Sanders, K., Lin, CL., Smith, AJ, Cronin, N., Fisher, G., Eftychidis, V., ... Dillingham, MS

14 Pages·2017·2.51 MB·English

Sanders, K., Lin, C. L., Smith, A. J., Cronin, N., Fisher, G., Eftychidis,

V., McGlynn, P., Savery, N. J., Wigley, D. B., & Dillingham, M. S.
(2017). The structure and function of an RNA polymerase interaction
domain in the PcrA/UvrD helicase. Nucleic Acids Research, 45(7),
3875-3887. [gkx074]. https://doi.org/10.1093/nar/gkx074
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Publishedonline4February2017 NucleicAcidsResearch,2017,Vol.45,No.7 3875–3887
doi:10.1093/nar/gkx074
The structure and function of an RNA polymerase
/
interaction domain in the PcrA UvrD helicase
Kelly Sanders1,†, Chia-Liang Lin2,†, Abigail J. Smith1,†, Nora Cronin2, Gemma Fisher1,
Vasileios Eftychidis3, Peter McGlynn3, Nigel J. Savery1, Dale B. Wigley2 and Mark
S. Dillingham1,*
1DNA:ProteinInteractionsUnit,SchoolofBiochemistry,BiomedicalSciencesBuilding,UniversityofBristol,Bristol
BS81TD,UK,2InstituteofCancerResearch,ChesterBeattyLaboratories,237FulhamRoad,LondonSW36JB,UK
andSectionofStructuralBiology,DepartmentofMedicine,ImperialCollegeLondon,SouthKensingtonCampus,
LondonSW72AZ,UKand3DepartmentofBiology,UniversityofYork,WentworthWay,YorkYO105DD,UK
ReceivedSeptember23,2016;RevisedJanuary17,2017;EditorialDecisionJanuary18,2017;AcceptedJanuary25,2017
ABSTRACT ble for ATP-dependent DNA translocation and unwind-
ing, and that their targeting to different pathways is often
The PcrA/UvrD helicase functions in multiple path-
achieved via the modular addition of different specificity
waysthatpromotebacterialgenomestabilityinclud-
domains,eitherflankingorinsertedwithinthecorehelicase
ingthesuppressionofconflictsbetweenreplication domains(6).
and transcription and facilitating the repair of tran- An interesting example is provided by the UvrD heli-
scribed DNA. The reported ability of PcrA/UvrD to case (also annotated Helicase II, or PcrA in many Gram
bind and backtrack RNA polymerase (1,2) might be positivebacteriaincludingBacillussubtilis)whichhasbeen
relevant to these functions, but the structural ba- implicated in nucleotide excision repair (NER), mismatch
sis for this activity is poorly understood. In this repair, homologous recombination and rolling circle repli-
cation mechanisms (7–13). This multi-functionality is re-
work, we define a minimal RNA polymerase inter-
flected in the ability of UvrD/PcrA to interact physically
action domain in PcrA, and report its crystal struc-
and functionally with many different partner proteins in-
tureat1.5A˚ resolution.ThedomainadoptsaTudor-
cluding UvrB, MutL, MutS, RecA and RepC/D (13–20).
like fold that is similar to other RNA polymerase in- We and others have recently shown that PcrA/UvrD also
teraction domains, including that of the prototype
interactswithRNApolymerase(RNAP)(1,14,21),andthis
transcription-repaircouplingfactorMfd.Removalor interaction could be important for the UvrD-dependent
mutationoftheinteractiondomainreduces theabil- backtracking of stalled RNA polymerase (1). It was sug-
ity of PcrA/UvrD to interact with and to remodel gestedthatthisactivityhelpstorecruittheNERmachinery
RNA polymerase complexes invitro. The implica- tositesofUVdamage,actingasanalternativepathwayof
tionsofthisworkforourunderstandingoftheroleof transcription-coupledrepair(TCR)inadditiontothewell-
PcrA/UvrD at the interface of DNA replication, tran- characterized Mfd pathway (for reviews see (22–25)). This
abilityofUvrDtoremodelRNAP-DNAcomplexesmight
scriptionandrepairarediscussed.
also be relevant to the ability of PcrA/UvrD to suppress
conflictsbetweenreplicationandtranscription,aproperty
INTRODUCTION itshareswiththeverycloselyrelatedhelicaseRep(although
RepdoesnotinteractwithRNAP)(26–28).TheextremeC-
Helicases are ubiquitous, abundant and diverse enzymes
terminalregionofPcrA/UvrDisimportantforinteraction
playing a wide variety of different roles in cellular nu-
withbothRNAP(14,29)andUvrB(30).However,despite
cleic acid metabolism (3). Several superfamilies (SF) of
itsapparentroleasaproteininteractionhubthattargetsthe
theseenzymeshavebeendescribedonthebasisofprimary
helicase to physiological substrates, there is no clear phe-
structure and SFI and II, which are non-hexameric heli-
notype established for removal of the C-terminal domain
cases, are by far the largest groups (4). Bacterial cells typ-
(CTD), and its structure has never been resolved. In thir-
ically encode several SFI enzymes that function in differ-
teenstructuresofthePcrA/UvrDproteinfromvariousor-
entgenomereplication,maintenanceandexpressionpath-
ganisms, this region of the protein was either removed to
ways (5). Structural studies have shown that SFI enzymes
share highly conserved core helicase domains responsi-
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Email:[email protected]
†Theseauthorscontributedequallytothisworkasfirstauthors.
(cid:2)C TheAuthor(s)2017.PublishedbyOxfordUniversityPressonbehalfofNucleicAcidsResearch.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0/),which
permitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
3876 NucleicAcidsResearch,2017,Vol.45,No.7
aid crystallization or is disordered in the final model (31– containingonlytheproteinofinterestweredialysedat4◦C
36)(SupplementaryFigureS1). overnightagainststoragebuffer(20mMTris–HClpH8.3,
Inthisreport,wedefineasmallfoldedRNApolymerase 200mMKCl,1mMEDTApH8.0,2mMDTT,20%(v/v)
interactiondomaininGeobacillusstearothermophilusPcrA glycerol).UvrD(cid:2)CwaspurifiedfromBL21DE3cellstrans-
and have solved its structure at high resolution. Based on formedwithpETDUET-UvrD asdescribed(15).
1–647
itssimilaritytootherTudor-likedomains,weidentifycon- Biotinylated PcrA and biotinylated PcrA-Ct (including
served residues on its surface that are likely to be directly residues653–724ofthenativeprotein)wereproducedusing
involved in the interaction with RNAP. Mutation of these vectorsandpurificationprotocolsthathavebeendescribed
residues, or the complete removal of the interaction do- previously (14). An equivalent vector for expression of bi-
main, substantially reduces the ability of PcrA/UvrD to otinylatedPcrA-sCtwascreatedbydeletingashortregion
bind to RNAP and to remodel transcription elongation ofthePcrA-Ctconstruct.Thisyieldedavectorforoverex-
complexesinvitro. pressionoftheextremeC-terminalregionofPcrA(residues
673–724) fused to an N-terminal AviTag sequence (MSG
LNDIFEAQK*IEWHEGGG;theasteriskindicatesthe
MATERIALSANDMETHODS biotinylatedlysine).Thisproteinwasoverexpressedandpu-
Proteinexpressionandpurification rifiedusingthesamemethodasforthePcrA-Ctconstruct.
Constructsfortheexpressionofthehistidine-taggedPcrA
His-tagged Escherichia coli RNAP holoenzyme was pu- C-terminus were produced by cloning synthetic DNA (In-
rified as described (37). GreB protein was a gift from vitrogen) into the pET47b vector (Novagen). The PcrA-
Terence Strick. Purified ParB protein was a gift from CtconstructexpressesaproteinwithanN-terminalhexa-
James Taylor. Biotinylated BSA protein was purchased histidinetagfusedviaa3Ccleavablelinkertoresidues653–
from ThermoScientific. UvrD and UvrDK708A were puri- 724 of Geobacillus stearothermophilus PcrA. The sequence
fied from BL21(DE3) cells transformed with pETDUET- of the tag is MAH HHH HHS AAL EVL FQG *PGG
UvrD (15) or pETDUET-UvrDK708A, which was made Gwheretheasteriskindicatesthepositionof3Ccleavage.
frompETDUET-UvrDbysitedirectedmutagenesis.Cells The PcrA-sCt construct only codes for residues 673–724
were grown at 37◦C to an A600 of ∼0.5 before being used of the native PcrA protein, but is otherwise equivalent to
toinoculate1lofLB+100(cid:2)g/mlampicillintoanA600 of PcrA-Ct.Pointmutationsweremadeinalloftheabovevec-
∼0.025.WhencellsreachedanA600 of0.2theyweretrans- torsusingtheQuikChangeIIkit(Invitrogen)andthecon-
ferredto18◦CandwhentheA600reached∼0.5,1mMIPTG structswereverifiedby DNAsequencing(Sequencingser-
wasaddedtoinduceproteinexpression.Cultureswerethen vice, University of Dundee). His-tagged PcrA-Ct and his-
grown overnight at 18◦C and the cells were harvested by tagged PcrA-sCt were overexpressed in BL21(DE3) with
centrifugationat4◦C.Thecellpelletwasresuspendedin20 appropriate antibiotics and harvested using the same pro-
mloflysisbuffer(20mMTris–HClpH8.3,10%(v/v)glyc- tocol as for full-length PcrA (14). Following sonication,
erol,200mMNaCl,5mMEDTApH8.0,0.5mMEGTA the proteins were bound to a 5 ml HisTrap column (GE
pH8.0,1mMDTT)containing4mgoflysozymeandin- Healthcare) in a buffer containing 50 mM Tris–Cl, pH7.5
cubatedonicefor30min.250(cid:2)l4%sodiumdeoxycholate and 200 mM NaCl, and eluted over a 20–500 mM imida-
wasaddedandcellswerethenincubatedoniceforanother zolegradient.Whereappropriate,thehis-tagwasremoved
30 min. To increase the UvrD solubility, NaCl concentra- withHRV3Cproteaseovernightat4◦C(ThermoScientific,
tionwasincreasedto∼450mMbyadding1.2ml5MNaCl manufacturer’sinstructions)whiledialysingagainstabuffer
andstirringfor15minat4◦C.Thecellswerelysedbyson- containing50mMTris–Cl,pH7.5,1mMEDTA,200mM
icationandthesolublefractionwasrecoveredbycentrifu- NaCl and 20 mM imidazole. The cleaved protein was re-
gation.InordertoprecipitatetheUvrD,saturatedammo- passed over the HisTrap column to remove HRV 3C pro-
niumsulphatewasaddedgraduallytothesupernatantun- tease contamination and PcrA S-Ct was collected in the
til30%saturationwasreached.Theproteinwasallowedto flow-through. The cleaved (or uncleaved) protein was fi-
precipitatefor1hinanicewaterbathandwasrecoveredby nallypurifiedusingaSuperdex75gelfiltrationcolumn(GE
centrifugationat4000rpmfor30min.Thepelletwasresus- Healthcare)inabuffercontaining50mMTris–Cl,pH7.5,1
pendedinbuffer(20mMTris–HClpH8.3,20%(v/v)glyc- mMEDTA,1mMDTTand200mMNaCl.Peakfractions
erol,400mMNaCl,2mMEDTApH8.0,0.5mMEGTA were pooled and concentrated using a 3 kDa cut-off spin
pH 8.0, 1 mM DTT) and was slowly diluted in buffer A concentrationdevice.Whereappropriate,removalofthetag
(20mMTris–HClpH8.3,20%(v/v)glycerol,1mMEDTA was verified by separation using a 10–20% Tris–tricine gel
pH8.0,0.5mMEGTApH8.0and1mMDTT)untilthe bycomparisonwiththehis-taggedprotein.Theconcentra-
saltconcentrationwasapproximately200mMNaCl(or100 tion of protein was determined by spectrophotometry us-
mMfortheUvrDK708Amutant).Thiswasthenloadedonto ingatheoreticalextinctioncoefficientof11000M−1cm−1.
a 5 ml Heparin column (GE) on an A¨KTA FPLC. The Theproteinwassnapfrozenandstoredat–80◦Cinabuffer
protein was eluted from the column using a NaCl gradi- containing50mMTris–Cl,pH7.5,1mMEDTA,200mM
entinbufferA.Fractionscontainingtheproteinofinterest NaCl,1mMDTTand10%glycerol.
were combined and diluted in buffer A until the salt con-
centration was approximately 200 mM NaCl (or 100 mM
for the UvrDK708A mutant). Protein was loaded onto a 1
ml MonoQ column (GE) on an A¨KTA FPLC. The pro-
teinwaselutedusingaNaClgradientinbufferA.Fractions
NucleicAcidsResearch,2017,Vol.45,No.7 3877
Crystallization,structuredeterminationandstructureanaly- Constructionoftemplatesforinvitrotranscriptionassays
sis
Linear DNA templates for in vitro transcription reactions
The PcrA-sCt protein (with the his-tagged removed) was were constructed by PCR amplification from pSRT7A1
concentrated to 20 mg/ml in a buffer of 50 mM Tris-Cl (49)usingPfuDNApolymerase.Templatescontainingthe
(pH 7.5), 1 mM EDTA, 200 mM NaCl and 10% glycerol. T7A1promoterwereamplifiedusingtheupstreamprimer
Crystalsweregrownusingthesitting-dropvapordiffusion 5(cid:5)-ACCTGACGTCTAAGAAACC-3(cid:5)andthedownstream
methodat18◦Cbymixing0.2(cid:2)lproteinsolutionwith0.2 primer 5(cid:5)-ATTACTGGAGGGGATGGGG-3(cid:5) to produce
(cid:2)l precipitant solution containing 3.5 M sodium formate a 236 bp product. On this template transcription can be
pH7.0.Crystalswereharvestedandfrozenusingtheprecip- stalled by nucleotide starvation at +20 by omitting UTP.
itantsolution.Amercuryderivativewaspreparedbysoak- Transcriptioncanalsobechasedtothetemplateendtopro-
ingthecrystalsfor4hincryosolution(3.5Msodiumfor- duce a transcript of 60 nt. Linear biotinylated DNA tem-
matepH7.0)containing10mMethylmercuryphosphate, platewasmadeinasimilarmannerusinga5(cid:5)-biotinylated
and then freezing. X-ray diffraction data were collected at upstream primer and DNA templates were purified us-
100 K using a Rigaku FR-X X-ray generator and PILA- ing the QIAEX II DNA extraction kit (Qiagen). Plasmid
TUS 300K detector. The data were processed and scaled pSRTB8B3+500 was created by inserting a 504 bp frag-
using the HKL3000R program (38). A single mercury site mentamplifiedfromtheE.colirpoBgenebetweentheNcoI
wasfoundbydirectmethodsinSHELXD(39).Heavy-atom and XhoI sites located between the T7A1 promoter and
refinementandphasing(usingSIRAS)wereperformedus- thetandemlyrepeatedBbvCIsitesofpSRTB8B3(50).On
ingSHARP(40).Solventflippinganddensitymodification theresultingtemplatetranscriptionfrom theT7 promoter
wereperformedusingSOLOMON(41)andParrot(42),re- can be stalled at +20 by UTP starvation, or allowed to
spectively. An initial model was built automatically using runtoadownstreamterminatortoproducea764nttran-
Buccaneer(43)andthefinalstructuremodelwasmanually script.Abiotinylatedclosed-circularplasmidtemplatecar-
rebuiltusingCoot(44)withrefinementinRefmac(45)and ryingabiotin-dTatposition+585onthetranscribedstrand
Phenix (46). Diffraction data and refinement statistics are wasgeneratedbyannealingabiotin-dT-containingoligonu-
listedinTable1,andtheco-ordinatesofthestructurehave cleotideintoBbvCI-nickedplasmidpSRTB8B3+500,asde-
beendepositedatthePDBunderIDcode5DMA.Figures scribedin(50).
showingtheconservationofresiduesinPcrAmappedonto
thestructurewerecreatedusingConSurf(47)andPyMOL. In vitro transcription time course and transcript release as-
Themultiplesequencealignmentwasbasedon150unique says
sequences that were the most similar to the G. stearother-
For time course assays, transcription initiation complexes
mophilusPcrAC-terminususingtheConSurfdefaultinput
wereformedbyincubating20nME.coliRNApolymerase
settings. holoenzymewith2ng/(cid:2)lDNAtemplatefor5minat37◦C
in repair buffer (40 mM HEPES, pH 8.0, 100 mM KCl,
Pulldownassays
8 mM MgCl , 4% glycerol (v/v), 5 mM DTT, 100 (cid:2)g/ml
2
Pulldown assays were performed as in previous work (14) BSA). Transcription elongation complexes stalled at +20
usingeitherstreptavidin-coatedmagneticbeads(NewEng- were then formed by nucleotide starvation: the preformed
land Biolabs) for biotin-tagged bait proteins or substitut- transcriptioninitiationcomplexesweremixedwithanequal
ing Ni2+-NTAmagnetic beads (NewEngland Biolabs) for volume of NTP stall mix in repair buffer (final concentra-
his-tagged bait proteins. Briefly, DNA/RNA-depleted ex- tions100(cid:2)MApU,10(cid:2)MATP,10(cid:2)MGTP,2(cid:2)MCTP,
tracts of B. subtilis 168 were produced as described previ- 0.5(cid:2)Ci/(cid:2)l[(cid:4)-32P]CTP)andincubatedfor5minat37◦C.
ously(14).B.subtiliswaschosenasthesourceforthebait Aliquotsofthestalledelongationcomplexeswereincubated
proteinsbecausethePcrAfromthisorganismishighlysimi- withUvrDoritsderivativesattheconcentrationsindicated
lar(85%)toitsorthologuefromG.stearothermophilus,and for 5 min at 37◦C. Transcription elongation was then al-
because the annotated proteome is important for the pro- lowedtocontinuefor5minat37◦Cbyaddinga‘chase’of
teomics analysis (below). Nevertheless, it should be noted 100(cid:2)MNTPs,togetherwith10(cid:2)g/mlrifamipicintoensure
thatourpulldownexperimentsmaybesusceptibletofalse thatonlyasingleroundoftranscriptiontookplace.Reac-
negatives because of the different Bacillaceae species used tionswerestoppedwithanequalvolumeofureastopmix
forbaitandpreyproteins.Thepreparedextractswerethen (7 M urea, 10 mM EDTA, 1% SDS, 2× TBE, 0.05% bro-
usedasthepreyinpulldownexperiments.Purifiedbaitpro- mophenolblue,0.05%xylenecyanol).Sampleswereheated
teinswereincubatedatnearsaturatingconcentrationswith for3minat90◦Candresolvedona15%polyacrylamide/7
magnetic beads to allow binding. The baited beads were Mureadenaturinggel.GelswereanalyzedusingaMolecu-
washedandaddedtoB.subtiliscellextractstoallowbind- larDynamicsTyphoonPhosphorImagerandImageQuant
ing with partner proteins. The beads were separated from software.ForcomparisonofRNAPremodelingactivityas
the cell extract and washed before bait and prey proteins showninFigure5,thedatawerenormalizedacrossmulti-
were harvested from the beads by boiling in SDS-PAGE plegelsbydeterminingthetotalintensityoftheremodeling
sample buffer. The pulldown experiments were analysed products for any given protein at any given timepoint (see
bySDS-PAGEfollowedbyeitherwesternblottingwithan blackbarinFigure5A)anddividingthisvaluebythehigh-
anti-RNAP (cid:3) antibody (8RB13, Abcam (48)), or by mass estvalueobservedforthewildtypeactivityoneachgel(typ-
spectrometry.Detailsofsamplepreparationformassspec- icallythefinaltimepoint).Thisvaluethereforerepresentsa
trometrycanbefoundintheSupplementaryInformation. relativeremodelingactivitycomparedtomaximalwild-type
3878 NucleicAcidsResearch,2017,Vol.45,No.7
Table1. X-raydatacollectionandrefinementstatisticsforPcrA-sCt
Hg Native
Datacollectionstatistics
Wavelength(A˚) 1.54 1.54
Spacegroup P3221 P3221
Celldimensions(a,b,c)(A˚) 50.4,50.4,40.4 50.5,50.5,40.2
Resolution(A˚) 50–1.7(1.8–1.7)a 50–1.5(1.6–1.5)a
Observed/uniquereflections 41952/6980 92776/9252
Dataredundancy 6.0(3.0) 10.0(5.1)
Completeness(%) 99.3(98.7) 99.9(98.9)
Rsym(%) 7.5(15.8) 3.4(18.5)
I/(cid:5)(I) 33.6(8.1) 61.6(7.7)
Refinementstatistics
Resolutionrange(A˚) 30.0–1.5
Reflections(work/test) 9167/438
Rwork/Rfree(%) 19.5/22.2
Numberofatoms(protein/water) 407/65
AverageB-factor(protein/solvent)(A˚2) 15.2/25.5
RMSDinbondlength(A˚)/bondangle(◦) 0.007/1.193
aValuesinparenthesesrefertothehighestresolutionshell.
activity.Theerrorbarsrepresentthestandarderrorofthe ATPaseassays
meanforwildtype(sixexperiments)ormutantUvrD(four
TheATPaseactivityofUvrDanditsderivativeswasmea-
experimentseach)respectively.
sured using an enzyme linked assay in which ATP hydrol-
Fortranscriptreleaseassays,stalledtranscriptionelonga-
ysisiscoupledtoNADHoxidationessentiallyasreported
tion complexes were formed as described above, but using
previously (51). However, ATPase assays were modified in
biotinylated template DNA. DNA containing the stalled thatreactionswerecarriedoutat37◦Cusing1nMUvrD,
complexes was bound to streptavidin paramagnetic beads
2 mM ATP and 2 (cid:2)M ssDNA (47 nt). The ATPase activ-
(NEB)byincubatingeachreactionwithbeadstakenfrom
ityofPcrAanditsderivativeswasmeasuredusingthesame
an equal volume of bead suspension (and washed twice
withrepairbuffer)for10minat20◦C.Thebeadswerethen linkedassay,accordingtothemethoddescribedin(34).
washedthreetimesinequalvolumesofrepairbuffertore-
moveunboundDNAandRNAP.1(cid:2)MUvrDorUvrD(cid:2)C TFOdisplacement(DNAtranslocase)assays
wasaddedto20(cid:2)laliquotsofthereactionwhereindicated
TFO assays were carried out essentially as described in
andreactionswereincubatedfor5minat37◦C.Transcrip-
(49). Assays were carried out on a linear plasmid tem-
tionreactionswerechasedbyadding100(cid:2)MNTPsand10
plate,pSRTB2EV,aderivativeofpSRTB2(50),inwhichan
(cid:2)g/ml rifamipicin for 5 min at 37◦C. A magnet was used
EcoRV site had been introduced downstream of the TFO
toseparatepelletandsupernatantfractionsandthesuper-
bindingsitebysite-directedmutagenesis.Thistemplatewas
natantwasaddedto20(cid:2)lureastopmix(fractionS).The
linearisedbyEcoRV,creatingabluntend33bpdownstream
pelletwasresuspendedin20(cid:2)lrepairbufferandaddedto
of the triplex end. Assays were performed in repair buffer
20(cid:2)lureastopmix(fractionP).
andtheTFOcontainingDNAtemplatewasincubatedwith
Fortranscript-releaseexperimentsinwhichbacktracking 1(cid:2)MUvrDoritsderivativesand100(cid:2)MNTPmix.0.25
was analyzed with GreB, reaction volumes were scaled up mg/mlProteinaseKand10mMCaCl wereaddedtothe
and 1 (cid:2)M UvrD or its derivatives were added to 100 (cid:2)l 2
GSMBstopbufferandreactionswereincubatedfor30min
aliquots containing stalled transcription elongation com- at20◦Cbeforeloadingontothegeltoeliminatebandshift-
plexes. Transcription reactions were chased as described
ingbyUvrD.
above. A magnet was used to separate pellet and super-
natant fractions and 20 (cid:2)l of the supernatant was added
to 20 (cid:2)l urea stop mix (fraction S). The beads were re- RESULTS
suspended in 80 (cid:2)l repair buffer and a 20 (cid:2)l sample was
Structure of a Tudor-like RNA polymerase interaction do-
added to 20 (cid:2)l urea stop mix (fraction P). The remainder
maininPcrA
ofthebeadsuspensionwassplitinto20(cid:2)laliquots.1(cid:2)M
GreB was added whereindicated and reactionswereincu- We have shown previously that the CTD of G. stearother-
bated for 5 min at 37◦C. Then 100 (cid:2)M NTPs were added mophilusPcrA(PcrA-Ct;residues653–724)isnecessaryand
whereindicatedandreactionswereincubatedfor5minat sufficientforinteractionwithRNApolymeraseusingaffin-
37◦C. Reactions were stopped with 20 (cid:2)l urea stop mix. ity pulldown assays from extracts of B. subtilis (14) (see
Samples were heated for 3 min at 90◦C and resolved on a SupplementaryFigureS1).Secondarystructurepredictions
15% polyacrylamide/7 M urea denaturing gel. Gels were using Jpred (52) suggested that this region of the protein
analysed using a Molecular Dynamics Typhoon in Phos- includesasignificantregionofnativelydisorderedprotein
phorImagermodeandImageQuantsoftware. thatispoorlyconserved.However,thisisfollowedbyavery
highlyconservedregionthatispredictedtoconsistentirely
ofbeta-sheet(residues673–724).Furthermore,thedomain
NucleicAcidsResearch,2017,Vol.45,No.7 3879
identification algorithms Ginzu (53) and Phyre2 (54) both complex of the Mfd Tudor domain bound to RNAP, be-
predict that this beta-sheet region will fold into a Tudor- cause PcrA/UvrD has been reported to function in an al-
likedomain.Despiteverylowprimarystructurehomology, ternativeTCRpathway(1).ThestructureofThermusther-
these algorithms identify RapA and CarD respectively as mophilus Mfd bound to RNAP shows that it binds to the
templatesforhomologymodelling.Interestingly,theseare so-called(cid:3)1region(60)andthisisalsotrueoftheTudordo-
bothbacterialRNAPinteractionpartnerscontainingcon- mainfoundinMycobacteriumtuberculosisCarD(58,61).In
served Tudor-like domains (55,56). Therefore, we hypoth- bothofthosestructures,theTudordomainformsacontinu-
esized that the extreme C-terminus of PcrA adopts a Tu- ousanti-parallelbeta-sheetwiththepartnerprotein,which
dorfoldandthatthisinteractsdirectlywithRNAP.Totest is further stabilized by side-chain interactions. Conserved
this idea, we designed a new shorter version of the PcrA residuesthatareimportantforinteractionwithRNAPin-
CTD using the homology models as a guide. This con- cludealysineresidue(K360inT.thermophilusMfd)thatis
struct, which we call PcrA-short Ct (PcrA-sCt), includes somewhatsimilarlypositionedtotheaforementionedK712
residues673–724 ofthenativePcrAprotein.Moreover,to inPcrA(57).However,itshouldbenotedthatthereisnoap-
producetheCTDofPcrAingreaterquantitiesthanwehad parentsequencehomologybetweentheTudor-likedomains
achieved previously using a biotin-tag (14), we engineered ofPcrAandMfd.
plasmids for expression of PcrA-Ct and PcrA-sCt with a
cleavablehistidinetagattheN-terminus.Usingthissystem,
ConservedaminoacidsrequiredforbindingRNAP
we were able to obtain large amounts of highly pure pro-
tein either with or without the tag (Supplementary Figure Several highly conserved residues cluster together on the
S2). To confirm that the shorter CTD construct retained surfaceofthePcrATudorfoldthatfrequentlyformsapro-
the ability to interact with RNAP, we performed affinity tein:protein interface in other systems (Figures 1 and 2).
pulldown experiments using the his-tagged PcrA-sCt pro- Wereasonedthattheseresiduesmightbedirectlyinvolved
tein as bait and nucleic acid-depleted cell extracts as prey in the interaction with RNA polymerase. To test this hy-
(14).Asexpected,theoriginalPcrA-Ctconstructefficiently pothesis,weindividuallymutatedseveralofthem(H681A,
pulled down RNAP from a B. subtilis extract in a dose- W684A, K712A and L714A) and tested the ability of the
dependent manner. In agreement with our hypothesis, the resulting CTD constructs to interact with RNAP in pull-
shorterPcrA-sCtconstructretainedtheabilitytopulldown down assays. The mutant PcrA-sCt proteins were purified
RNAP,andtheefficiencywascomparabletoPcrA-Ct(Sup- usingthesamemethodasforwildtypeandtheirCDspec-
plementary Figure S2). For reasons that will be discussed trawereallcharacteristicof(cid:3)sheetasexpected,suggesting
below,wealsoinvestigatedwhetherthePcrACTDwasable normalglobalfolding(SupplementaryFigureS4).Thepull-
tobindtoDNA.However,nointeractionbetweenthePcrA downassaysrevealedthateachofthesinglemutantproteins
CTD and either single- or double-stranded DNA was de- hadaseverelyreducedabilitytobindtoRNAP(Figure1C).
tectedusinggelshiftassays(SupplementaryFigureS3). Indeed,westernblottingofthegelswithananti-RNAPsub-
Crystals of PcrA-sCt (with the histidine tag removed) unitantibodysuggestedthebindingwasmarginallyabovea
were obtained using the sitting drop vapor diffusion ‘nobait’controlandcomparabletoasecondnegativecon-
method. The structure was solved using a heavy atom trolexperimentusingB.subtilisParB(aproteinnotknown
derivativeatafinalresolutionof1.5A˚ (R =22.2%)(Fig- tobindRNAP).
free
ure 1). The final model includes all of the residues of the TheK712AandL714Amutationswerealsomadeinthe
native PcrA sequence (W673 to V724) and an N-terminal context of a full length biotinylated PcrA construct used
glycinefromthetaglinkerregion.Aspredicted,theextreme in our previous studies. These mutant proteins displayed
C-terminalregionofPcrAadoptsaTudor-likefoldconsist- a greatly reduced ability to interact with RNAP, but the
ing of five anti-parallel beta strands that form a twisted (cid:3) bindingwasreproduciblyhigherthanbackground(Figure
sheet. It closely resembles other bacterial Tudor domains 3A).Moreover,ashasbeenshownpreviously(14),thecom-
such as those found in RapA, CarD and Mfd (Figure 2) pletedeletionoftheCTDfromthefulllengthproteindra-
(57–61).AsearchwithDALI(62)alsorevealsstrongsimi- matically reduces binding to RNAP. In order to validate
laritytoNusG(63)andtoeukaryotic‘histonereaders’in- theseexperimentsandtotestforproteininteractionsofthe
cluding PHF1 (64). These histone readers are responsible CTDinanunbiasedfashion,wealsoanalysedthepulldown
for the recognition of methylated lysine residues in chro- experiments using mass spectrometry. Relative quantifica-
matin.ManystructuresofTudordomainsinteractingwith tion of prey proteins was performed by comparing total
theirpartnerproteinsorpeptideshaveshownthatonepar- ion scores using the ‘no bait’ pulldown as a control (Fig-
ticular face of the fold is often responsible for the interac- ure3BandSupplementaryTableS1).Therelativeionscore
tion(65),althoughthereareapparentexceptionsincluding for the PcrA bait acts as an internal control, showing en-
RapA(59)(Figure2).Inhistonereaders,thisfaceincludes richment over control for full length constructs, and a re-
an‘aromaticcage’thattypicallyacceptsthemethylatedside duced enrichment for the PcrA-Ct construct as would be
chain of a lysine residue. Interestingly, some key residues expectedbasedonthedifferentpolypeptidelengths.There-
thatformthisaromaticcageappeartobeequivalentinPcrA sultsforthewildtypePcrAbaitreproducedourpreviously
(egW684,F705).However,PcrAalsofeaturesachargedly- publishedexperimentsshowingthatitinteractswithmany
sine residue (K712) in this region. Although not common proteinsinthecellextract(seealso(14)andSupplementary
forTudordomainsingeneral,thislysineresidueisstrongly Table S1 for full details). Prominently these include both
conservedwithinPcrA/UvrDorthologues(Figure1D).We coreandaccessorysubunitsofRNApolymerase((cid:4),(cid:3),(cid:3)(cid:5),
wereespeciallyinterestedtocompareourstructurewiththe (cid:6),(cid:7)),as well as a variety of sigma factors. Other enriched
3880 NucleicAcidsResearch,2017,Vol.45,No.7
A B C
M 1 2 3 4 5 6 7 8
W684 H681A W684A K712A L714A bait
K712 none ParB Ct sCT sCT- sCT- sCT- sCT-
kDa
H681 250
150
100
L714 75
50
37 ParB
25
20
15 Ct
10
~180o
anti-β
D
1 4 2 4
8 8 1 1
6 6 7 7
Figure1. ThecrystalstructureofanRNAPinteractiondomaininPcrAhelicase.(A)CrystalstructureoftheTudor-likeCTDofPcrA.Theco-ordinates
havebeendepositedatthePDBunderPDBIDcode:5DMA.Thestructureiscoloredaccordingtoresidueconservationwiththeleastconservedresiduesin
cyan,throughwhite,tomostconservedresiduesinmagenta.Theproteinisshowninribbonsformatwithsidechainsonlyshownfortheaminoacidswhich
wereselectedforsite-directedmutagenesisstudies.(B)Asecondviewofthestructure,facingtheputativeRNAPbindingsurface.(C)Affinitypulldown
assaysusingthehis-taggedproteinsindicatedasbaitandBacillussubtiliscellextractasprey.TheupperpanelshowsanSDS-PAGEgelanalysisofthecell
extractfollowingpulldownusingbaitedmagneticbeads.Thebaitproteinisindicatedatthetopofthegelandthearrowindicatesthepositionofthe(cid:3)and
(cid:3)(cid:5)subunitsofRNAP.Thelowerpanelshowswesternblotanalysisofthesamegelusingamonoclonalantibodyagainstthe(cid:3)subunitofRNApolymerase.
Inadditiontoamockpulldown,thepulldownwithParB(acentromerebindingproteinthatisnotknowntointeractwithRNAP)isincludedasasecond
negativecontrolfornon-specificpulldownofRNAP.(D)WeblogoformatmultiplesequencealignmentoftheCTDof∼250PcrA/UvrDhomologues.The
residuesthatweremutatedinthisstudyarehighlightedusingtheirresiduenumbersfromtheG.stearothermophilusPcrAprotein.
proteinsofinterestincludeYvgS/HelD(aSF1helicasealso teracts directly with RNAP (66). In distinct contrast, the
known to associate with RNAP), UvrB (a known PcrA removalormutationoftheCTDofPcrAdidnotaffectthe
bindingpartnerinvolvedinNER),DNApolI(aDNAre- apparentinteractionwithDNApolIorLigA,whereasthe
pairspecificandbypasspolymerase)andLigA(anNAD+- CTD alone bound poorly to these proteins, showing that
dependent DNA ligase originating from the same operon they must interact mainly with the N-terminal region of
asPcrA)(Figure3B).Ingoodagreementwiththewestern PcrA.Importantly,thisobservationconfirmsthatthemu-
blottinganalysis,massspectrometryshowedthatmutation tant PcrA proteins remain largely folded, as would be ex-
(K712AorL714A)orremovalofthePcrACTDsubstan- pected based on CD analysis of the CTD variants alone
tiallyreducedoralmosteliminatedtheinteractionwithcore (see above). This assertion is further supported by analy-
RNAPsubunitsrespectively.Thesedatashowthatthemu- sisoftheDNA-dependentATPaseactivity,whichissimilar
tated residues are critical for the RNAP binding function or slightly better than wild type for both mutant proteins
oftheCTD,andthattheapparentresidualbindingwehave (Table2).
observedprobablyoccursatadifferentsiteinthePcrApro-
tein.Interestingly,aconcomitantreductioninseveralother
interactionpartnersincludingUvrBandYvgSwasalsode- The UvrD CTD is important for the remodeling of RNAP
tected. This is consistent with two possibilities that can- transcripts
notbedistinguishedbasedontheseexperiments.Eitherthe
Duetodifferencesinthemannerinwhichitwasdiscovered
CTDofPcrAisimportantfordirectinteractionwithallof
inthemodelorganismsB.subtilisandE.coli,thehelicase
theseproteins,ortheotherproteinsareassociatedwiththe
studied here is often annotated as PcrA in Gram-positive
RNAP subunits that are pulled down by PcrA. In this re-
organisms and UvrD in Gram–negative organisms. It was
spectitshouldbenotedthatPcrA/UvrDinteractsdirectly
therefore of considerable interest to us that E. coli UvrD
with RNAP and UvrB (1,14,15), and that YvgS/HelD in-
was recently shown to induce backtracking of RNAP in
NucleicAcidsResearch,2017,Vol.45,No.7 3881
A B C
D E F
Figure2. ComparisonofTudor-likedomainsandtheirinteractionswithpartnerproteins.(A)StructureoftheCTDofPcrA(PDB:5DMA;thiswork).
(B)SuperimpositionoftheRNAPinteractiondomainofE.coliMfd(cyan)(PDB:2EYQ;(57))withacomplexbetweentheMfdRID(blue)andthe
(cid:3)1domainofRNAPpolymerase(brown)(PDB:3MLQ;(60)).(C)ThecomplexofCarD(red)boundtotheRNAP(cid:3)subunit(brown)(PDB:4KBM;
(58)).(D)ComplexbetweenthesecondTudordomainofRapA(orange)andthe(cid:3)(cid:5)subunitofRNAP(blue)(PDB:4S20;(59)).(E)Complexbetweenthe
Tudor-likedomainofNusG(green)andribosomalproteinS10(yellow)(PDB:2KVQ;(63)).(F)ThePHF1protein(turquoise)incomplexwithapeptide
(white)showninstickformatandcontainingatrimethylatedlysineresidue(red)(PDB:4HCZ;(64)).
Table2. ssDNA-dependentATPaseactivityofPcrAandUvrD Ininitialexperiments,bothcircularandlinearDNAtem-
plates were used, and these were also biotinylated so that
Protein ATPase(s−1)a
displacement of RNAP could be monitored by examin-
WildtypePcrA 12.1 ± 0.8 ing the release of the transcript into the supernatant us-
PcrAK712A 15.8 ± 0.7 ing a pulldown approach (Figure 4A). In the absence of
PcrAL714A 18.9 ± 1.8
WildtypeUvrD 81.1 ± 3.0 UvrD,theRNAPformslongtranscriptsindicativeofpro-
UvrD(cid:2)C 194.9 ± 7.5 cessive transcription elongation (Figure 4B; lanes 2, 3, 7
UvrDK708A 82.8± 19.8 and 8). Shorter RNA transcripts are formed in a UvrD-
dependent manner regardless of whether the template is
aATPhydrolysiswasmeasuredusingacoupledassayasdescribedinthe circular or linear, and these transcripts generally remain
MaterialsandMethodsunderconditionsofsaturatingATPandssDNA.
in the pellet fraction (Figure 4B). This is consistent with
Thevaluesreportedarethemeanturnovernumberandthestandarderror
ofthemeanforthreeindependentexperiments. UvrD causing backtracking of RNAP as reported previ-
ously(1),andthiswasconfirmedbyaddingthetranscript
cleavagefactorGreB(whichremovestheextruding3(cid:5) por-
vitro,causingittoslidebackwardsontheDNAandbring- tion of the nascent RNA) and then chasing the reaction
ingaboutthedisplacementofthe3(cid:5) endoftheRNAfrom withNTPstogeneratefulllengthtranscripts(Supplemen-
the active site (1). To reproduce this activity and to probe taryFigureS5,lanes1–13,seefigurelegendfordetails).In
the potential role of the CTD, in vitro transcription reac- additiontobacktracking,wealsoobservedefficientUvrD-
tions were performed in which wild type or mutant UvrD dependentdisplacementofshortRNAtranscriptsintothe
wasincubatedwithRNAP(bothfromE.coli)thathadbeen supernatant,andthisphenomenonwasonlyobservedwith
stalled20ntdownstreamoftheT7A1promoterbyomission thelineartemplate(Figure4B,seeasterisks).Interestingly,
ofUTP.Thereactionwasthen‘chased’withNTPs,andri- thesizeofthesereleasedtranscriptsisequivalenttothesize
fampicinwasaddedtoensuresingleroundconditions.
3882 NucleicAcidsResearch,2017,Vol.45,No.7
bait ofthemajorGreB-cleavageproducts(SupplementaryFig-
A ureS5,comparelanes9and11),whichareindicativeoffa-
A A vored backtracking positions for RNAP on the template.
Mprey none BSA FL-PcrA FL-K712 FL-L714 Ct sCt ΔC ItphnlaattnheetwhaeebrdseeisnotcbaesnecorefvfeUrdov.mrSDut,hcsheevpterraoranmlsoctrrtaiepnrtstscorrietphstueslettnhfdarotomafrtethrleaontnesgfmeerr-
ofRNAPfromanendofoneDNAmoleculetoanendof
another(67).Interestingly,additionofUvrDabolishesthis
PcrA end-to-endtransfer,possiblybybacktrackingRNAPaway
PcrAΔC from the DNA end, or by dissociating it from the DNA
altogether.Together,theseexperimentsshowthatUvrDis
capable of remodeling transcription elongation complexes
in vitro, by promoting both the reversible backtracking of
RNAP,andtheprematurereleaseofshortRNAtranscripts
Ct fromlineartemplates.
sCt We next performed experiments on free linear template
DNAusingmutantUvrDproteinsinwhichtheCTDwas
anti-β eitherremovedormutated(Figure5).Theverystrongcon-
servationofprimarysequenceintheCTD(Supplementary
Figure S1) allows the facile design of mutations in E. coli
UvrD(UvrD(cid:2)CorUvrDK708A)thatareequivalenttothose
we have studied in PcrA. In the absence of UvrD, RNAP
transcribedthetemplateDNAtoproduceproductswhich
B 40 30 wild type
PcrA-Ct correspondtotranscriptiontotheendofthetemplate(Fig-
PcrA-ΔC ure 5A; lane 1 +60). As expected, in the presence of wild
30 K712A
20 L714A type UvrD a series of shorter RNA transcripts were ob-
served(Figure5A,comparelane1withlanes2–5).Thisef-
20
fect was UvrD dose-dependent, but less efficient than has
e 10 been reported previously (1). On these substrates, UvrD-
r 10
o dependentRNAPremodellingwassubstantiallyreducedin
c
S the absence of the CTD (Figure 5A and B, compare lanes
on 0 PcrA 0 α β β' δ ω 2–5withlanes6–9).However,itwasstillpossibletoobserve
e I RNAP subunit backtrackingofRNAPonlinearDNAtemplatesusingthis
elativ 100 12 100 30 mneutitcanbte,aedssp(eScuiapllpylewmheenntabroyuFndigtuorestSr5ep).tTavhideidnecleotaiotendomftahge-
R CTDisparticularlyeffectiveatdecreasingtheprematurere-
75 75
8 20 leaseofRNAintosolutionthatisobservedonlineartem-
plates(Figure5AandSupplementaryFigureS5,seeaster-
50 50
isks). Mutation of the highly conserved lysine in the CTD
4 10 (K708A)alsoreducedtheremodelingofRNAP-RNAcom-
25 25
plexesrelativetowildtype,albeitnottothesameextentas
doesthecompleteremovaloftheCTD(Figure5AandB).
0 0 0 0
YvgS UvrB PolI LigA Together,theseexperimentsindicatethattheCTDisim-
portant but not essential for catalysing backtracking and
Figure3. MutationofthePcrACTDreducesbindingtoRNAP.(A)Affin- RNAreleasefromRNAPduringtranscriptioninvitro.To
itypulldownassaysusingthebiotinylatedproteinsasbaitandB.subtilis eliminatethepossibilitythatthemutantproteinsweresim-
cellextractasprey.TheupperpanelshowsanSDS-PAGEgelanalysisof
ply unfolded, or that the observed remodeling defects re-
thecellextractfollowingpulldownusingbaitedmagneticbeads.Thearrow
signifiesthepositionofthe(cid:3)and(cid:3)(cid:5)subunitsofRNAP.Thelowerpanel flected a reduced ability of the mutant proteins to move
showsanalysisofthesamegelusingamonoclonalantibodyagainstthe(cid:3) along DNA we assayed for ATPase and DNA translocase
subunitofRNAP.NotethattheK712AandL714Amutationsweremade activity. These experiments showed that UvrD proteins in
inthecontextoffulllength(FL)PcrA.(B)Quantificationofthepulldown whichtheCTDhadbeeneithermutatedordeletedretained
experimentsusingmassspectrometryusingthebaitproteinsindicatedand
ssDNA-dependent ATPase and translocase activities that
a‘nobait’controlasthereference.TheRelativeIonScorevalueshownis
thetotalionscoreforthepreydividedbytheequivalentvalueinthecon- were either comparable to, or even better than, wild type
trol:ameasureofrelativeabundanceoftheprey.TheenrichmentofPcrA activity (Table 2 and Figure 5C). This is broadly consis-
inthepulldownsamplesreflectsthepresenceofthebaitproteinandactsas tentwithpreviousexperimentsonaUvrDproteinwitha40
aninternalcontrol.SelectedresultsareshownforthesubunitsofRNAP
amino acid C-terminal deletion which displayed wild type
andforseveralotherproteinsthatarediscussedinthetext.Furtherand
ATPaseandhelicaseactivity(68).
moredetailedresultsareshowninSupplementaryTableS1.
NucleicAcidsResearch,2017,Vol.45,No.7 3883
A B
1 2 3 4 5 6 7 8 9 10 Lane
bead 60 nt circular linear Template
+ + + + UvrD
all all
t P S P S t P S P S Fraction
S S
T7A1
+20 stall
end-to-end
transcripts
terminator
(+764)
bead +60
*
+20 stall
*
T7A1
+20
Figure4. UvrDremodelsRNAPtranscriptsoncircularandlinearDNAtemplates.(A)SchematicofthelinearandcircularDNAtemplatesusedfor
transcriptionreactionsinthiswork.(B)RemodelingassayforbothcircularandlinearDNAtemplatesasindicated.Reactionswereperformedeitherwith
orwithout1(cid:2)MUvrDasdescribedintheMaterialsandMethods.BiotinylationtagsinthetemplateDNAmoleculesallowthepulldownofthetemplate
intoapelletfraction(P)leavingasupernatantfraction(S)whichonlycontainstranscriptsreleasedintosolution.Thestalllanesshowthestalledtranscript
productat+20withouttheadditionofchasenucleotides.Allotherlanesshowtranscriptsformedfollowingre-initiationoftranscriptionfrom+20witha
nucleotidechase.AsteriskshighlightthepositionoftheprincipaltranscriptsthatarereleasedintosolutionbytheactionofUvrD
DISCUSSION organisms. However,themechanisms by whichtheydo so
areonlyrecentlybecomingapparentandmayvarywidely.
Superfamily I helicases are highly abundant and often
In E. coli, the SF1 helicases Rep, UvrD and DinG have
multi-functionalenzymesplayingdiverserolesinbacterial
allbeenshowntoresolvereplication:transcriptionconflicts
nucleic acid metabolism. Interestingly, several recent stud-
(26,27,70).Repfunctionsasacomponentofthereplisome
ieshaveshownthattheseenzymesfunctionattheinterface
itself,byassociatingwiththereplicativehelicaseDnaB,and
ofDNAreplication,transcriptionandrepair.Inrapidlydi-
loss of this interaction results in transcription:replication
viding bacterial cells, the replication and transcription of
conflicts (26,71). In distinct contrast, UvrD interacts di-
DNA occur on the same template at the same time. Con-
rectly with RNAP (1), and an equivalent interaction has
flicts between the two systems are inevitable and can lead
also been demonstrated in the orthologous PcrA enzyme
togenomicinstability,andsocellshavedevelopedsystems
(14,21,72). However, recent work in B. subtilis shows that
thateitherreducetheiroccurrenceorminimizetheirimpact
deletionofthePcrACTDdoesnotaffectitsessentialrolein
(reviewed in (69)). For example, some Superfamily I heli-
resolvingreplication:transcriptionconflicts,whereaselimi-
caseshavebeenshowntohelpthereplisomebypassphysical
nationofitsATPase/helicaseactivitydoes(27).Although
barriersincludingtranscriptioncomplexesinvariousmodel