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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: storz@helix.nih.gov.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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)).
Cell. Authormanuscript; available in PMC 2011 July 10.
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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.
Cell. Authormanuscript; available in PMC 2011 July 10.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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
Cell. Authormanuscript; available in PMC 2011 July 10.
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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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