Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification

12 Pages·2000·0.73 MB·English

RNA(2000),6:668–679+CambridgeUniversityPress+PrintedintheUSA+
Copyright©2000RNASociety+
Probing the structure of RNAIII, the
Staphylococcusaureusagrregulatory RNA,
and identification of the RNA domain involved
in repression of protein A expression
YVONNE BENITO,1 FABRICEA. KOLB,2 PASCALE ROMBY,2 GERARD LINA,1
JEROME ETIENNE,1 and FRANÇOIS VANDENESCH1
1EA1655,FacultédeMédecineLaennec,69372LyonCedex08,France
2UPR9002duCentreNationaldelaRechercheScientifique,InstitutdeBiologieMoléculaireetCellulaire,
67084StrasbourgCedex,France
ABSTRACT
RNAIII,a514-ntRNAmolecule,regulatestheexpressionofmanyStaphylococcusaureusgenesencodingexoproteins
andcell-wall-associatedproteins.WehavestudiedthestructureofRNAIIIinsolution,usingacombinationofchem-
icalandenzymaticprobes.Amodelofthesecondarystructurewasderivedfromexperimentaldatawiththehelpof
computer simulation of RNAfolding. The model contains 14 hairpin structures connected by unpaired nucleotides.
Thedataalsopointtothreehelicesformedbydistantnucleotidesthatcloseoffstructuraldomains.Thismodelwas
generally compatible with the results of in vivo probing experiments with dimethylsulfate in late exponential-phase
cultures. Toe-printing experiments revealed that the ribosome binding site ofhld, which is encoded by RNAIII, was
accessibletotheEscherichiacoli30Sribosomalsubunit,suggestingthattheinvitrostructurerepresentedatrans-
latableformofRNAIII.Wealsofoundthat,withinthe39endofRNAIII,theconservedhairpin13andtheterminatorform
anintrinsicstructuraldomainthatexertsspecificregulatoryactivityonproteinAgeneexpression.
Keywords: agrlocus;chemicalandenzymaticprobing;invivoprobing;regulation;RNAIII
INTRODUCTION toxin and enterotoxins), and represses the expression
of exponential-phase surface proteins such as protein
InStaphylococcusaureus, expression of several viru-
A and coagulase+ RNAIII appears to be the specific
lence factors is regulated by the global regulon agr+
regulator,asclonedRNAIII,defectiveindelta-hemolysin
Theagrsystemiscomposedoftwodivergentoperons
production, and expressed in an RNAIII-deletion mu-
designated P2 and P3+ The P2 operon combines a
tant,restoredboththepositiveandnegativeeffectson
density-sensing cassette (agrD and B) and a two-
theexpressionofexoproteinsandsurfaceproteins(Jan-
componentsensorytransductionsystem(agrAandC),
zon &Arvidson, 1990) and in anagr-null strain (Korn-
both of which are required for autocatalytic activation
blum et al+, 1990; Novick et al+, 1993)+ RNAIII acts
ofthepromoterP2andfortranscriptionfromthediver-
primarily on target gene transcription initiation and, in
gentpromoterP3(Jietal+,1995;Linaetal+,1998)+The
thecaseofalpha-hemolysin,independentlystimulates
effector of the S.aureusagrsystem is a 514-nt tran-
translation by a mechanism that appears to involve
scriptoftheP3operon,designatedRNAIII,whichcon-
direct interaction between the 59 end of RNAIII and a
tains the 26-amino-acid delta-hemolysin gene (hld)+
stretchofapproximately80bpwithintheribosomebind-
RNAIII stimulates the expression of postexponentially
ingsiteofthehlatranscript(Morfeldtetal+,1995)+The
expressed extracellular toxins and enzymes such as
mechanism by which RNAIII controls the transcription
alpha and beta-hemolysins, enzymes (lipases, prote-
of its target genes is unknown+ One possibility is that
asesandnucleases)andtoxins(toxicshocksyndrome
RNAIII acts via trans-acting factors, as RNAIII is po-
tentially capable of forming stable secondary struc-
turesthatmaycreateproteinbindingsites(Novicketal+,
Reprint requests to: Francois Vandenesch, EA1655, Faculté de
1993)+Theimportanceofthe59and39regionsofRNAIII
MédecineLaennec,RueGuillaumeParadin,69372LyonCedex08,
France;e-mail:denesch@univ-lyon1+fr+ for its regulatory function has been tested by deletion
668
StructureofS.aureusagr-RNAIII 669
analysisandbystudyingtheabilityofRNAIIIhomologs ionspriortomodification+WealsoverifiedthattheRNAs
from non-aureus staphylococcal species to comple- foldedintoahomogenouspopulationbyusinggelelec-
ment agr-deficient strains of S.aureus(Benito et al+, trophoresis in nondenaturing conditions+ A secondary
1998;Tegmarketal+,1998)+The39endofRNAIIImay structuremodelforRNAIIIwasderivedfromtheexper-
beimportanttorepressproteinAexpression,andmay imental data with the help of a computer simulation of
therefore be an intrinsic domain (Novick et al+, 1993; RNA folding (Gultyaev et al+, 1995)+ This procedure
Benito et al+, 1998)+ As mentioned above, besides its folds the molecule by generating consecutive transi-
regulatory function, RNAIII is also an mRNA that en- tions between favorable intermediate structures, that
codes the delta-hemolysin gene+Translation of RNAIII is,bysimulatingfoldingpathwaysratherthanbysearch-
intodelta-hemolysinwasdelayedby1hfollowingtran- ingfortheglobalminimumstate(Gultyaevetal+,1995;
scription and this delay was abolished by deletion of Gerdesetal+,1997)+Thesecondarystructuremodelis
the 39 half of RNAIII, suggesting that a conformational characterized by 14 hairpin structures and three heli-
changeoftheRNAisrequiredfortranslation(Balaban ces (A–C) which involve distant nucleotides (Fig+ 2)+
& Novick, 1995)+ Secondary structure predictions indi- The model can be divided into three mains domains:
catethatthe59and39endsofRNAIIImaypair,thereby the 59 end domain (nt 1–31), a central domain delimi-
sequestering the ribosomal binding site ofhld(Novick tated by helix B (nt 32–382) and the 39 end domain
et al+, 1993)+ Taken together, these data indicate that (nt 383–514)+
thestructureofRNAIIIplaysanessentialrolenotonly The59enddomaincontainsawell-characterizedstem-
inhldtranslation but also in regulatory functions+ loopmotif+Thishairpinstructure1bearsaGNRAloop
Here, we probed the secondary structure of S.au- thatisknowntoadoptaparticularstructureclosedbya
reus RNAIII in vitro, using chemical and enzymatic shearedG-Abasepair(Heus&Pardi,1991;Pleyetal+,
probes+ The results were compared with those of in 1994)+ The poor accessibility of G9 and A12 towards
vivo probing of RNAIII produced during the late expo- chemicalsandenzymessupportstheexistenceofsuch
nential phase+The proposed model of RNAIII second- a G-A base pair (Fig+ 1A,E)+ Two long-range inter-
ary structure is characterized by 14 hairpin structures actionsinvolvingresiduesA24-G30/U452-458(helixC)
andthreelong-rangeinteractionsthatbringthe39and and U32-A42/U371-G382 (helix B) can be proposed+
59 ends of RNAIII into close proximity+ A number of Both helices are susceptible to RNase V1 hydrolysis
hairpin structures appear to be conserved within the and several nucleotides are weakly or not reactive at
Staphylococcusspecies+ One, located in the 39 end of one of their Watson–Crick positions+ However helix C
S.aureusRNAIII, efficiently repressed protein A ex- presentsaweakstabilityasevidencedbytheconcom-
pression in vivo+ The proposed model for the second- itantpresenceofseveralsingle-strand-specificRNase
ary structure of RNAIII is discussed in the light of its cleavages (Fig+ 2)+ Thus, helix C brings in close prox-
regulatory functions+ imitythe39and59domainswhereashelixBdelimitates
the central domain+
Inthislargecentraldomain,thereactivitypatternsof
RESULTS
nucleotides in hairpin 2–11 were characteristic of hair-
pin structures (Fig+ 2), as most of the nucleotides lo-
Structural probing of
catedintheloopswerereactiveattheirWatson–Crick
StaphylococcusaureusRNAIII
positions+ This was accompanied by single-strand-
WeinvestigatedtheconformationofRNAIII(nt1–514) specific RNase cleavages (Figs+ 1 and 2)+ Further-
with various chemical and enzymatic probes (Ehres- more, the stems were all cleaved by RNase V1, and
mann et al+, 1987) (Fig+ 1)+ We chose an identical pH, most of the nucleotides were weakly or not reactive
temperature, ionic strength, and divalent ion concen- towards chemicals+ This is particularly well-illustrated
trationforthedifferentprobestominimizetheeffectsof for the stem-loop motifs 3, 4, 5, and 7–11 (Fig+ 2)+ He-
theseparametersonconformationalchanges+Thefour lix2,whichisA-Urich,formsametastablehelicalstruc-
basesweretestedatoneoftheirWatson–Crickpairing ture,whichmayaccountfortheconcomitantpresence
positions by using DMS (N1A . N3C) and CMCT of RNase V1 and RNase T2 cleavages and for the
(N3U . N1G)+ Single-stranded regions were probed reactivityofseveralnucleotidesatWatson–Crickposi-
with RNases T2 and T1, and helical regions were de- tions (Fig+ 2)+ The long hairpin structure 6, character-
tectedwiththedouble-strandedspecificRNaseV1+In- izedbyastemrichinA-Ubasepairsandinterruptedby
formationonthe39and59endsofRNAIIIwasobtained an internal loop, required magnesium for stabilization:
using enzymes, DMS (for N3C), and nickel complex most of the residues within the helix became strongly
(which modifies N7G)+ Nickel complex is sensitive to reactive in the absence of magnesium (Fig+ 1B)+ The
stacking of base rings; for example, the N7 position of central domain also contained a branched structure
aguaninewithinahelixisnotreactiveunlessthedeep constitutedbyhairpins7–9andclosedbyhelixA(Fig+2)+
groove is widened (Chen et al+, 1993)+ The RNAmol- ThishelixinvolvingthetwodistantregionsG207–U216
ecules were renatured in the presence of magnesium andA310–U319 is supported by the presence of sev-
670 Y.Benitoetal.
eral RNase V1 cleavages in both strands, and by the the helix became reactive under semidenaturing con-
nonreactivity of several residues towards chemicals ditions (Fig+ 2D)+ The stem, rich in A-U base pairs, is
(Figs+ 1 and 2)+ The single-stranded conformation of interruptedbytwobulgednucleotidesandclosedbya
the interhelical regions is supported mainly by the re- 13-nt long loop+ Finally, hairpin 14, corresponding to
activity of nucleotides at their Watson–Crick position the terminator, is rich in G-C base pairs and is one of
(Figs+ 1 and 2)+ This was notably the case for nucleo- the most stable stem-loops of RNAIII+ Its existence is
tides A42–U47, U65–A68, A105–A109, C138–C140, supported by the presence of RNAse V1 cuts on both
A157–U163,A218–A220,U258–A260,U321–U327,and sides of the helix, and by the fact that none of the
U350–C353+ cytosines at position N3 and the guanines at position
The 39 end domain is constituted by three hairpins, N7 (located in the helix) were reactive towards chem-
12–14, and by a long single-stranded region involving icals(Fig+1C)+Inaddition,cytosines495–496and498–
residues459–483(Fig+2)+Thestem-loopstructure13 500attheirN3positionswerehighlyreactivewithDMS,
is clearly identified, but its folding required the pres- and several RNaseT2 cleavages occurred in the loop
enceofmagnesium,becausemanynucleotideswithin (Fig+ 1C)+
FIGURE1. Probing the structure of RNAIII in vitro and in vivo+ Selected autoradiograms showing enzymatic (A) and
chemical(B,C)probingperformedat378ConinvitrotranscribedRNAIII+A:RNaseT1(T1),RNaseT2(T2)andRNaseV1
(V1)hydrolysison59-labeledRNAIII,shortmigration+LanesI,I9:incubationcontrolsinbufferusedforRNaseT1,andRNase
V1hydrolysis,respectively;lanesT1:0+05and0+15UofRNaseT1;lanesT2:0+02and0+04UofRNaseT2;lanesV1:0+1
and0+2UofRNaseV1;lanesT1andU2:RNasesT1andU2indenaturingconditions,respectively;laneL:alkalineladder+
B: DMS and CMCT modifications, long migration+ Lane I: incubation control in buffer used for DMS; lane DMS: native
conditions,1mLofDMSdiluted1:16;lanesCMCT:nativeconditions(N)andsemidenaturingconditions(SD),2mLofCMCT
(100mg/mL);lanesG,A,T,andCcorrespondtosequencingladders+C:DMSmodificationofcytosinesatN3on39-end-
labeledRNAIII,shortmigration+LaneI:incubationcontrol;lanesDMS:nativeconditions,1,2,and4mLofDMSdiluted1:8;
lanesU2andLasinA+DandE:selectedautoradiogramsshowingDMSandCMCTmodificationsoninvitrotranscribed
RNAIII(asinB)andacomparisonwithDMSmodificationonRNAIIIinvivo,shortmigration+Thedifferencesinreactivities
aredenotedbyarrows+LanesI:incubationcontrolsinbufferusedforDMS;lanesDMSinvivo:25mLofDMSin20mLof
staphylococcalcultureatlateexponentialgrowthphase;lanesDMSinvitro:nativeconditions(N),semi-denaturingcondi-
tions(SD),1mLofDMSdiluted1:16;lanesCMCT:nativeconditions(N),semi-denaturingconditions(SD),1mLofCMCT
(100mg/mL);lanesG,A,T,andCcorrespondtosequencingladders+(Figurecontinuesonfacingpage.)
StructureofS.aureusagr-RNAIII 671
Probing the structure of the 39 end of RNAIII main alone, all these nucleotides were reactive at one
of their Watson–Crick positions and were cleaved by
Becauseithadbeensuggestedthatthe59and39ends
RNaseT2(Fig+3A),indicatingthatthisregionissingle
of RNAIII would pair, and because specific regulatory
stranded+
activitywasattributedtothe39end(Novicketal+,1993;
Benito et al+, 1998), the conformation of this region
(nt394–490)wasprobedseparatelywithchemicaland
In vivo DMS probing of RNAIII
enzymatic probes (Fig+ 3)+ Interestingly, the enzymatic
cleavageandchemicalreactivitypatternsofnt409–483 The structure of RNAIII was studied in vivo by using
in the 39 domain were identical to those in the entire DMS, which alkylates cytosine at N3 and adenine at
RNAIIImoleculeandarethusconsistentwiththeexis- N1 (Mayford & Weisblum, 1989)+ In a typical experi-
tenceofstem-loopstructure13(Figs+2and3)+Indeed, ment,S.aureusstrain RN6390 was grown to the late
RNase V1 cuts occurred at positions 410–418, 421– exponentialphaseandtreatedwithDMS+SitesofRNAIII
422, 439, 443–444, and 447–450, whereas the main modificationweredetectedbytheprimerextensionre-
RNase T2 cleavage sites were located in the hairpin action on RNAextracts and were compared to modifi-
loop at positions 423–430 and 434–437 (Fig+ 3)+ Fur- cations observed in vitro+ As shown in Figure 1D,E,
thermore, the nucleotides in the external loop were all reactivitywithRNAIIIinvivoandinvitrowasessentially
highly reactive with chemical probes at their Watson– identical, as only nucleotides in the hairpin loop and
Crickposition(Fig+3)+ThepresenceofseveralRNase interhelical regions were highly sensitive to DMS+ Dif-
V1 cuts in the upper part of the stem (A419–U424/ ferencesbetweentheinvitroandinvivomappingdata
A438–U443) and the reactivity of several uridines mainly concerned the reactivity of the adenine resi-
pointed to the formation of a metastable helix+ duesinvolvedintheproposedlong-rangeinteractions,
The main reactivity changes observed between the particularlyhelixB(U371–G382/U32–A42)+Thesead-
entire RNAIII molecule and the 39 end domain alone enines were more sensitive to DMS alkylation at their
were located at nt U452–U466+ Indeed, in the 39 do- N1 position in vivo than in vitro (Fig+ 1D)+Another sig-
FIGURE1. (continued.)
672 Y.Benitoetal.
FIGURE2. Secondary structure model of RNAIII showing the enzymatic cleavages (A) and reactivity of nucleotides to
chemicalsprobes(B)+Thehairpinstructuresarenumbered1to14;longdistanceinteractionsaredesignatedA,BandC;
theShine–Dalgarnosequenceandthestartandstopcodonsofhldareinboldface+A:RNaseT1cleavagesaredenoted
by( ),RNaseT2by( ),thick,medium,orthinarrowsforstrong,moderateorlowreactivity,respectively;RNaseV1
cleavagesaredenotedbyblack,gray,oremptytrianglesforstrong,moderate,orlowreactivity,respectively+B:Reactivity
of Watson–Crick positions to DMS (N1A, N3C) and CMCT (N3U, N1G) are represented as follows: reactive in native
conditions(darkenedblackorgreycirclesforstrong,moderateorlowreactivity,respectively);reactiveonlyinsemidena-
turing conditions (squares); more reactive in semidenaturing conditions (cross); nonreactive in both native and semi-
denaturing conditions (no symbol)+ Dots indicate not determined due to unspecific cleavages or pauses of RT in the
incubation control+ Guanines reactive at position N7 to NiCr are denoted by black squares, and empty squares denote
unreactive guanines+ Information about guanine accessibility at N7 position is only provided for region 410–512+ The
differencesbetweenthereactivityofadenineatN1andcytosinesatN3toDMSinvitroandinvivoareindicatedasfollows:
increasedreactivityinvivobyanasteriskandprotectioninvivobyblacktriangles+Thesedatawereobtainedfromfour
independentexperiments+
nificantdifferencebetweeninvivoandinvitromapping only 1 h later (Balaban & Novick, 1995)+ This delay is
resultsconcernedseveraladenineresidueslocatedat not dependent on a specific timing signal during the
the ribosome initiation site+ Indeed, adenines 58, 71, growthcycle,andisabolishedbydeletionofthe39end
and 72 were clearly nonreactive in vivo, whereas ad- of RNAIII (Balaban & Novick, 1995)+ We therefore
enines 58 and 57 became more reactive at their N1 probed the accessibility of the hldribosome binding
positions in vivo (Figs+ 1E, 2)+ site in vitro by using a toe-printing assay (Hartz et al+,
1988)+Inthisprimerextensioninhibitionassay,forma-
tionoftheternarycomplexcomposedofthe30Ssub-
unit, RNAIII, and the initiator tRNAMet blocks the
Ribosomes bind toS.aureusRNAIII
elongation of a cDNA primer by reverse transcriptase
Although RNAIII is transcribed in the mid-exponential (Hartzetal+,1988)+Indeed,the30S/tRNAMet complex
f
phase of growth, it is translated into delta-hemolysin incubated with the refolded RNAIII produced a strong
StructureofS.aureusagr-RNAIII 673
FIGURE3. Probing the secondary structure of in vitro-transcribed
RNAIII 39 domain+ A: autoradiogram showing enzymatic cleavage
sitesonthe39domain+RNaseV1(V1)andRNaseT1(T1)hydro-
lysis,shortmigration+LanesI,I9:incubationcontrols;lanesT1:0+05
and0+15UofRNaseT1;lanesV1:0+1and0+2UofRNaseV1;lanes
G,A,T,Careforsequencingladders+B:secondarystructuremodel
ofthe39enddomainwiththeenzymaticcleavagesandthechemical
reactivities to DMS and CMCT+ Symbols as in Figure 2+ The data
correspondtothreeindependentexperiments+
toe-print at A100, 15 nt downstream of the initiation ribosomes bind efficiently to hldRBS and that the in
codon(Fig+4),aspreviouslyshown(Hartzetal+,1988)+ vitrostructurerepresentsatranslatableformofRNAIII+
The yield of the toe-print was identical when RNAIII
was denatured prior to the experiment (Fig+ 4)+ Given
the possibility that the primer used for reverse tran- The 39 domain of RNAIII represses
scriptionperturbedthestructureofRNAIII,formationof proteinAgene expression
theternary30S/RNAIII/tRNAMetcomplexwasalsostud-
iedinfilterbindingassays(notshown)+Againthedata We have shown here that the 39 end of RNAIII is an
showed no major differences between native and de- independent structural domain+ Furthermore, we and
natured RNAIII+ Altogether, these results indicate that others have previously shown that the 39 end of the
674 Y.Benitoetal.
structure of RNAIII has been proposed (Novick et al+,
1993)+This model was based on minimum energy so-
lutionsandwascharacterizedbyextensivelong-range
interactions between the 59 and 39 domains (Novick
et al+, 1993)+ This prompted us to analyze the confor-
mationofRNAIIIinvitrobyusingseveralchemicaland
enzymatic probes, and in vivo by using DMS+ These
experimental data formed the basis for a secondary
structuremodelderivedwiththehelpofcomputersim-
ulation of RNAIII folding (Gultyaev et al+, 1995)+ The
presentsecondarystructureofRNAIIIischaracterized
byanumberofhairpinstructures(Fig+2),mostofwhich
hadbeenpredicted(Novicketal+,1993;Tegmarketal+,
1998)+Wealsobringevidencethatmostofthesestem-
loopstructuresoccurredinvivo+ThealignmentofRNAIII
fromS.aureuswith that of non-aureusstaphylococcal
FIGURE4. Accessibility of the ribosome binding site in RNAIII in species (S.epidermidis, S.warneri, S.simulans, and
vitro+Theexperimentswereconductedinparallelonnativeandde-
S.lugdunensis)revealsthatthestem-loopmotifs1,7,
natured (unfolded) RNAIII molecules+ The position of the toeprint
(A100) is indicated+ Lane c: incubation control of free RNAin the 13, and 14 are predicted to be highly conserved by
presenceof30Ssubunit;lanes30S:experimentinthepresenceof computer folding, and the hairpins 6, 8, 9, 11, and 12
increasingconcentrationsof30Ssubunits(125,250and625nM);
must be present in all RNAIII but their sequences di-
lanesG,A,TandCareforsequencingladders+
verged(Vandeneschetal+,1993;Tegmarketal+,1998)+
Interestingly,severalbasepaircompensatorychanges
are found in helices 1, 7, 13, and 14 amongStaphylo-
coccusspecies(Fig+6)+Thusthehighconservationof
RNAIIIisimportantforrepressingproteinAgene(spa)
hairpins1,7,13,and14suggestsacommonfunction+
expression,andmaythusbeafunctionaldomain(No-
Forinstance,withtheexceptionofS.epidermidisRNAIII,
vick et al+, 1993; Benito et al+, 1998)+ To further inves-
which carries a 5-nt-long loop (Fig+ 6), hairpin 1 con-
tigate this question, we subcloned selected fragments
tains a conserved GAGA loop motif+ It is well known
of RNAIII under the control of the RNAIII promoter on
thatstablestem-loopstructureslocatedatthe59endof
the staphylococcal plasmid pE194 and introduced the
resulting constructs in S.aureusstrain WA400 (agr2, mRNAmayincreaseitsstability(forareview,seeBech-
hofer, 1993)+ It is therefore tempting to propose a 59
lackingRNAIII)+Downregulationofspaexpressionwas
stabilizer function for this conserved hairpin motif in
achieved normally in WA400/pE194 expressing either
the RNAIII fragment comprising residues 391–514
(Fig+5,lane4)orresidues403–455and484–514(both
RNAfragments contain hairpin 13 and the terminator)
(Fig+ 5, lane 5)+ Interestingly, WA400/pE194 express-
ing a fragment of RNAIII that contains only the tran-
scriptionterminator(nt484–514)wasstillabletorepress
spaexpression,albeittoalesserextent(Fig+5,lane3)+
DISCUSSION
The secondary structure model
ofS.aureusRNAIII
S.aureusRNAIII regulates the expression of several
genes encoding exoproteins and cell-wall-associated
FIGURE5. EffectoftruncatedRNAIIIonproteinAgenetranscrip-
proteins(Novicketal+,1993)+RNAIIIalsoencodesdelta-
tion+NorthernblotanalysisofRNAIIIandproteinAmRNAexpres-
hemolysin, but inactivation of the open reading frame sion+ RNAs from postexponential phase cultures were hybridized
does not affect the regulatory function of RNAIII (Jan- withprobescorrespondingtoRNAIIIandproteinAgene(spa)+Lane1:
RN6390(agr1);lane2:WA400(agrmutant);lane3:WA400/pLUG310
zon & Arvidson, 1990; Novick et al+, 1993)+ There is
(terminatortranscriptionalone,nt484–514);lane4:WA400/pLUG300
alsoevidencethatRNAIIImayundergoconformational (39endRNAIII,nt391–514);lane5:WA400/pLUG315(hairpinstruc-
changes that activate either its translation or its regu- ture13transcriptionterminator,nt403–455::484–514)+RNAIIItran-
scripts are not seen in lanes 4 and 5 because of their very small
latoryfunctions(Novicketal+,1993;Balaban&Novick,
sizes+Positionsofmigrationof16SrRNA(1541nt)and23SrRNA
1995)+ A computer-simulated model of the secondary (2904nt)indicated+
StructureofS.aureusagr-RNAIII 675
FIGURE6. SequenceofseveralconservedhairpinstructuresofRNAIIIfromS.aureus(typesI–IV),andcomparisonwith
four different species,S.epidermidis(e),S.warneri(w),S.simulans(s), andS.lugdunensis(l)+ Nucleotide insertion is
shown by an open arrow, and D is for deletion+The hairpin structures have been assigned according to the secondary
structuremodelpresentedinFigure2+
RNAIII+ The central domain (nt 32–382) is closed by proposed model of RNAIII, the two long-range inter-
helix B (Fig+ 2)+Analogous long-range interaction may actions B and C hide part of the hla-mRNA binding
also arise in S.epidermidis, S.simulans, and S.war- sequence, indicating that conformational changes of
neriassuggestedbycomputerfolding,despitethefact RNAIII are required for efficient binding+ Interestingly,
that the sequence is not conserved (data not shown)+ the greater accessibility of adenines 372–378 in vivo
The central domain encloses the open reading frame suggeststhathelixBisdestabilized,likelyastheresult
encodingfordelta-hemolysinthatispresentinallRNAIII, ofhlamRNAbindingtothe59regionofRNAIII,explain-
exceptS.lugdunensis,withahighdegreeofsequence ing the apparent discrepancy with the in vitro data+
identity(Vandeneschetal+,1993;Tegmarketal+,1998)+ Different pairing pathways that result in the formation
Thecentraldomainalsocontainsabranchedstructure of stable antisense RNA and mRNA complexes have
constituted by three hairpins, 7–9, closed by a long- been described (for a review, see Zeiler & Simons,
range interaction (helix A)+ The existence of helix A, 1998)+ In most cases, binding initiates between two
which was clearly demonstrated in vitro, was also loops, or between a loop and a single-stranded RNA
supported by the in vivo probing+ In this branched segment involving a restricted number of nucleotides,
structure, hairpin 7 is of particular interest+ Several and more stable complexes are subsequently formed
base pair compensatory changes are found among by pairing of distal RNAsegments+ In region 42–72 of
Staphylococcusspecies, and the loop contains a con- RNAIII, several nucleotides show high accessibility to
served sequence (UCCCAA) that resembles that of single-strand-specific probes, indicating that they are
theloopsofhairpin13andtheterminator(Fig+6)+This potential sites for initial pairings+Afragment of RNAIII
highconservationindicatesthathairpin7isapotential containingnt1–380,whichpotentiallyformshelixB,is
recognition site fortrans-acting factor(s)+ indeedcompetentforbindingtohlamRNAinvitro(Mor-
feldt et al+, 1995)+
The central domain also contains the ribosomal ini-
The central domain may undergo tiationsiterequiredforthetranslationofdelta-hemolysin,
specific conformational changes which is the only known translation product of RNAIII+
Thus,thetwofunctionsofRNAIII(translationofhldand
The 59 side of the central domain of RNAIII contains translational regulation of hla) are mutually exclusive+
theregulatoryregion(nt19–87)involvedintheactiva- Structural probing of RNAIII in vitro revealed that the
tionofalpha-toxintranslation(Novicketal+,1993;Mor- RBSsequenceandtheAUGinitiationcodonofhldare
feldtetal+,1995)+BindingofRNAIIItotheleaderregion both accessible to single-strand-specific probes+ The
ofalpha-toxinmRNAinducedaconformationalchange toe-printing and filter binding assays showed that the
ofthehlamRNA,renderingtheribosomalinitiationsite ribosome efficiently binds both to native RNAIII and to
available for translation (Morfeldt et al+, 1995)+ In the thedenaturedmoleculeatthehldinitiationsite(Fig+4)+
676 Y.Benitoetal.
Footprinting experiments indicated that the initiating showed an almost identical nucleotide reactivity pat-
ribosome protects ;40–50 nt (Murakawa & Nierlich, tern, a finding consistent with the existence of a stem-
1989; Huttenhofer & Noller, 1994)+ As the 39 side oc- loop motif, as further supported by the in vivo probing
curredatposition115fromtheadenineoftheinitiation (Fig+ 3)+ The stem of hairpin 13 is interrupted by two
codon (as determined by the toe-printing approach), bulged residues (U417 andA418)+ Despite the appar-
weassumethattheribosomemaycoverpartofstem- entlylowerstabilityoftheupperstem,theprobingdata
loop structure 2 from the 59 side+ Many groups have pointtotheexistenceofa13-nt-longloop(Fig+2)+More-
investigatedtheeffectsofRNAstructureontranslation over,this39domainexertedspecificregulatoryactivity,
initiation (e+g+, for a review, see de Smit & van Duin, as the last 124 nt cloned downstream of the P3 pro-
1994)+However,chemicalprobingindicatedthatstem- moter in an RNAIII-deficient strain were sufficient to
loopstructure2ismetastable,andtheribosomeisthus repress spaexpression (Fig+ 6)+ It is remarkable that
able to bind to the unfolded form+ It was also shown onlyhairpin13(nt403–455)andtheterminatorhairpin
that a 5-bp hairpin located 3 nt upstream of the SD (nt 484–514) were required for efficientspatranscrip-
sequenceoftheT4rIIBgenecanbeincreasedto9bp tioncontrol,asdeletionoftheconservedresiduesU456
without having an inhibitory effect (Shinedling et al+, to U489 did not affect regulation (Fig+ 2)+ It has previ-
1987)+Therefore,ourdataindicatethattheinvitrostruc- ouslybeenshownthatRNAIIIfromS.lugdunensisdoes
tureofRNAIIIcorrespondstoitstranslatableform+One not repress spa transcription, while a chimeric con-
intriguing finding is that translation ofhldin vivo is de- structconsistingofthe59endofS.lugdunensisRNAIII
layedby1hfollowingtranscription(Balaban&Novick, and the 39 end ofS.aureusRNAIII repressedspaex-
1995)+ It was postulated that a conformational change pression more efficiently in a S.aureusbackground
ofRNAIIItoatranslatableformprecededitsregulation (Benitoetal+,1998)+Severalsequencedifferenceswere
of target genes (Balaban & Novick, 1995)+This appar- observedbetweenS.lugdunensisandS.aureusRNAIII
ently conflicts with the fact that the in vitro structure is in the hairpin loop of the terminator and stem-loop 13
efficiently recognized by the ribosome+ It is possible (Fig+ 6)+ Conversely, other coagulase-negative staph-
that, owing to transcription–translation coupling, some ylococci in which the last 150 nt of RNAIII (including
transientinhibitorystructuresoccurringinvivoseques- stem-loop13,Fig+6)aremorestronglyconservedwere
ter the ribosome binding site or influence the kinetic able to repressspaexpression (Tegmark et al+, 1998)+
foldingpathwayofRNAIIIinvivo+Interestingly,theover- These data point to a shared function of the 39 end
all pattern of nucleotide reactivity to chemical probes domainofseveralRNAIIIspeciesinsparegulation+We
was very similar in vivo and in vitro+ One major differ- also found that the last 31 nt of RNAIII (containing the
encewaslocatedwithintheRBS:inparticular,thetwo transcriptionterminator)partiallyrepressedspaexpres-
adenines(71and72)oftheShine–Dalgarnosequence sion,suggestingthattheterminatoralsoparticipatesin
becameprotectedinvivo(Fig+2),aphenomenonwhich theregulatorymechanism+Whetherthe39domainreg-
may correlate with occupancy by the ribosome of its ulates transcription directly or indirectly is unknown+
initiationhldbinding site+ Indeed, in the initiation com- One plausible explanation is that hairpin 13 and the
plex,base-specificprotectionmainlyoccursintheRBS terminator of RNAIII act via a ribonucleoprotein com-
and at the initiation codon, reflecting the formation of plex+Apossible role of RNAin modulating the activity
stable base pairings with the 39 end of 16S rRNAand of DNAbinding proteins has been reported (Retallack
the anticodon sequence of the tRNAMet, respectively & Friedman, 1995; Sledjeski & Gottesman, 1995)+ It is
f
(Huttenhofer & Noller, 1994)+ However, in vivo probing also noteworthy that both hairpin loops contain a con-
of RNAIII took place in the late exponential phase of served CCCAAsequence that may provide a bipartite
growth when RNAIII is also involved in transcriptional protein binding site (Fig+ 6)+
regulation of target genes+ It is likely that RNAIII un- In summary, RNAIII has the unique property of act-
dergoesspecificconformationalchangesthatarenec- ing as a messenger RNAand having pleiotropic regu-
essaryfordefinedfunctions+Thus,severalpopulations latory functions+ The secondary structure model of
of RNAIII may coexist in vivo but are difficult to distin- RNAIII proposed here should help to clarify functional
guish by chemical mapping+ domains+Wedemonstratedherethatthe39domainof
RNAIII contains an independent structure and has a
regulatory function in proteinAsynthesis+
The 39 end domain is sufficient for
the control of proteinAexpression
MATERIALSAND METHODS
We also identified an intrinsic structural domain in the
39 end of RNAIII, with hairpin 13 at its center+ Stem-
Bacterial strains and growth conditions
loop13hasbeenpredictedtobepartiallybasepaired
withtheregionencompassingnt130–98(Novicketal+, Escherichiacoli TG1 and S.aureus RN4220, a nitroso-
1993)+ However, structural mapping of the full-length guanidine-inducedmutantcapableofacceptingE.coliDNA
RNAIII and of the RNAIII domain from nt 391–490 (Kreiswirthetal+,1983),wereusedforplasmidamplification
StructureofS.aureusagr-RNAIII 677
and genetic manipulations+S.aureusRN6390 derives from Bam1081andagr-sa757);thesetworegionswereseparated
8325-4andisourstandardagr1strain+S.aureusWA400is by a short cloning sequence that contained the restriction
aderivativeof8325-4inwhichtheP2operonisfunctionalbut sitesStuIandBamHI+Toexpresshairpinstructure13under
theP3operonisdeletedandreplacedbythechloramphen- thecontroloftheP3promoter,thecorrespondingsequence
icoltransacetylasegene(cat86)(Janzon&Arvidson,1990) (nt 1168–1116) was amplified using primers Stu1168 and
(seeTable 1)+ Staphylococci were grown either on BM agar Bam1116, digested by StuI and BamHI, and ligated to
plates(1%peptone,0+5%yeastextract,0+1%glucose,0+5% pLUG310digestedbythesameenzymes+
NaCl, 0+1% K2HPO4), or in brain-heart infusion (BHI), with Forinvitrotranscription,theRNAIIIcodingsequence(cor-
erythromycin(5mgmL21)whenappropriate+ respondingtont1–514ofRNAIII)wasamplifiedusingprim-
ersStu1572andBam1055+ThePCRproductwasdigested
byStuIandBamHIandligatedtothepUT7vector(Serganov
Molecular cloning and plasmid construction
etal+,1997)cleavedwiththesamerestrictionenzymes+The
Total DNA and plasmid DNA were prepared with standard resultingplasmidwasintroducedbytransformationinE.coli
methods (Sambrook et al+, 1989)+ Transformation of E.coli TG1+ Similarly, the 39 end of RNAIII (nt 391–490) lacking
TG1wasperformedwithcellsrenderedcompetentbytreat- the transcription terminator was amplified by using primers
ment with CaCl2, andS.aureusRN4220 and WA400 were Stu1180andBam1079andwasthenclonedontopUT7vec-
transformedbyelectroporation(Bio-Radgenepulser)+ torinE.coliTG1+
RNAIIIwasexpressedinStaphylococcusbyusingplasmid
pE194(Horinouchi&Weisblum,1982),whichhadbeenmod-
Northern blots
ifiedbyaddinganEcoRVrestrictionsitenexttotheXbaIsite
formingpLUG274+PCRwasperformedfromtheRN6390agr Electrophoresis of total RNA through 1% agarose gel con-
sequence (GenBank accession number X52543)+ To clone taining2+2Mformaldehyde,vacuumtransfertonylonmem-
the39domainofRNAIIIundercontroloftheP3promoter,the brane, hybridizations with specific digoxigenin-labeled RNA
promotersequencewasamplifiedusingprimersagr-sa1819/ probes and luminescent detection were carried out as de-
Stu1569 (Table 2), and the 39 end of RNAIII was amplified scribed(Benitoetal+,1998)+PCRprimersfortheproduction
using primers agr-sa757/Stu1180+ The PCR products were of agrRNAIII and protein A-specific gene probes were se-
digested byStuI and ligated together before being reampli- lectedfromGenBankaccessionnumberX52543andA04518
fied with the external primers agr-sa1819 and agr-sa757+ respectively+
The resulting PCR product was digested with HindII and
XbaI (site present on each primer) and ligated to the modi-
Preparation of RNA
fiedpE194vectordigestedbyEcoRVandXbaI+Theligation
product was electroporated to RN4220 and the resulting RNAIIIandthe39domainofRNAIIIweretranscribedinvitro
plasmid (pLUG300) was introduced into strain WA400+ To with T7 RNA polymerase from BamHI linearized pLUG322
express intramolecular sequences of RNAIII under the con- andpLUG281,respectively+TranscriptionofRNAIIIyieldeda
troloftheP3promoter,weconstructed,usingasimilarmethod, run-offproductof522ntincludingfiveadditionalnucleotides
plasmidpLUG310thatcontainedtheP3promoterregionlinked atthe59endandthreeatthe39end,whichallcamefromthe
to the transcriptional terminator (amplified using primers vector+The39domaincorrespondedtont391–490ofRNAIII+
TABLE1+ Maincharacteristicsofstrainsandplasmids+
Strainsorplasmids Relevantcharacteristics Referenceorsource
S.aureusstrains
8325-4 NCTC8325curedofthreeprophages Novick,1963
RN4220 restriction-mutantof8325-4 Kreiswirthetal+,1983
RN6390 derivativeof8325-4,agrpositive Pengetal+,1988
WA400 8325-4DRNAIIIregion::cat86 Janzon&Arvidson,1990
LUG450 WA400/pLUG300 thiswork
LUG460 WA400/pLUG310 thiswork
LUG467 WA400/pLUG315 thiswork
E.coliplasmids
pUT7 pUC119::T7promoter Serganovetal+,1997
PLUG322 pUT7::RNAIIInt1–514 thiswork
pLUG281 pUT7::RNAIIInt391–490 thiswork
Staphylococcalplasmids
pE194 3+728kbS.aureusplasmid,inducible Horinouchi&Weisblum,1982
MLSresistance(erm)
pLUG300 pE194::P3promoterlinkto39-endRNAIII thiswork
(nt391–514)
PLUG310 pE194::P3promoter-TT(nt484–514) thiswork
pLUG315 pLUG310::hairpin13(nt403–455) thiswork