, aswell asat filtering noise.

Moreover, sRNA-based regulation isthought to be ultra-sensitive to changesin sRNAand

mRNAlevelsaround the critical threshold, especially in the case ofmultiple, redundant

sRNAsasin the V.choleraeQrrquorum sensing system, which isproposed to lead to

switch-like all ornothing behavior(Lenz et al., 2004).

Additional featuresofdifferent subsetsofthe RNAregulatorsprovide otheradvantages.

Some riboswitcheslead to transcription termination orself-cleavage and some base pairing

sRNAsdirect the cleavage oftheirtargets, rendering theirregulatory effectsirreversible. For

the cis-encoded antisense sRNAsand the CRISPR RNAs, the extensive complementarity

with the target nucleic acidsimpartsextremely high specificity. In contrast, the ability of

trans-encoded sRNAsto regulate many different genesallowsthese sRNAsto control entire

physiological networkswith varying degreesofstringency and outcomes. The extent and

quality ofbase pairing withsRNAscan prioritize target mRNAsfordifferential regulation

and could be used by cellsto integrate different statesinto gene expression programs

(Mitarai et al., 2007). In addition, when multiple target mRNAsofa given sRNAare

expressed in a cell, theirrelative abundance and binding affinitiescan strongly influence

expression ofeach otherthrough cross-talk (Levine et al., 2007; Mehta et al., 2008; Shimoni


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et al., 2007). Conversely, competition between different sRNAsforHfq ora specific mRNA

islikely to alterdynamicswithin a regulatory network. Finally, base pairing flexibility

presumably also allowsrapid evolution ofsRNAsand mRNAtargets.

Moreover, while not an advantage perse, RNAregulatorsusually act at a level

complementary to protein regulators, most often functioning at the post-transcriptional level

asopposed to transcriptionfactorsthat act before sRNAsorenzymessuch askinasesor

protea

sesthat act a

fte

rsRNAs

.D

iff

erent combination

so

fthe

se p

rotein and R

NAregulato

rs

can provide a variety ofregulatory outcomes, such asextremely tight repression, an

expansion in the genesregulated in response to a single signal orconversely an increase in

the numberofsignalssensed by a given gene (Shimoni et al., 2007).

EvolutionofRegulatoryRNAs

We do not yet knowwhetherall bacteria contain regulatory RNAsorwhetherwe are

coming close to having identified all sRNAsand riboswitchesin well-studied bacteria.

Given the redundancy in the sRNAsbeing found, the searchesforcertain classesofsRNAs,

in particularsRNAsencoded in intergenic regionsand expressed undertypical laboratory

conditions, appearnearsaturation in E.coli. However, othertypesofsRNAs, such ascis-

encoded antisense sRNAsand sRNAswhose expression istightly regulated, may still be

missing from the listsofidentified RNAregulators.

Are RNAregulatorsremnantsofthe RNAworld orare the genesrecent additionsto

bacterial genomes? We propose that the answerto thisquestion isboth. Some ofthe

regulatorssuch asriboswitchesand CRISPR systems, which are very broadly conserved, are

likely to have ancient evolutionary origins. In contrast, while regulation by base pairing may

long have been in existence, individual antisense regulators, both cis-and trans-encoded

sRNAsmay be recently acquired and rapidly evolving. Thisisexemplified by the poor

conservation ofsRNAsequencesacrossbacteria. Forexample, the PrrRNAsof

Pseudomonasbearalmost no resemblance to the equivalent RyhB sRNAofE.colialthough

both are repressed by Furand act on similartargets(Wilderman et al., 2004). One might

imagine that the expression ofa spurioustranscript, eitherantisense orwith limited

complementarity to a bona fide mRNA, which providessome selective advantage could

easily be

fixed in a population.

It isintriguing to note that distinct RNAregulatorshave been used to solve specific

regulatory problems, emphasizing the pervasivenessand adaptability ofRNA-mediated

regulation. Forexample, in B.subtilis, the glmSmRNAisinactivated by the self-cleavage of

the glucosamine-6-phosphate-responsive cis-acting riboswitch (Collinset al., 2007),

whereasin E.coli, the glmSmRNAispositively regulated by the two trans-acting sRNAs

GlmYand GlmZ (Urban and Vogel, 2008). Asanotherexample, RyhB-like trans-encoded

sRNAsrepressthe expression ofiron-containing enzymesduring iron starvation in various

bacteria, while the cis-encoded IsiR sRNAofSynechocystisrepressesexpression ofthe IsiA

protein, a light harvesting antenna, underiron replete conditions(reviewed in (Mass et al.,

2007)).

ApplicationsofRegulatoryRNAsThe central rolesplayed by RNAregulatorsin cellularphysiology make them attractive for

use astoolsto serve asbiosensorsorto control bacterial growth eitherpositively or

negatively. EndogenousRNAscould serve assignalsofthe environmental statusofthe cell.

Forexample, the levelsofthe RyhB and OxySsRNAs, respectively, are powerful indicators

ofthe iron statusand hydrogen peroxide concentration in a cell (Altuvia et al., 1997; Mass

and Gottesman, 2002). CRISPR sequencesprovide insightsinto the history ofthe


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extracellularDNAencountered by the bacteria and have been used to genotype strains

during infectiousdisease outbreaks(reviewed in (Hebert et al., 2008; Sorek et al., 2008)).

Regarding the control ofbacterial cell growth, one can imagine howriboswitchesmight be

exploited asdrug targetsgiven theirpotential to bind a wide variety ofcompounds

(reviewed in (Blount and Breaker, 2006)). Similarly, since interference with the functionsof

some ofthe sRNAsisdetrimental to growth and several sRNAscontribute to virulence,

these regulatorsand theirinteracting proteinsalso could be targeted by antibacterial

therapies. Alternatively, ectopic expression ofspecific regulatory RNAsmight be used toincrease stressresistance and facilitate bacterial survival in variousindustrial orecological

settings.

RNAalso presentsa powerful system forrational design asit ismodular, easily synthesized

and manipulated, and can attain an enormousdiversity ofsequence, structure, and function.

Although lessdeveloped than in eukaryotes, the application ofsynthetic RNAsisbeing

explored in bacteria (reviewed in (Hebert et al., 2008; Isaacset al., 2006)). Forexample,

riboswitch elementshave been engineered to use novel ligands, and sRNAshave been

designed to base pairwith novel transcripts. Engineered CRISPR repeatspresent an obvious

mechanism by which to repressuptake ofspecific DNAsequences. Limitationsto these

approachesinclude incomplete repression observed forthe synthetic riboswitchesand base

pairing sRNAsthusfar, offtarget effects, aswell asproblemsin delivering the RNA

regulatorsinto cellswhere they might be ofgreatest utility. Nevertheless, synthetic RNAshave potential to provide a variety ofuseful toolsand therapeuticsin the future.

Perspectives

In summary, RNAmoleculesserve a wide range ofregulatory functionsin bacteria and

modulate almost every aspect ofcell metabolism. Examplesofthese RNAregulatorswere

known long before the discovery ofsimilarregulatorsin eukaryotes, though the large

numbersofriboswitches, sRNAs, and CRISPR RNAs, aswell astheircorrespondingly large

importance to cellularphysiology and defense mechanisms, were not anticipated. Many

bacteria are facile experimental systemsand have small genomes, which aid computational

predictionsand robust model development. In addition, hundredsofbacterial genome

sequences, representing a broad diversity ofspecieswith a variety oflifestylesand

ecological niches, are available. These factorsmake bacteria an ideal system in which todelve deeply into mechanistic, physiological and evolutionary questionsregarding

regulatory RNAs.

Acknowledgments

We regret that we were only able to cite a subset ofthe most recent publicationsdue to the broad scope ofthe

review. Readersare referred to numerousreviewsformore in depth coverage ofspecific topics. We appreciate the

commentsfrom so many ofourcolleagues. Supported by the Intramural Research Program ofNICHDand a

fellowship from the PRAT program ofNIGMS(L.S.W.).

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