Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria

14 Pages·2014·2.44 MB·English

NucleicAcidsResearch,2014 1
doi:10.1093/nar/gku793
Ms1, a novel sRNA interacting with the RNA
polymerase core in mycobacteria
Jarmila Hnilicova´1,†, Jitka Jira´t Mateˇjcˇkova´1,†, Michaela Sˇikova´1, Jirˇ´ı Posp´ısˇil1, Petr Halada2,
Josef Pa´nek3 and Libor Kra´sny´1,*
1DepartmentofMolecularGeneticsofBacteria,InstituteofMicrobiology,AcademyofSciencesoftheCzech
Republic,Prague14220,CzechRepublic,2DepartmentofMolecularStructureCharacterization,Instituteof
Microbiology,AcademyofSciencesoftheCzechRepublic,Prague14220,CzechRepublicand3Departmentof
D
Bioinformatics,InstituteofMicrobiology,AcademyofSciencesoftheCzechRepublic,Prague14220,CzechRepublic o
w
n
lo
a
ReceivedMay29,2014;RevisedAugust19,2014;AcceptedAugust20,2014 d
e
d
fro
m
ABSTRACT surviveinthehumanbodyfordecadesasalatentinfection h
ttp
withnosymptoms(1). s
Small RNAs (sRNAs) are molecules essential for a Mycobacteria as well as other bacteria need to quickly ://a
numberofregulatoryprocessesinthebacterialcell. adapt to changing conditions to survive. When the sur- cad
Here we characterize Ms1, a sRNA that is highly ex- roundingenvironmentisfavorable,bacteriagrowexponen- em
pressed in Mycobacterium smegmatis during sta- tially; in harsh conditions, bacteria enter stationary phase ic.o
tionary phase of growth. By glycerol gradient ul- ofgrowthandwaitforbetterconditions.Successfuladapta- up
tracentrifugation, RNA binding assay, and RNA co- tiondependsonchangesingeneexpression.Akeymolecule .co
m
immunoprecipitation, we show that Ms1 interacts participating in this process is RNA polymerase (RNAP) /n
with the RNA polymerase (RNAP) core that is free that is itself regulated by various auxiliary factors. Bacte- ar/a
toofr.thTehpisrimcoanrtyrassigtsmwaitfhactthoer (sσitAu)aotiroanniynomthoesrtσotfhaecr- ruσinafilatRsc:tNo(cid:2)rA2s(cid:3)Pt(cid:3)hia(cid:2)s(cid:4)ta.rmeTcuholegtinsRiuzNbeuAdniPiftfeecrnoeznryetmpaersoscomocmoiatpteeorssseewdqiouthfencdocierfsef,esrauenbnd-t rticle-a
dsanpifdefectriheeinsscwienhtieenrraethciettioiisnnt6reSerqaRcuNtiirAoensthtohafettihpnerteetsrwaeocntcsteywpoeiftshσoAR.fNTsAhRPe- TMswh.ietscnmhuienmggmbbeaerttiowsfehσeansfat2hc6teosσresσfvaafcartcioetrsosar(sm2r,oe3gn)ugwlabhtaielcsetgeEerisnacelhesexprpieccrheieisass-iceoo.ngli.. bstract/42
NAs(Ms1or6SRNA)withRNAPpossiblyreflectsthe hasseven(3).Typically,bacteriahaveoneprimary(house- /18
difference in the composition of the transcriptional keeping) σ factor responsible for the majority of gene ex- /11
7
machinerybetweenmycobacteriaandotherspecies. pression. This primary σ factor is called σ70 in E. coli or 6
3
Unlike Escherichiacoli, stationary phase M.smeg- σA in M. smegmatis and Bacillus subtilis (4). When con- /2
4
matiscellscontainrelativelyfewRNAPmoleculesin ditions become unfavorable and bacteria enter stationary 35
complexwithσA.Thus,Ms1representsanoveltype phase, the transcription of σ70/σA-dependent genes is re- 070
duced and genes recognized by alternative stress σ factors b
ofsmallRNAsinteractingwithRNAP. y
areactivated(5). g
u
Gene expression is also regulated by small RNAs (sR- es
NAs).sRNAsusuallyhavealengthof50–300ntandmost t o
n
INTRODUCTION of them base-pair with mRNA and regulate mRNA sta- 09
bility or the efficiency of mRNA translation (6). It is esti- A
p
Mcluydceobleatchtaelrihauamreananpaitmhpoogertnasn-Mt gyrcooubpacotferbiuamctetruibaetrhcuatloisnis- matedthatabacterialcellsuchasE.coliencodeshundreds ril 2
of different sRNAs (7). Only a limited number of studies 0
1
andMycobacteriumleprae-andnonpathogenicsaprophytic have mapped sRNAs and addressed their function in my- 9
species,suchasMycobacteriumsmegmatis.Approximately cobacteria (8–14). In exponentially growing M. tuberculo-
one third of the world’s population is infected by M. tu- sis, 17% of total non-rRNA transcripts originate from in-
berculosisandalmost9millionpeopledevelopedtheactive
tergenicregionsandrepresentsRNAs(15).Thisnumberin-
disease in 2012. According to WHO, tuberculosis caused
creasestoalmost60%instationaryphasecells,mainlydue
1.3milliondeathsin2012.Inaddition,multi-drugresistant
totheaccumulationofahighlyabundantsRNAdesignated
tuberculosisispresentinmostcountriessurveyed.Anim-
MTS2823 (or ncRv13661 according to the new nomencla-
portant feature of M. tuberculosis is that the bacteria can
*Towhomcorrespondenceshouldbeaddressed:Tel:[phone];Fax:[phone];Email:[email protected]
†Theauthorswishittobeknownthat,intheiropinion,thefirsttwoauthorsshouldberegardedasJointFirstAuthors.
(cid:3)C TheAuthor(s)2014.PublishedbyOxfordUniversityPressonbehalfofNucleicAcidsResearch.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0/),which
permitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
2 NucleicAcidsResearch,2014
ture (16)). MTS2823 is present in stationary phase cells in ure5C)wereconductedinaTecanInfinite200Proreader
amounts comparable to rRNAs (8). An even higher accu- andgrowthwasmonitoredfor24h.
mulation of MTS2823 sRNA was observed in mice dur- pJAM2-σA(31)(agiftfromV.Nagaraja,IndianInstitute
ingchronicinfection.WhenMTS2823wasartificiallyover- of Science, India) contains an inducible acetamidase pro-
expressed in exponential phase cells, the transcription of moter that allows overexpression of σA upon the addition
∼300genesdecreased(8).AlthoughMTS2823ishighlyex- ofacetamide.pJAM2-σA ortheemptypJAM2(32)vector
pressed, affects mycobacterial gene expression and has a were electroporated into M. smegmatis mc2 155, yielding
possible role during infection, the mechanism of its func- strainsLK1304andLK1302.Acetamideatafinalconcen-
tionisunknown.Wepreviouslyindependentlyidentifieda trationof0.2%wasaddedtobothstrainsattheentryinto
homologofMTS2823,Ms1,asmallRNAinM.smegmatis stationaryphase(OD =1.6)andthecellsweregrownfor
600
thatishighlyabundantinstationaryphase(17). additional6h(experimentsshowninFigure6).
Ms1wasoriginallyidentifiedbyinsilicosearchesfor6S Ms1,Ms1nb(thelatterisamutantMs1lackingtheinter-
D
RNAhomologs.6SRNAsfoldintoasecondarystructure nalbubble)and80bprrnBpromoter(33)DNAsequences o
w
that mimics an open promoter (13,18–20) and this struc- (see Supplementary Data) were synthetized by Invitrogen n
ture binds to RNA polymerase in complex with the pri- (GeneArt Strings DNA Fragments). By BamHI/KpnI di- loa
mary σ factor (RNA polymerase holoenzyme). 6S RNA gestion, the acetamidase promoter was deleted from the de
d
preventsthebindingoftheRNAPholoenzymetopromoter pJAM2 plasmid and in its place the rrnB promoter fused fro
sequences and reduces its transcriptional activity (21–24). to Ms1 or Ms1nb was cloned using a GeneArt Seamless m
h
We had originally hypothesized that Ms1 may be the my- CloningandAssemblyKit(Invitrogen;fordetailsseeSup- ttp
cnoobtaicntteerriaaclt6wSiRthNtAhe. HRNowAePvecr,owmeplsehxowcoendtathinaitnMg tsh1edporeis- panledmeelnectatrroypoDraattae)d.iPnltaostmhiedms cw2e1r5e5vsetrriafiiend,ybieyldsienqgusetnrcaiinngs s://ac
maryσ factor(17).Inaddition,Ms1hasalengthof∼300 overexpressing either Ms1 (LK1323), or its mutant form ad
e
nt,while97%of>3500 known6SRNAsequences(either Ms1nb(LK1337)orwithoutanyoverexpression-acontrol m
ic
predictedorvalidated)havealengthinthe150–210ntrange strainwithanemptyplasmid(LK1302). .o
and no known 6S RNA has a length of ∼300 nt (Rfam up
.c
database,(25)).6SRNAshavebeenfoundinmanybacterial o
RNAisolation,RNaseHtreatmentandPAGE m
species(20,26–28);mycobacteriaareanexception.Despite /n
a
severalstudiesidentifyingsmallRNAsinmycobacteria(8– The same protocol (adapted from (34)) was used for E. r/a
14),itisstillunclearwhethermycobacteriapossess6SRNA coli,M.smegmatisandB.subtilisRNAisolation.Briefly,8– rtic
ornot. 20 ml cells from the indicated growth phases were quickly le
-a
Here we use M. smegmatis as a model organism and pelleted and immediately frozen. The pellet was then sus- b
s
searchforthebindingpartnerofMs1.WeshowthatMs1is pendedin240(cid:5)lTE(pH8.0)plus60(cid:5)llysisbuffer(50mM tra
c
asRNAthatdirectlyinteractswiththetranscriptionalma- Tris–HClpH8.0,500mMLiCl,50mMethylenediaminete- t/4
chinerybutinadifferentwaythan6SRNA-Ms1bindscore traaceticacid(EDTA)pH8.0,5%sodiumdodecylsulphate 2/1
RNApolymeraseinsteadofRNAPholoenzyme.Thus,Ms1 (SDS))and600(cid:5)lacidicphenol(pH∼3):chloroform(1:1). 8/1
representsanovelclassofsmallRNAs.Finally,wediscuss Lysates were sonicated in a fume hood, centrifuged, the 17
6
possible reasons why mycobacteria may differ from most aqueousphaseextractedtwomoretimeswithacidicphenol 3
otherbacterialspeciesintheinteractionofRNAPwiththe (pH∼3): chloroform and precipitated with ethanol. RNA /24
3
sRNA. wasdissolvedinwaterandDNasetreated(TURBODNA- 50
freeKit,Ambion).Five(cid:5)goftotalRNAweremixedina 70
b
MATERIALSANDMETHODS 1:1 ratio with the sample buffer (95% formamide, 20 mM y
EDTApH8.0),heatedfor1minat90◦C,thenkeptonice gu
Bacterialstrains,growthconditions,plasmids e
andelectrophoresedin7Murea7%polyacrylamidegels. st o
For detailed descriptions of individual strains see List of To test whether the prominent 300 nt band in the M. n
0
strainsandplasmidsinSupplementaryData.M.smegmatis smegmatis total RNA consists of Ms1, we used RNase 9
mc2 155andFLAG-taggedRpoBstrain(29)(strainname: H treatment that degrades RNA in RNA/DNA duplexes. Ap
MR-sspB;kindlyprovidedbyD.Schnappinger,WeillCor- We set up 9 (cid:5)l reactions with 3.5 (cid:5)g total RNA, 2 (cid:5)l ril 2
nellMedicalCollege,NewYork,USA)weregrownat37◦C 5xRT buffer (from SuperScriptIII CellsDirect cDNA Syn- 01
inMiddlebrook7H9mediumwith0.2%glyceroland0.05% thesisSystem,Invitrogen)and1(cid:5)l100(cid:5)MDNAoligonu- 9
Tween80andharvestedinexponentialphase(OD ∼0.5) cleotides (anti-Ms1 oligo 5(cid:2)-GTCGTGGCCGTCCGCT
600
or4–6haftertheentryintostationaryphase(OD ∼2.5– TTTCGAAACTACGC-3(cid:2),ns-oligo 15(cid:2)-CGGGTCACAG
600
3)unlessstatedotherwise.TransformationsofM.smegma- CCCAACGTAACTGCCTCAAC-3(cid:2)andns-oligo 25(cid:2)-AA
tismc2 155cellswereperformedbyelectroporation.When GACTTCGACGTGCGCGACCACCGCAAAC-3(cid:2)). Re-
requiredforselectionoftransformants,mediaweresupple- actions were incubated for 1 min at 95◦C, 1 min at 60◦C
mentedwithhygromycin(50(cid:5)g/ml)and/orkanamycin(20 andcooledonice.One(cid:5)l(2U)ofRNaseH(fromSuper-
(cid:5)g/ml).Wild-typeE.coliK12KW72(30),kindlyprovided ScriptIII CellsDirect cDNA Synthesis System, Invitrogen)
by Tamas Gaal, University of Wisconsin-Madison, USA) wasthenaddedandthereactionswereincubatedfor10min
and B. subtilis 168 strains were grown in LB medium and at37◦C,mixed1:1withsamplebuffer(95%formamide,20
thecellswerecollectedinexponentialphase(OD ∼0.5) mM EDTA pH 8.0), heated for 1 min at 90◦C and elec-
600
or3–4hafterentryintothestationaryphaseofgrowthun- trophoresedin7Murea7%polyacrylamidegels.Thegels
lessstatedotherwise.Growthphenotypeexperiments(Fig- werestainedfor20minwithGelRed(LabMark),10000×
NucleicAcidsResearch,2014 3
dilutedin1×TBE(Tris-borate-EDTA)gelrunningbuffer ImmunoprecipitationandRT-qPCR
andvisualizedusingaUVtransilluminator.RNAisolation
E. coli and M. smegmatis cell lysates were prepared in the
after the glycerol gradient or immunoprecipitation is de-
same way as the lysates used for the glycerol gradient ul-
scribedintherelevantsections.
tracentrifugation, and 300 (cid:5)g (protein) of lysates were in-
cubated for 2 h at 4◦C with 20 (cid:5)l of Dynabeads Protein
A (Invitrogen) coated either with4 (cid:5)g mouse monoclonal
5(cid:2)RACE antibody to σ70/σA [clone name 2G10], 2–8 (cid:5)g mouse
monoclonalanti-(cid:3)subunitofRNAPantibody[clonename
Five(cid:5)goftotalRNAwastreatedwith2UofTobaccoAcid
Pyrophosphatase (TAP; Epicentre) for 1 h at 37◦C. RNA 8RB13] (both from Santa Cruz) or 10 (cid:5)g mouse nonspe-
cific IgG (Sigma-Aldrich) used as a negative control. The
wasextractedwithacidicphenol(pH∼3):chloroform(1:1),
precipitated with ethanol and a 5(cid:2)-adaptor DNA/RNA capturedcomplexeswerewashed4×with20mMTris–HCl
poelirgocnausceleloetttideres)(5w(cid:2)-AasTCligGaTteadggtcoactchuega5a(cid:2)a-e3n(cid:2)d,sD. NRANAinwuaps- pbpuHarfft7es.r.9Af,o1qr5u50amrmteiMnroafKtt9Ch5el◦,bC1eaamdnMsdweMeluregteCidnlc2purabonatdeteidndisvinwidSeerDdeSidnestatoemcttpwelode Downlo
thenextractedandreversetranscribedintocDNA(Super- a
by western blotting. The remaining three quarters of the d
ScriptIII, Invitrogen) with an Ms1-specific reverse primer e
p(5r(cid:2)i-mCeGrTaCndCtGheC5T(cid:2)T-ATTTCCGGTAAAGAGCCTAACCC-3T(cid:2))G.TAhAeAsa-m3(cid:2)eforervwearrsde b20eamdsMwTerreis–reHsuCslppenHde7d.9,in12m00M(cid:5)Ml 1g%ClS2DanSd, 1v5o0rtmexMedKwCithl, d from
200 (cid:5)l acidic phenol (pH∼3):chloroform (1:1) for 10 min. h
primer were used for polymerase chain reaction (PCR) ElutedRNAwasprecipitatedwithethanol,dissolvedinwa- ttp
wucitths wTaerqeDseNqAuenpcoeldymaenrdasem(aBpipoetdootlos).thTeheMP.CsRmepgrmoda-- terandDNasetreated(TURBODNA-freeKit,Ambion). s://ac
RNAwasvisualizedona7Murea7%polyacrylamidegel a
tis mc2 155 genome (GenBank # NC 008596.1). The d
by staining with GelRed (LabMark). Alternatively, RNA e
transcription start sites for other species were retrieved m
was reverse transcribed into cDNA (SuperScriptIII, Invit- ic
from previous publications: MTS2823 (TSS 4 100 669, .o
rogen)usingrandomhexamersandamplifiedbyquantita- u
M.tuberculosisH37Rv)(8)GenBank#AL123456.2,Mcr8 p
tive reverse transcription PCR (RT-qPCR) in a LightCy- .c
(TSS4,073,797,MycobacteriumbovisBCGPasteur1173P2 o
cler 480 System (Roche Applied Science) in duplicate re- m
genome)(10)GenBank#AM408590.1,igMAV 0468–0469 actionscontainingLightCycler(cid:2)R 480SYBRGreenIMas- /na
(TSS 458,799, Mycobacterium avium MAH104) (35) Gen- terand0.5(cid:5)Mprimers(each).FortheEc 6SRNAprimer r/a
Bank#CP000479.1. pair, dimethyl sulfoxide (DMSO) was added to a 1% final rtic
le
concentrationtooptimizeamplificationefficiency.Primers -a
b
were designed with Primer3 software and their sequences stra
Glycerolgradientultracentrifugationandwesternblotting areintheSupplementaryprimerlist.Negativecontrols(no c
templatereactionsandreactionswithRNAasatemplateto t/4
E. coli and M. smegmatis stationary phase cells were pel- controlforcontaminationwithgenomicDNA)wererunin 2/1
leted and resuspended in 20 mM Tris–HCl pH 7.9, 150 eachexperiment,thequalityofthePCRproductswasde- 8/1
mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT), termined by dissociation curve analysis and the efficiency 176
0.5 mM phenylmethylsulfonyl fluoride (PMSF) and Cal- 3
oftheprimersdeterminedbystandardcurves.Thepropor- /2
biochem Protease Inhibitor Cocktail Set III protease in- 4
tions of coimmunoprecipitated RNAs were quantified on 3
hibitors,sonicated15×10swith1minpausesoniceand thebasisofthethresholdcycle(Ct)foreachPCRproduct 507
centrifuged.Proteinextracts(1.5mg)wereloadedonalin- 0
that was normalized to input values according to the for- b
ear10–30%glycerolgradientpreparedingradientbuffer(20 y
mula2(Ct(immunoprec)–Ct(input)). g
mMTris–HClpH7.9,150mMKCl,1mMMgCl ,1mM u
2 e
DanTdTf,ra0c.5tiomnMatePdMbyScFenatnrdifuCgaaltbiioonchatem32p0r0o0terpasme(in13h0ib0it0o0r×s) FLAG-tagpulldown st on 0
g)for17husinganSW-41rotor(Beckman).Thegradient 9
was divided into 20 fractions, RNA from individual frac- Lysates from MR-sspB (FLAG tag on (cid:3)) and the wt mc2 Ap
ptiroencsipwitaasteedxtbryacettehdawnoitlhaancdideilcecpthroepnhoolr(pesHed∼i3n):7chMlourorefoar7m%, 1as55thsteralyinsa(tneesgfaotrivgelycocenrtorollg)rwaderieenptrecpenatrreidfuingatthioens,am30e0w(cid:5)agy ril 201
polyacrylamidegels.Thegelswerestainedfor20minwith of lysate incubated with 10 (cid:5)l MS2 resin (Sigma-Aldrich) 9
GelRed(LabMark)10000×dilutedin1×TBEgelrunning for 1 h at 4◦C, captured complexes were washed 4× with
bufferandimagedusingtheUVtransilluminator.Proteins 20mMTris–HClpH7.9,150mMKCl,1mMMgCl2 and
were analyzed by sodium dodecylsulphate-polyacrylamide proteins/RNAisolatedanddetectedinthesamewayasthe
gel electrophoresis (SDS-PAGE) and Coomassie staining immunoprecipitations.
(SimplyBlue,Invitrogen)ordetectedbywesternblottingus-
ing mouse monoclonal antibodies to σ70/σA [clone name
BiotinylatedRNApulldown
2G10]ortothe(cid:3)subunitofRNApolymerase[clonename
8RB13] and secondary antibodies conjugated with a flu- Ms1andMs1nbtemplateswereamplifiedfromInvitrogen
orophore dye and quantified with an Odyssey reader (LI- DNA(GeneArtStringsDNAFragments)withtheprimer
CORBiosciences).6SRNA,Ms1,σA,σ70andRNApoly- carryingtheT7promoterattheir5(cid:2)end(seeSupplementary
merase((cid:3)subunit)werequantifiedfromRNAgels/western Primer List). RNAs were prepared with a T7 RiboMAX
blotsusingtheImageJsoftware. Express Large Scale RNA Production System (Promega)
4 NucleicAcidsResearch,2014
andtheir3(cid:2)endswerebiotinylatedwithanRNA3(cid:2)EndBi- andresolvedthemelectrophoreticallyonaPAGEgel(Fig-
otinylationKit(ThermoScientific).Afterbiotinylation,2.5 ure1A).ThegelwasstainedwithGelRed,anintercalating
(cid:5)gofeachRNAwasdenaturedfor2minat90◦Candre- fluorescentnucleicacidgelstainthatismoresensitivethan
folded for 20 min at RT in 200 (cid:5)l folding buffer (100 mM ethidiumbromideandabletodetectnanogramsofssRNA
KCl,10mMMgCl ,10mMTris–HClpH7).RNAswere (seeSupplementaryFigureS1A).ByusingthistotalRNA
2
incubated in the same buffer for 1 h at 4◦C with 100 (cid:5)l stainingwewereabletodirectlycomparetheamountofin-
magnetic streptavidin-coated beads (Sigma-Aldrich) while dividual small RNAs with 5S rRNA. While the 6S RNAs
beinggentlyagitated.Thebeadswerethenwashedwithly- ofE.coliandB.subtiliswereprominentlyvisible,noRNA
sis buffer (20 mM Tris-HCl pH 7.9, 150 mM KCl, 1 mM ofsimilarlengthwasdetectedinM.smegmatis.Instead,we
MgCl ) and incubated with ∼400 (cid:5)g of cell extract (pre- observeda∼300ntsRNAexpressedmainlyduringstation-
2
pared in the same way as for glycerol gradient ultracen- ary phase and its amount was comparable to the quanti-
trifugation)for1hat4◦C,washed4×withlysisbuffer,re- ties of 6S RNAs in E. coli and B. subtilis. The length of
suspendedinSDSsamplebuffer,heatedfor5minat95◦C thisRNAcorrespondedtoMs1.Toconfirmthatthe∼300 Do
w
and the eluted proteins were detected by SDS-PAGE and nt band was indeed Ms1, we incubated total RNA with n
lo
Coomassiestaining(SimplyBlue,Invitrogen). Ms1-specificorcontrolDNAoligonucleotidesandtreated a
d
theRNAsampleswithRNAseHthatspecificallydegrades e
d
RNA within RNA/DNA duplexes. When incubated with fro
QuantificationofMs1andnorthernblotting an oligonucleotide complementary to Ms1, RNAse H di- m
Cells from stationary phase were used to determine the gested the ∼300 nt band and this band disappeared from http
nceulml bneurmboefrsviawbelree cceollusntaenddaspufroifllyowtos:ta1l00RN(cid:5)Al .alViqiuaobtles tthheegMels;1twseoqcuoenntcreoldoidlignoontuaclfefeocttidtehsen∼ot30co0mnptlseRmNenAta(rFyitgo- s://ac
a
of cells from serial dilutions (10−5 to 10−8) were plated ure1B). de
on Middlebrook 7H10 agar. The plates were incubated We then quantified the amount of Ms1 in mycobacte- mic
at 37◦C for 5 days before counting the colony forming rial cells using total RNA staining (GelRed, Supplemen- .ou
units (CFU) and calculating CFU/ml. The obtained data taryFigureS1A)andnorthernblothybridization(Supple- p.c
were used in subsequent calculations of amounts of Ms1 mentaryFigureS1B).InbothcaseswecomparedtheMs1 om
per cell. The quantity of Ms1 in the total RNA sample amount extracted from a known number of cells to seri- /na
(stained with GelRed) was determined by comparing the ally diluted standards of known concentration. From the r/a
Ms1 band density to a standard curve derived from the GelRedstainedgels,wecalculatedtheamountsofMs1and rtic
RThibeoRquualenrtiLtaotwionRawnagsedRoNnAe wLiatdhdeQru(aTnhteitrymOoneScsioenfttwifiacr)e. 5∼S28rR00NmAo(laescualceosnpterrolc)eilnl,srteastpioenctairvyelpy.hFasreomtotbhee∼n4o0r0thaenrnd le-abs
(Biorad). The Ms1 quantity determined by northern blots(SupplementaryFigureS1B)weobtainedacompara- tra
blottingwasdonebycomparingthesignalintensityofMs1 bleresultof∼600Ms1moleculespercell.Comparedtoex- ct/4
with a standard curve derived from serial dilutions of in ponentialphase,theMs1amountincreased∼130-foldupon 2/1
vitrotranscribedMs1.Northernblottingwasperformedas entryintostationaryphase(SupplementaryFigureS1B). 8/1
described previously (17). Briefly, RNAs were resolved on BesidesMs1,wealsodetecteda<100ntbandinstation- 17
a 7% polyacrylamide gel and transferred onto an Amer- ary phase (Figure 1A). Another prominent band of ∼130 63/2
sham Hybond-N membrane. Probes were 5(cid:2) 32P-labeled nt was detected in exponential phase. The identity of the 43
oligonucleotides(anti-Ms1:seeRNAisolation;anti-5S:5(cid:2)- sRNAs represented by these bands is currently unknown 50
7
CTGGCAGGCTTAGCTTCCGGGTTCGGGATG-3(cid:2)), andtheyarethesubjectsofanotherstudy.Hence,weestab- 0 b
and signals were captured in Fuji MS phosphor storage lishedthat Ms1 isa highly abundant sRNA present in the y g
screens and scanned with a Molecular Imager FX (BIO- cellinquantitiescomparabletothe6SRNAfoundinother ue
RAD)andquantifiedwithQuantityOnesoftware(Biorad). speciesandwedecidedtofurthercharacterizeMs1. st o
ForcalculatingtheincreaseofMs1expressioninstationary n 0
phaserelativetoexponentialphase,theMs1northernblot Ms1’spositiononthechromosome,itssyntenyandsecondary 9 A
signalwasnormalizedto5SrRNA. structureareconservedinmycobacteria pril 2
We began characterizing Ms1 by performing 5(cid:2)RACE 01
RESULTS (RapidAmplificationofthe5(cid:2)cDNAEnd)withtotalRNA 9
isolated from the M.smegmatis stationary phase cells and
Ms1ismycobacterialsRNAexpressedinaquantitycompa-
identifiedthefirstnucleotideofMs1tobeanadeninetran-
rableto6SRNAs
scribedfromposition6242368intheM.smegmatisgenome
First,wewantedtoexploretherelativeamountsofMs1in (Figure 1C). Upstream of this nucleotide we detected se-
M. smegmatis and compare it to the amounts of known quencesresemblingmycobacterial−10and−35consensus
6S RNAs in E. coli and B. subtilis (recently reviewed in promoterhexamersfortheprimaryσ factor(36).Thepu-
(23,24)).6SRNAinE.coli(184nt)andthemain6SRNA tative promoter sequence of Ms1 is conserved among M.
in B. subtilis (represented by the 190 and 201 nt doublet) smegmatis, M. bovis, M. avium and M. tuberculosis (Fig-
arehighlyexpressedduringstationaryphaseandbothare ure 1C). Although the transcription start sites of Ms1 ho-
clearly visible when total RNA is resolved on denaturing mologs in M. bovis and M. avium (10,35) were mapped
PAGEs(13,20).WeisolatedRNAsfromM.smegmatis,B. previously to the same or nearly identical positions, the
subtilisandE.colifromexponentialandstationaryphases 5(cid:2)terminus of MTS2823, the Ms1 homolog in M. tubercu-
NucleicAcidsResearch,2014 5
B ns-oligo 2 - - - - +
ns-oligo 1 - - - + -
A anti-Ms1 oligo - - + - -
B.s. E.c. M.s. RNase H - + + + +
EX ST EX ST EX ST total RNA + + + + +
1000 nt
600 nt
400 nt
Ms1
300 nt Ms1
200 nt
6S RNA
D
o
100 nt 5S rRNA 5S rRNA wn
lo
a
pre-tRNA de
tRNA pre-tRNA d fro
tRNA m
h
ttp
C s
conse nsus promoter sequence: TTGACAG 16-19bp TATAAT 6 242 368 Ms1 ://aca
M. smegmatis mc155 GGATTTTGGCCCTTGCTGTGACTCGGGACACGTAGTACAAAGGAGGCACGGAAGCTTGGCGAGGCCAAG de
CAGAATTGGGCCTTGCTGTGTTAAAGGTCTAGTAGTACAAAGGAACCACGGAAGCCCGGTGAGGCCAAG m
MM.. tbuobveisrc BuCloGsis H37Rv CGAAGCAAATTTTTGGGGGGCCCCTTTTGGCCTTGGTTGGTCTGACAAAGGGGGTTCCTTAAGGTTAAGGTTAACCAAAAAAGGGGAAAACCCCAACCGGGGAAAAGGCCCCCCGGGGTTGGAAGGGGCCCCAAAAGG ic.ou
p
M. avium **** ********** ** * ************* ****** ** ********* .c
o
m
D MSMEG_6173 Ms1MMSSMMEEGG__66117744 E ori /na
Ms1 r/a
morpholoagsicsaolc diaiftfeedre pnrtoiatetioinn, tIcralRn sfcarmipitlyio pnraol treeingulator, rticle
similar to HAD family hydrolase -a
b
s
tra
c
Figure1. MycobacterialMs1sRNAisexpressedinamountscomparableto6SRNAs.(A)TotalRNAwasisolatedfromBacillussubtilis(B.s.),Escherichia t/4
caonldi(sEta.cin.)eadnwditMhyGcoelbRaecdte.rIinumMs.msmegemgmataisti(sM,a.ns.)∼in30e0xpnotnsRenNtiAalw(EaXs)porersestnattiionnsatraytio(SnTar)ypphhasaes.eRceNllAssinwaemreoruensotslvceodmopnardaebnlaettuoriBng.spuobltyilaicsroyrlaEm.idcoelige6lSs 2/18
RNAs.(B)Beforeloadingontothegel,totalRNAfromM.smegmatisstationaryphasewasincubatedeitherwithacomplementaryDNAoligonucleotide /11
(anti-Ms1oligo)ornonspecificcontrololigonucleotides(ns-oligo1andns-oligo2)andtreatedwithRNaseH.(C)ThefirstnucleotideofMs1isadenine 76
tMrayncsocbraibcetedrifurmomtupboerscituiolonsi6s,2M42yc3o6b8acintertihuemgbenovoimsBe.CTGheanpdutMatyivceob−a1c0tearinudm−a3vi5umpr.oTmhoet5er(cid:2)esneqduseenqcueesn(cfersamofedp)reavrieoupselryfeidctelnyticfioendseMrvse1dhionmMo.losgmseignmthaetisse, 3/24
3
speciesarehighlightedinbold.Theconsensuspromotersequencewasadoptedfrom(36).(D)TheflankinggenesofMs1inM.smegmatisareshown.(E) 5
0
SchemeofMs1’spositioninthegenomeofM.smegmatiswithrespecttotheoriginofreplication(ori). 7
0
b
y
g
losis, was originally mapped further upstream (see Figure 1EandSupplementaryTableS1).Thispositionisindepen- u
e
1C) and no match for the promoter consensus sequence dentofgenomesize(SupplementaryFigureS2B).Next,we st o
upstreamofthisnucleotidewasreported(8).Thestrength alignedallMs1homologoussequencesandcomparedtheir n
0
of the putative Ms1 promoter as well as the identity of predicted secondary structures (40). In silico, we created a 9
the σ factor recognizing this promoter need to be further ‘common’ structure that is prevalent in all Ms1 homologs Ap
tested, but Ms1 is likely a single gene transcription unit. (see Supplementary Figure S3). These sequences fold into ril 2
The putative rho-independent Ms1 terminator was found alongdouble-strandedhairpinwithabubbleinthemiddle 01
9
by TransTermHP (37) and is located 304 nt downstream andtwoshorthairpinsclosetotheends.Theinternalbubble
ofthe5(cid:2)terminus.WeidentifiedhomologsofMs1inmany andtheadjacentdoublestrandedregionsarethemostcon-
mycobacterialspeciesandalsoinNocardiaandRhodococ- served elements. Although the primary sequences of Ms1
cus (Supplementary Table S1), which belong to the group homologsarenotidentical,theirpredictedsecondarystruc-
of actinobacteria. Based on sequence similarity searches tures are similar, which suggests that these sRNAs could
(38), Ms1 was found in 33 actinobacterial species; for the havethesamefunction(s).
phylogenetic tree (39) of Ms1 homologs see Supplemen-
tary Figure S2A. When Ms1 is present, its genomic con-
Ms1ispresentinalargemacromolecularcomplex
text (Figure 1D) is partially conserved also in other acti-
nobacterialspecies(SupplementaryTableS1).Inaddition, As a first approach to explore whether or not Ms1 is part
Ms1isveryoften(in84%ofcases)locatedatthesamepo- ofaproteincomplex(analogousto6SRNAorribosomal
sitiononthechromosome:closetothereplicationstartsite RNAs),wefractionatedM.smegmatisandE.coli(asacon-
(ori)withthedirectionoftranscriptiontowardori(Figure trol)stationaryphasecelllysatesbyglycerolgradientultra-
6 NucleicAcidsResearch,2014
centrifugation. After ultracentrifugation, large complexes down RNAP (Supplementary Figure S4A) from an M.
suchasribosomesaresedimentedatthebottomofthetube smegmatisstationaryphaselysateandisolatedRNAassoci-
whereas e.g. 6S RNA is found in the middle of the gradi- atedwithRNAP.InteractionofRNAswithFLAG-tagged
ent. Importantly, molecules that are in the same complex RNAPwasassessedusingRT-qPCR,withprimersspecific
must be found in the same fractions of the gradient. This for Ms1, as well as 16S rRNA, rpoC and mysA mRNAs
methodwaspreviouslyusedtoidentifyproteinsthatarein asnegativecontrols.WhileMs1interactedwiththeFLAG-
complex with E. coli 6S RNA (21). The majority of Ms1 tagged RNAP, we observed no interaction of 16S rRNA,
sedimented in fractions 10–13 (Figure 2A) indicating that rpoCormysAmRNAs(SupplementaryFigureS4B).This
Ms1 was part of a large protein complex. Using western experiment, however, did not enable us to distinguish the
blotting, we found the RNAP (cid:3) subunit in Ms1 fractions; RNA polymerase core from the holoenzyme ((cid:3) is present
however, no σA was found there (Figure 2A), since σA is inbothcomplexes).
notinthecomplexwithMs1(17).Usingmassspectrometry TodistinguishwhetherMs1bindstothecoreorholoen-
weidentifiedallabundantproteinspresentintheMs1frac- zyme (σA-containing) form of RNAP we performed im- Do
w
tions(Figure2BandSupplementaryTableS2)-thepotential munoprecipitationswithtwospecificantibodies.Bothanti- n
interactingpartnersofMs1sRNA.Importantly,theRNA bodiesefficientlyimmunoprecipitatedE.coliandM.smeg- loa
polymerase(cid:3),(cid:3)(cid:2)and(cid:2)subunits,butnoσAorotherσ fac- matis proteins (see Figure 4A showing the amounts of de
d
tors,wereamongtheproteinsenrichedintheMs1fractions. pulled-down primary σ factors and RNAP core (cid:3) sub- fro
Finally,incontrasttoE.coliwhere6SRNAco-sediments units). The anti-σA/σ70 antibody (2G10) recognized both m
with σ70 and (cid:3) (Figure 2C, fractions 8–12), no highly ex- free σA/σ70 as well as σA/σ70 in complex with RNAP http
pthreesMse.dsmsReNgmAatsipseσcAifi-cRaNllyApPeahkoelodeinnzythmees(aFmigeufrrea2ctAio,nfrsaacs- (aRntNiA(cid:3))Pwhaosloperenvziyomusel)y. rTehpeoratnedti-tRoNmAaPinlaynbtiibnoddtyhe(8RRNBA13P, s://ac
tions7–8). coredevoidofσ factors(41,42)andweverifiedthisforsta- ad
e
tionaryphaseM.smegmatis(Figure4B,lane3)andE.coli m
ic
Ms1interactswithRNApolymerasecoredevoidofσ factors cmeellrsas(Fesiguubruen4itBs,((cid:2)la,n(cid:3)e,(cid:3)1)(cid:2)).wInefaodudnidtioonnlytotwcooroethReNrpAropteoilnys- .oup
.c
To identify which of the proteins from the Ms1 fractions thatimmunoprecipitatedwiththeanti-RNAP(8RB13)an- o
m
truly interacted with Ms1, we pulled down Ms1 binding tibodyinE.coli(andnoneinM.smegmatis)andidentified /n
a
partnersdirectly.First,wepreparedMs1sRNAbyinvitro thembymassspectrometry(seeSupplementaryTableS4)as r/a
transcription. In addition, we also prepared Ms1 with a NusG(transcriptionelongationfactor)(43)andtheDNA- rtic
deleted internal bubble-designated ‘Ms1nb’ (Figure 3A). binding factor CbpA (44). Importantly, no σ factors were le
-a
We then ligated biotinylated Cytidine (Bis)phosphate to detectedeitherinM.smegmatisorinE.coliimmunoprecip- b
s
the 3(cid:2)ends of both RNAs, and attached these biotinylated itations.WethenisolatedthecoimmunoprecipitatedRNA tra
c
RNAstostreptavidinbeads(Figure3B).Wesubsequently andvisualizedthisRNAeitheronPAGEgels(Figure4C, t/4
incubatedtheMs1andMs1nb-coatedbeadswiththelysate lane7)ormeasureditsamountbyRT-qPCR(Figure4D). 2/1
from M. smegmatis cells from exponential and stationary We found that almost 40% of the Ms1 in stationary cells 8/1
phases. Ms1 is expressed mainly in stationary phase but was bound to mycobacterial core RNA polymerase (Fig- 17
6
we avoided using only stationary phase lysate because it ure 4D, the input represents the total amount of Ms1 iso- 3
/2
contains abundant endogenous Ms1 that could compete latedfromthecelllysate;notethattheamountofdetected 4
3
for interacting proteins with biotinylated Ms1 and there- Ms1 depends on the amount of the antibody used). No 50
fore we might miss some of the interacting proteins. Us- other small RNAs such as 5S rRNA, tRNAs or the <100 70
ing this approach, we pulled down significant amounts of ntand∼130ntRNAsidentifiedinFigure1Awerebound by
g
thecoresubunitsofRNAPwithMs1,butconsiderablyless to the RNAP core (Figure 4C, lane 7), indicating that the u
e
wThitihsiMndsi1cnabte,sfrtohmatbthoethinetxeprnoanlebnutibabllaenwdhsitcahtiiosnsatrryucpthuarasellsy. Mcosn1t-rRolNRANPAcso(rtewiontmerRacNtiAosn:imsyspsAeceifincc.oAdicncgorσdAin,grplyo,Cthernee- st on
conserved among Ms1 homologs is important for the in- coding RNAP subunit (cid:3) and 16S rRNA) also did not in- 09
A
teractionwithRNApolymerase.NoRNApolymerasein- teractwithRNAP(Figure4D).Inagreementwithprevious p
teractedwiththecontrolbeadswithoutRNA(Figure3C). results((17)andglycerolgradientultracentrifugation,Fig- ril 2
In addition to core RNAP subunits, Ms1 pulled down a ure 2B), we did not see any binding of Ms1 to σA or the 01
∼80kDabandconsistingoftwoproteinsaccordingtothe RNAP-σAholoenzyme(Figure4C,lane6,Figure4D).This 9
massspectrometryanalysis:transcriptionterminationfac- is in sharp contrast with the situation in E. coli, where al-
tor Rho and polyribonucleotide nucleotidyltransferase in- mostall6SRNAwaspulleddownwiththeantibodyagainst
volvedinRNAdegradation(seeSupplementaryTableS3). σ70,as6SRNAinteractswiththeRNAP-σ70complex(21)
Importantly, no σ factor or any other proteins found in (Figure4C,lane2,Figure4D).Furthermore,inE.colino
the glycerol gradient Ms1 fractions interacted with Ms1, 6SRNAcoimmunoprecipitatedwiththeanti-RNAPanti-
although we cannot exclude the possibility that a highly body(Figure4C,lane3).Thus,incontrastto6SRNA,Ms1
substochiometricpresenceofσ factors(withrespecttothe interactsspecificallywiththeRNAPcoredevoidofσ fac-
othercoresubunitsofRNAP)mightbebelowourdetection tors,representinganoveltypeofRNAP–sRNAinteraction
limit. module.
To verify that Ms1 associates with RNAP, we utilized a
M. smegmatis strain carrying the FLAG-tag on (cid:3) (RpoB)
(29), one of the core subunits of RNAP. Via (cid:3) we pulled
NucleicAcidsResearch,2014 7
A Mycobacterium smegmatis
top Ms1 RNA bottom
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
400 nt
300 nt Ms1 RNA
200 nt
5S rRNA
100 nt
tRNA
D
o
w
RNAP ( ) n
A loa
d
1 e
relativeamount0.05 MRANs1A PR N(A) d from h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ttp
s
://a
c
a
Ms1 RNA d
B e
9 10 11 12 13 14 15 m
saucceAt y(l2-/-porxoopgioluntyalr-actoee ennzyzmymee A) RNAP ( ´ ) ic.ou
p
carboxylase Rho transcription termination factor .c
o
propionyl-CoA carboxylase m
glutamine synthetase 1 /n
alcohol dehydrogenase RNAP ( ) ar/a
S30AE ribosomal protein (RafS) rtic
le
-a
b
s
tra
c
t/4
2
C Escherichia coli /1
8
top 6S RNA bottom /11
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6
400 nt 3/2
300 nt 4
3
5
0
200 nt 7
6S RNA 0 b
y
g
u
100 nt 5S rRNA es
t o
n
tRNA 0
9
A
p
RNAP ( ) ril 2
70 01
1 9
relativeamount0.05 6RS7N0 ARPN A( )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure2. Ms1ispresentinalargeproteincomplex.LysatesfromstationaryphaseMycobacteriumsmegmatis(A)orEscherichiacoli(C)cellswere
fractionatedbyultracentrifugationinglycerolgradients;individualfractions(1–20;toptobottom)werecollectedandtheRNAspresentineachfraction
wereresolvedondenaturingpolyacrylamidegelsandstainedwithGelRed.TheRNAP(cid:3)subunitandprimaryσfactorswerevisualizedbywesternblotting.
RelativeamountsofsRNAs(Ms1and6SRNAvisualizedbyGelRed)andproteinsdetectedbywesternblottingareshownbelow.(B)ProteinsfromMs1
fractionswereseparatedbySDS-PAGEandstainedwithCoomassie.RNAPsubunitsbutnoσ factorswereamongthemostabundantproteinsinMs1
fractions.Fordetailsonthemassspectrometryanalysis,seeSupplementaryTableS2.Thisexperimentwasperformed3×withidenticalresults.
8 NucleicAcidsResearch,2014
B EX ST C EX ST
A empty Mbse1adMss1nbempty bMse1adMss1nb [kMDWa] empty Mbse1adMss1nbempty bMse1adMss1nb
Ms1 1000 nt
´
600 nt 160

110
400 nt Rho factor
80
Ms1nb 300 nt PNPase
60
200 nt 50

40
D
30 o
w
n
20 lo
a
100 nt de
d
sRtreNpAt aavtitdaicnh beeda tdos from
h
ttp
s
Figure3. Ms1pullsdownRNApolymerasecore.(A)PredictedstructuresofMs1anditsmutantvariantlackingthecentralbubble,Ms1nb.BothMs1and ://a
c
Ms1nbRNAswerepreparedinvitro,biotinylatedandcoupledtostreptavidinbeads.(B)OnetenthoftheRNA-coatedstreptavidinbeadswererunona a
d
urea-polyacrylamidegelasacontrolfortheefficiencyofbiotinylationandcoupling.(C)TheMs1-andMs1nb-beadswereincubatedwithMycobacterium e
m
smegmatisexponentialorstationaryphaselysates.ProteinsthatassociatedwithMs1andMs1nbwereseparatedbySDS-PAGE,stainedwithCoomassie ic
andMs1-interactingproteinsanalyzedbymassspectrometry.CoresubunitsofRNAPinteractedwithMs1sRNA,butconsiderablylesswithMs1nband .o
notwithemptybeads.Thisexperimentwasrepeated3×withidenticalresults. up
.c
o
m
Ms1overexpressioninexponentialphasedoesnotaffectσA IncreasedlevelofσAinstationaryphaseofgrowthdiminishes /na
bindingtoRNApolymerase theamountofMs1-RNApolymerasecomplex r/a
rtic
WeaskedwhetherMs1sRNAoverexpressioninexponen- AsMs1seemstonotinfluencethelevelofRNAPincom- le
tialphasecoulddisrupttheassociationoftheRNAPcore plexwithσA,weaskedthereversequestion:CanσA affect -ab
withσA.Toinvestigatethis,wetransformedM.smegmatis theamountofMs1boundtotheRNAPcore?Totestthisin stra
with a plasmid carrying Ms1 under the control of a ribo- vivo,weoverexpressedσAinstationaryphasecellsfromthe ct/4
somal RNA promoter (rrnB) that is strongly active in ex- pJAM2-σAplasmid,whichcontainstheσA(mysA)geneun- 2
/1
ponential phase (9,33). In vitro, Ms1 with a deleted inter- der an inducibleacetamidase promoter (31). As a control, 8
/1
nal bubble (Ms1nb) interacted much less with core RNA weusedcellswithanemptypJAM2.Acetamidewasadded 1
7
pfoorleymweeraalsseocpormeppaarreeddtaostthreaiinntwacitthMMs1s1(Fnbiguurned3eCr)t.hTehrernreB- aσtAtheexpernetsrsyioinn,tocesltlastiwoenraeryhaprhvaessete(dOaDft6e0r0 ∼61h.6a)ntdotihneduince- 63/24
promoter to test the importance of this predicted struc- creaseintheσAproteinlevelwasconfirmedbywesternblot- 35
0
tural element in vivo. Both RNAs were expressed in high ting(Figure6A).Interestingly,overexpressionofσAalsoin- 70
amountsinexponentialphase(Figure5A).Next,wedeter- creasedtheexpressionofMs1∼2.5-fold(Figure6B),which by
minedwhethertheartificiallyoverexpressedMs1couldin- is in agreement with the presence of the putative σA-like gu
e
teractwiththeRNAPcoreinexponentialphase.Ms1inter- promoter sequence upstream of Ms1 in the genome (Fig- s
actedwiththeRNAPcoreinasimilarmannertoendoge- ure1C).WethenimmunoprecipitatedtheRNAPcoreand t on
nous Ms1 in stationary phase (Figure 5B, ∼20% of total measuredtheamountofMs1coimmunoprecipitatedfrom 09
Ms1presentinbacterialcellwaspulleddownwithRNAP cellswithpJAM2-σAoverexpressingσAandcompareditto A
p
when2(cid:5)gofanti-RNAPantibody(8RB13)wereused).In cells with pJAM2. In cells overexpressing σA we observed ril 2
addition,<5%ofthetotalMs1nbassociatedwithRNAP, an∼8-foldreductionintheamountsofMs1boundtothe 0
1
9
indicatingthatthepresenceofthecentralbubbleincreases RNAP core (Figure 6C). Although this reduction can be
bindingtoRNAP.Incontrasttothepreviousobservations partially explained by the increase in the total amount of
withaM.tuberculosisstrainwithanoverexpressedMs1ho- Ms1inpJAM2-σA cells,the8-folddecreaseinMs1bound
molog(8),Ms1overexpressiondidnotslowthegrowthof tocoreRNApolymerasesuggeststhatσAnegativelyaffects
M.smegmatis.StrainswithelevatedlevelsofMs1orMs1nb theMs1–RNAPinteraction.
did not exhibit any significant difference in growth rate
fromthestraincarryinganemptycontrolvector(pJAM2)
TheamountofRNApolymeraseholoenzymedecreasesinsta-
(Figure5C).Moreover,theoverexpressionofMs1didnot
seemtoaffecttheamountoftheRNAP(cid:3)subunit(i.e.the tionaryphase
amount of RNAP core) that was bound to σA, indicating Finally, we asked why mycobacterial Ms1 interacts with
that the level of RNAP holoenzyme is unchanged despite coreRNAPwhereas6SRNA,whichispresentinthema-
theelevatedMs1level(Figure5D). jorityofotherspecies,bindstotheRNAPholoenzyme.A
possible explanation could be that mycobacteria may dif-
NucleicAcidsResearch,2014 9
A B stationary ph
E.coli M. smegmatis
E.c. M.s.
ex st ex st
P G P G
0% INPUT70A/nti- nti-RNAP ontrol IgG 0% INPUT70A/nti- nti-RNAP ontrol IgG 0% INPUTA70/nti- nti-RNAP ontrol IgG 0% INPUT70A/nti- nti-RNAP ontrol IgG anti-RNA control Ig anti-RNA control Ig
8a a c 8a a c 8a a c 8a a c ´ ´
RNAP ( ) RNAP ( )
70 A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

C E.coli M. smegmatis CbpA
D
10%INPUT 70Aanti-/anti-RNAPcontrol IgG 10%INPUT70Aanti-/anti-RNAPcontrol IgG 1000 nt D80 NusG 1 2 3 4 ownloaded fro
m
600 nt
340000 nntt 6700 antibodies: https
Ms1 200 nt ut50 a(notin-RlyN MA.P s m8 eggmatis) ://aca
6S RNA % inp40 aannttii--R7N0/APA 2 g dem
ic
5S rRNA 100 nt 30 control IgG .ou
20 p.c
o
10 m
/n
a
1 2 3 4 5 6 7 8 0 r/a
6S RNA rpoD rpoC rR16NSA Ms1 mysA rpoC r1R6NSA rtic
le
Escherichia coli Mycobacterium smegmatis -a
b
s
coimmunoprecipitated RNA tra
c
Figure4. Ms1interactswithcoreRNApolymerase.(A)AntibodiesagainstRNAP(anti-RNAP;clonename8RB13-itrecognizesthecoreformofRNAP) t/42
andσA/σ70 (anti-σA/(cid:6)70,clonename2G10-itrecognizesalsotheholoenzymecontainingσA/σ70)efficientlyimmunoprecipitatedproteinsfromboth /1
8
MycobacteriumsmegmatisandEscherichiacoli.Immunoprecipitatedproteinswerevisualizedbywesternblotting.ex:exponentialphase;st:stationary /1
phase.‘ControlIgG’isamousenonspecificIgGusedasanegativecontrol.Theexperimentwasrepeated3×withidenticalresults.(B)TheRNAP 17
antibody(8RB13)immunoprecipitatedthecoreformofRNApolymerase.ImmunoprecipitatedproteinsfromE.coli(E.c.)andM.smegmatis(M.s.) 63
stationaryphaselysateswereseparatedbySDS-PAGEandstainedwithCoomassie.‘ControlIgG’standsforamousenonspecificIgGusedasanegative /2
4
control.AdditionalE.coliproteinswereidentifiedbymassspectrometry(seeSupplementaryTableS4).Noσfactorsweredetected.Nosignificantbands 3
5
besidesRNAPsubunitswerefoundforM.smegmatis.(C)RNAscoimmunoprecipitatedwithRNAP(2(cid:5)gofantibody)andσA/σ70 antibodieswere 0
7
resolvedonPAGEandstainedwithGelRedor(D)quantifiedbyRT-qPCRandnormalizedtotheinput.InimmunoprecipitationsquantifiedbyRT- 0
qPCR,twoamountsoftheanti-RNAPantibody(8RB13)wereused(2and8(cid:5)g);theamountofcoimmunoprecipitatedMs1increasedwiththeincreased by
amountoftheRNAPantibody,suggestingthattheconcentrationoftheRNAPantibodywasnotsaturating.E.coli6SRNAcoimmunoprecipitatedwith g
u
σ70 whichisincomplexwithRNApolymerase.NoneofthecontrolmRNAs-twomRNAs:rpoD(σ70 mRNA),mysA(σA mRNA),rpoC(RNAP(cid:3)(cid:2) e
s
subunitmRNA)and16SrRNAwerecoimmunoprecipitatedwiththeantibodiesused.‘ControlIgG’ismousenonspecificIgGusedasanegativecontrol. t o
ErrorbarsareSEM(standarderrorofthemean)fromatleastthreeindependentexperiments. n 0
9
A
p
finersitnattihoeniarraympohuanset.sTohfetrheefoRreNwAepcoolmympaerraedsethhoeloamenozuynmtes FRuNrAthPercmoroereh,adinanlaetveesntalotiwoenracrhyapnhceastoef(o1r2mhahinotlooeint)z,ytmhee ril 20
oftheRNAPcorethatcoimmunoprecipitatedwithσ70/σA withσA inM.smegmatisasweobservedadecreaseinthe 19
(the ratio of RNAP to σA) in exponential versus station- totalamountofσAinthecells(relativetotheRNAP(cid:3)sub-
ary phase both in M. smegmatis and in E. coli by analyz- unit,Figure7A)whereasinE.colithetotallevelofσ70did
ing the data from Figure 4A. While in E. coli, a similar notchangeeven16hafterenteringstationaryphase(com-
amountofRNAP(monitoredbythelevelof(cid:3))coimmuno- paretheamountofσ70totheRNAP(cid:3)subunit,Figure7A),
precipitated with σ70 both in exponential and stationary suggestingthatthedeclineinσA wasspecificforM.smeg-
phases(comparelanes2and6inFigure4A),inM.smeg- matis.ThisdifferenceintheσAlevelinM.smegmatisisnot
matisfewerRNAPscoimmunopreciptatedwithσAafterthe visible when the cells are harvested in the early stationary
transition from exponential to stationary phase (lanes 10 phase (Figure 4A). This means that the adaptation to sta-
and 14 in Figure 4A). Thus, in M. smegmatis there was a tionaryphase(withrespecttoσA associationwithRNAP)
smaller amount of the σA-containing holoenzyme in sta- inM.smegmatisis2-fold:thebindingofσAtoRNAPisde-
tionaryphase whencomparedtoexponentialphase,while creasedandthetotallevelofσAdrops.Thus,M.smegmatis
inE.colitheamountoftheholoenzymewasaboutthesame.
10 NucleicAcidsResearch,2014
A exponential phase B C rrnB-Ms1 rrnB-Ms1nb pJAM2
wt pJArMr2nB-rrMnsB1-nMbs1 30 000..68
0
1000 nt D60.4
20 O
600 nt
400 nt ut
300 nt Ms1 np 0.2
Ms1nb % i
200 nt 10 0
0 5 10 15 20 25
[hrs]
D rrnB rrnB
100 nt 5S rRNA 0 pJAM2 -Ms1 -Ms1nb Do
P G P G G G G w
ranti-RNArnB-Mcontrol Igs1 rranti-RNAnB-Mcontrol Igs1nb RNAP σ(βA)σAanti- control Ig σAanti- control Ig σAanti- control Ig nloaded from
h
ttp
fFriogmurest5ra.inOsvcearrerxypinregspsiloansmoifdMstsh1aitnceoxnptoanineendtieailthpehraMse.s(1Ao)rTMotsa1lnRbNuAndwerasthiseorlrantBedpfrroommottheer.wTthceoRntNroAlsstwraeirnetthhaetncroensotalvineeddotnhedeenmaptutyripnJgApMoly2avcercytloarmaindde s://a
c
gelsandstainedwithGelRed.BothMs1andMs1nbwerehighlyexpressedinexponentialphaseofgrowth.(B)CoreRNAPwasimmunoprecipitated a
d
with2(cid:5)goftheanti-RNAPantibody(8RB13)fromstrainsoverexpressingMs1orMs1nb.CoimmunoprecipitatedRNAwasisolatedandtheamountof e
m
Ms1orMs1nbquantifiedbyRT-qPCR.‘ControlIgG’isamousenonspecificIgGusedasanegativecontrol.ErrorbarsareSEMfromthreeindependent ic
experiments.(C)Growthcurvesofthecontrolstrain(pJAM2)andstrainsoverexpressingMs1(rrnB-Ms1)orMs1nb(rrnB-Ms1nb)werecompared.The .o
graphshowsonerepresentativeexperiment;theexperimentwasrepeated3×.(D)σAwasimmunoprecipitatedfromthecontrolstrain(pJAM2)andstrains up
overexpressingMs1(rrnB-Ms1)orMs1nb(rrnB-Ms1nb)andtheamountofthecoimmunoprecipitatedRNAP(cid:3)subunitwasdeterminedbywestern .co
blotting.NodifferenceintheamountofRNAP-boundσAwasdetecteduponMs1orMs1nboverexpression.‘ControlIgG’isamousenonspecificIgG m
usedasanegativecontrol. /na
r/a
rtic
A A le
A M2 M2- E.coli M.sm -ab
A A s
pJ pJ ex st ex st tra
RNAP ( ) RNAP ( ) ct/4
A 70/ A 2/1
8
B C /11
7
n 3 6
o B 3
si 1 /2
s 4
e 3
Fold-change in Ms1 expr 012 pJAM2 pJAM2- A Relative amount of Ms1-bound to RNAP0000....02468 pJAM2 pJAM2- A C 6S RRNRNANAAPP RNAP 5070 by guest on 09 April 20
1
9
Figure 6. σA overexpression decreases the amount of Ms1-RNAP. (A) Ms1 RNA
ProteinlysatesfromMycobacteriumsmegmatiscarryingpJAM2-σAorthe
emptypJAM2plasmidwereculturedfor6hin0.2%acetamide(whichin-
ducesexpressionfromthepJAM2acetamidasepromoter)andharvested
instationaryphase.TheamountofσA andRNApolymerase(cid:3)subunit
(RNApolymerase(cid:3) servedasaloadingcontrol)wasdetectedbywest-
ernblotting.Uponinductionwithacetamide,theσAlevelincreasedinM.
Figure7. ModesofinteractionofsRNAswithbacterialRNAP.(A)The
smegmatisstationaryphasecellscarryingpJAM2-σAbutnotincellscar- levelofσA relativeto(cid:3)droppedinMycobacteriumsmegmatiscellshar-
ryingtheemptyvector.(B)Ms1sRNAexpressionincreases∼2.5-foldafter
vested12hafterentryintostationaryphase.InEscherichiacoli,therelative
theoverexpressionofσA(Ms1RNAlevelwasmeasuredbyRT-qPCRand proteinlevelofσ70to(cid:3)remainedunchangedevenafter16hinstationary
normalizedto16SrRNA).(C)TheamountofMs1coimmunoprecipitated phaseofgrowth.Theexperimentwasrepeated3×withidenticalresults.
withRNAPcoredecreasedmorethan8-foldaftertheoverexpressionofσA
(B)6SRNA(e.g.E.coli,Bacillussubtilis)bindstoRNAPcontainingthe
(coimmunoprecipitatedMs1wasfirstnormalizedtotheinputandthento mainσfactor.(C)Ms1(mycobacteria)bindstotheRNAPcoreintheab-
controlcellswiththeemptypJAM2;thegraphshowstheaveragesfrom senceofσ factorsandthepresenceofσAdecreasesthisinteraction.
twoindependentexperimentsandtheerrorbarsindicatetherange).