Deducing putative ancestral forms of GNRA/receptor interactions from the ribosome

15 Pages·2017·4.23 MB·English

480–494 NucleicAcidsResearch,2019,Vol.47,No.1 Publishedonline12November2018
doi:10.1093/nar/gky1111
/
Deducing putative ancestral forms of GNRA receptor
interactions from the ribosome
Erin R. Calkins, Paul Zakrevsky, Vasken L. Keleshian, Eduardo G. Aguilar, Cody Geary and
Luc Jaeger *
DepartmentofChemistryandBiochemistry,BiomolecularScienceandEngineeringProgram,UniversityofCalifornia,
SantaBarbara,CA93106–9510,USA
ReceivedJuly27,2018;RevisedOctober17,2018;EditorialDecisionOctober20,2018;AcceptedOctober22,2018 D
o
w
n
lo
a
d
ABSTRACT than being idiosyncratic, these contacts are often part of e
d
Stable RNAs rely on a vast repertoire of long-range recurrent RNA structural modules (or motifs), which are fro
well-defined three-dimensional (3D) conformers specified m
ilunltaerramctaicohnisnetorieassssiuscthinasthteheforlidbionsgoomfec.oTmhepluenxivceerl-- bmyopdaurletiscuislatrhese‘tdsooufbnleu-clolecokteiddebsu(leg.ge.’((72–b1p0)2)x.Obunlegeo)fmthoedse- https
sally conserved L39/H89 interaction is a long-range ule, which forms the binding site of protein S8 in bacte- ://a
c
GNRA-like/receptor interaction localized in proxim- rial16SrRNAs(8,11,12).Interestingly,thesamestructural ad
e
itytothepeptidyltransferasecenterofthelargesub- module is also found in helix H89 of the large subunit m
ic
unitoftheribosome.Becauseofitscentrallocation, rRNAsfromallkingdomsoflife,whereitformsauniver- .o
u
L39/H89 likely originated at an early evolutionary sally conserved loop receptor that binds to the universally p.c
conservedterminalloopL39(L39pentaloopinEukaryotes o
stageoftheribosomeandplayedasignificantrolein m
its early function. However, L39/H89 self-assembly andArchaeaandL39hexaloopinBacteria)(8,13–15)(Fig- /na
is impaired outside the ribosomal context. Herein, ure 1). This long-range interaction participates in the as- r/a
wedemonstratethatstructuralmodularityprinciples semblyprocessofthelargesubunitrRNAbydockingdo- rtic
main2todomain5andlikelystabilizesthepeptidyltrans- le
canbeusedtore-engineerL39/H89toself-assemble -a
ferase center (PTC) of the ribosome (16–18). The riboso- b
invitro.The newversions ofL39/H89 improveaffin- mal L39/H89 interaction has remarkable structural simi- stra
ity and loop selectivity by several orders of mag- laritieswithGNRAtetraloop/receptorinteractions,oneof ct/4
nitude and retain the structural and functional fea- the most widespread classes of long-range interactions in 7
/1
tures of their natural counterparts. These versions structuredRNAs(19–25)(Figure1).However,preliminary /4
8
of L39/H89 are proposed to be ancestral forms of investigationsofL39/H89inisolationhaveindicatedthatit 0/5
L39/H89thatwerecapableofassemblingandfolding is thermodynamically metastable and unlikely to promote 17
independentlyfromproteinsandpost-transcriptional efficient self-assembly outside its natural structural con- 366
text(8).Wehypothesizedthatancestralformsoftheribo- 4
modifications. This work demonstrates that novel some likely relied on more stable RNA/RNA interactions by
RNA modules can be rationally designed by taking g
for folding and assembling into their native structures. In- u
athdevapnrotasgpeecotfotfhceremaotidnuglanrewsybnitoacxheomf iRcNalAm. oItdoeflfseorsf dtiemeed,ttohebeLc3o9m/He 8d9epinentedreancttioonnmadigdhittiohnaavlefeavcotlovresdltikheropurgoh- est on
theancestralribosomeandincreasesthetoolkitfor teins and/or post-transcriptional modifications within the 05
A
RNAnanotechnologyandsyntheticbiology. contextoftheribosome(8,13,26).Therefore,anoutstand- p
ingquestioniswhetherstableandmoreancestralversions ril 2
0
of this universally conserved ribosomal interaction can be 1
INTRODUCTION 9
recovered by taking advantage of the present structural
RibosomalRNAs,ribozymesandriboswitchestakeadvan- knowledgeofRNA,andmorespecifically,GNRA/receptor
tage of extended networks of non-covalent tertiary con- interaction.
tacts to stabilize their native conformation (1–6). Rather
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Fax:[phone];Email:[email protected]
Presentaddresses:
ErinR.Calkins,DepartmentofBiologicalSciences,MoorparkCollege,7075Campusroad,Moorpark,CA93021,USA.
PaulZakrevsky,RNABiologyLaboratory,CenterforCancerResearch,NationalCancerInstitute,Frederick,MD21702,USA.
VaskenL.Keleshian,MayoClinic,BaldwinBuilding4thFloor,221FourthAvenueSW.Rochester,MN55905,USA.
EduardoAguilar,LaboratoryofVirologyandInfectiousDisease,230YorkAve,Box64,NewYork,NY10065,USA.
CodyGeary,Bioengineering,ComputerScience,andComputationandNeuralSystems,CaliforniaInstituteofTechnology,Pasadena,CA91125,USA.
(cid:2)C TheAuthor(s)2018.PublishedbyOxfordUniversityPressonbehalfofNucleicAcidsResearch.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense
(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalwork
isproperlycited.Forcommercialre-use,[email protected]
NucleicAcidsResearch,2019,Vol.47,No.1 481
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
a
r/a
rtic
le
-a
b
s
tra
c
t/4
7
/1
/4
Figure1. PrinciplesofRNAarchitectonicsandstructuralsyntaxfornaturalandrationallydesignedGUAA/receptorinteractions.(A)ThroughtheRNA 80
architectonicsapproach(39,42),RNAmodulessuchastheL39/H89(pink/red)andS8protein/S8bindingsite(turquoise/blue)interactionsareextracted /5
1
fromtheX-rayatomicstructureoftheribosome(e.g.Thermusthermophilus)andcomparedfortheirstructuralsimilarities.(B)Theseinteractionscanbe 7
3
incorporatedintothetectoRNAdimersystem(PDBID:2JYJ)(34)forstudyingtheirself-assemblypropertiesoutsidetheirnaturalribosomalcontext. 6
TwotectoRNAunits,eachcontainingaloopandareceptor,assembleinthepresenceofMg2+throughloop-receptorinteractions(24,30,31).(C)Structural 64
syntaxofRNAmodulesofincreasingcomplexitythatentersintothecompositionofnaturalloop/receptorinteractions(e.g.:i-iii)andthatcanbeused by
forrationallydesigningnewGNRA/receptorinteractions(iv-vi).Thenomenclatureforbasepairsandtertiarycontactsisaccordingtoreferences(7,8,82). g
u
(D)3DstructuresofnaturalandengineeredGNRAtetraloop/receptorinteractions:(i)GYRA/helixinteraction(from[pdbID:1s72]);(ii)GAAA/11nt- e
s
receptorinteractionfrom[pdbID:1hr2];(iii)GUAAG/H89-receptorinteractionfrom[pdbID:1s72];(iv)type2aGUAA/S8-receptorinteraction(S8from t o
[pdbID:2avy]);(v)type1aGUAA/S8-receptorinteraction(S8from[pdbID:2avy]);(vi)type3aGNRA/P12-receptorinteraction(P12from[pdbID: n
0
1us9]). 5
A
p
GNRA/receptorinteractionstypicallyinvolveaGNRA involves a nt platform and forms additional specific con- ril 2
0
terminal loop (with R standing for purine, N for any nu- tacts with its GGAA tetraloop (24,38). Using the archi- 1
9
cleotide and the first guanine forming a trans shallow- tectonicsapproach(39–42),wepreviouslydevelopedatec-
groove/Hoogsteen base pair with the fourth adenine) that toRNAdimersystemthatconsistsoftwounitsabletoself-
forms an ‘A-minor’ interaction (22) between the two last assemblethroughtwoloop/receptorinteractionsinasyner-
positions of the tetraloop and two base-pairs (usually two gisticmanner(Figure1B)(30,31,33,34).Thissystemproved
G:C bps) within a helix (19,27) (Figure 1). Moreover, ad- extremelyusefulforcharacterizingthebehaviorsofvarious
ditionalnon-canonicalbasepairsand/orstackinginterac- GNRAloop-receptorinteractions(24,30,31,33–35,43,44).
tions often contribute to an increase of stability and loop Herein, this tectoRNA system was used as a scaf-
specificity for the GNRA/receptor interaction (24,28–36). fold to retro-synthesize potential ancestral versions of the
Forinstance,inadditiontothe‘A-minor’sub-module,the L39/H89 ribosomal interaction (Figure 1). Although im-
GAAA/11ntinteractionincludesanucleotide(nt)platform portant RNA structures often display a high degree of se-
and UA handle sub-module that are responsible for the quenceconservation,ithasactuallybeenfoundthatstruc-
stacking and specific recognition of the second loop posi- ture is more conserved in the long run (45,46). Therefore,
tion(24,29,37).Similarly,theGGAA/R1interactionlikely considering that the L39 pentaloop from Eukaryotes and
482 NucleicAcidsResearch,2019,Vol.47,No.1
Archaea folds as a GUAA-like tetraloop and that R89 ing ImageQuant software (24,30) (Supplementary Figure
adopts the same structural module as the S8 RNA bind- S1C).Anon-linearfitfromtheexperimentalequation:f =
ing site (S8) (Figure 1A), we hypothesized that GUAA- [2(cid:3)M +K −(4M (cid:3)K +K 2)0.5]/2M ,wasusedtodeter-
0 d 0 d d 0
tetraloop/S8-like receptor interactions could be derived mineK ’sfortheequilibriumreactionP+ R↔ PR,where
d
from the natural L39/H89 interaction, and promote self- fisthefractionofRNAheterodimer,definedastheweight-
assemblywithinthecontextofthetectoRNAdimer.More- in-weight (w/w)ratioof thedimer(PR) tothetotalRNA
over, by using a structural syntax network that illustrates species (P+R+PR), (cid:3) is the maximum fraction of RNA
howsmallsubmodulescanbecombinedtogeneratelarger able to dimerize, and M is the total concentration of the
0
modular loop/receptor interactions, we explored the as- probe(55).With(cid:3)equalto1formostmoleculestested,the
sembly properties of rationally designed GNRA/receptor equation simplifies to K = [(M )(1−f)2]/f so that M /2
d 0 0
interactions with novel submodular combinations not yet isthevalueatwhich50%ofheterodimerisformed(24,31).
identifiedinnature(Figure1).Besidesdemonstratingthat Foreachsetofmolecules,K valuestypicallycorrespondto
d
retro-synthesized GUAA/receptor interactions can poten- theaveragecalculatedfromatleasttwoindependentexper-
D
tially mimic ancestral forms of the universally conserved iments.Forthosevaluesthatwerecalculatedfromthreeor ow
L39/H89interactionfromthelargeRNAsubunitoftheri- moreindependentexperimentsthestandarderrordeviation nlo
bosome (47), our study also expands the toolkit of RNA isusuallywithin30%butcanvaryfromonesampletothe ad
e
structural building blocks for RNA nanotechnology and other (Supplementary Table S3). K measurements below d
RNA synthetic biology applications (e.g. (6,36,39–41,48– 4000nMhavevariationsusuallywithdin15%orlesswhereas fro
m
50)). thoseabove4000nMcanbelessprecise,especiallyatcon- h
centration closer to the upper limit of detection (20 (cid:2)M). ttp
s
MATERIALSANDMETHODS The corresponding free energy of dimerization ((cid:2)G) be- ://a
tweentectoRNAreceptorsandRNAtetraloopprobesare ca
Rational structural design of new GNRA/receptor interac- determinedfromtheequation,(cid:2)G = RTlnK ,whereRis de
d m
tionsandtectoRNAsynthesis thegasconstant(1.985cal/K/mol)andTisthetemperature ic
ThebimoleculartectoRNAsystemusedinthisstudyassem- (283◦K).Theapparentfreeenergyvariationofdimerization .ou
blesthroughtwoGNRAloop/receptorinteractions(24,30) at 10◦C ((cid:2)(cid:2)G) can be derived from the equation, (cid:2)(cid:2)G p.co
accordingtotheatomicmodelstructuresavailable(Figure1 = (cid:2)G(PR)-(cid:2)G(reference) (Supplementary Table S4). In m
andSupplementaryFigureS1)(31,34).UsingtheRNAar- thisstudy,4000nMwasarbitrarilychosenasthereference /na
cnhoivteelctGonNicRsAgu/irdeecleipnteosr(3in9,t4e2ra),c3tiDonsstrduecstcurriablemdohdeerlesinfowrtehree tlohwreswhhoilcdh.aItllctohreretsepctoonRdNstAothheelinceaalrreescte‘protournsda’sKsedmvballeuewbiteh- r/article
built from RNA modules present in X-ray atomic struc- theircognateGURAtetraloops.GNRAspecificityprofiles -a
b
Mturoedse[PRDNBAIwDass:1uuse9ds,t1os7g2e,n2earvatye]wspiethciSfiwc,isssinPgdlbevniuewcleero(b5a1s)e. (tuesttileidzitnogmcoolroerecaosidlyedidbeanrtsif)ywbeirnedcinregapteadttefornrse(aScuhprpelceempetonr- strac
replacements (52) and image rendering was performed in taryFigureS1DandE).AttheexceptionofGAAA,allthe t/47
PyMOL(53).Properfoldingofthesecondarystructureof otherGNRAprobesweretestedfortheiraffinitytoeachre- /1/4
eachRNAconstructwascheckedusingUnafold(54). ceptor(SupplementaryTablesS3and4).AnUUCGprobe 80
RNA molecules (Supplementary Tables S1 and 2) were wasusedasanegativecontrolforindicatingthepositionof /51
transcribedfrompolymerasechainreactiongeneratedtem- themonomer.TheUUCGprobewasalsotestedwithsome 73
6
plates with T7 RNA polymerase, purified by denaturing ofthereceptorsinvestigatedinthisstudy.Itdidnotassem- 64
polyacrylamidegelelectrophoresis(PAGE)and3(cid:3)-[32P]pCp bleatRNAconcentrationsupto20(cid:2)M(datanotshown). by
labeledaspreviouslydescribed(30,31,33)(seeSupplemen- gu
e
taryData).AntisenseandprimerDNAsequenceswereor- s
deredfromIDT. Chemicalprobingwithdimethylsulfate(DMS) t on
0
5
TheS8tectoRNAmonomer(2(cid:2)M),withanelongated20 A
TectoRNA self-assembly and determination of equilibrium p
constantsofdissociation(Kd)andreceptorspecificity Sn2tB5’)t,awilafsoarspsreimmbelreedxwteinthsioonrw(Si8thtoauilt,GSuUpApAlemperonbtaer(y2T(cid:2)aMble) ril 20
1
K valueswerederivedfromthetitrationexperimentsper- (dimer contains equimolar amounts of GUAA tetraloop 9
d
formed at 10◦C as previously described (9,24,44) (Supple- and receptor) as described above in presence of 50 mM
mentary Table S3). In brief, self-assembly samples were HEPES, 100 mM KCl, 15 mM Mg(OAc) final. Follow-
2
prepared by mixing equimolar concentrations of recep- ingincubation,modifiedRNAsamplesweredirectlytreated
tor (R) and probe (P) tectoRNAs (ranging from 1 nM– with a solution of dimethylsulfate (DMS) diluted in 100%
20 (cid:2)M) in water, including 1nM of 3(cid:3)-[32P]pCp-labeled EtOH (30 mM or 60 mM final) and reacted at room tem-
probe tectoRNA (24). After denaturation (2 min, 95◦C; perature for either 4 min (monomer) or 8 min (dimer) at
3 min, 4◦C; 5 min, 30◦C), RNA samples were incubated 18◦C.Afterquenchingofthereaction,RNAsampleswere
20 min at 30◦C in association buffer (15 mM Mg(OAc) precipitatedandsubjectedtoprimerextensionusingSuper-
2
and 89 mM Tris-borate, pH 8.3) followed by equilibra- scriptIIIRT(Invitrogen)asdescribedintheSupplemental
tion for 30 min at 10◦C. TectoRNA assembly was mon- Informationandreference(56).Radio-labeled(32P)reverse
itored by native 7% (29:1) polyacrylamide gels at 10◦C transcription products of DMS-modified RNA were then
in association buffer. Monomers (Receptor [R]) and het- separated on 8% denaturing PAGE gel and visualized us-
erodimers (Receptor [R]xProbe [P]) were quantified us- ing a Typhon phosphorimager. Sequencing reactions were
NucleicAcidsResearch,2019,Vol.47,No.1 483
usedasmarkersandDMSexperimentswereconductedin the helix receptor were tested in the tectoRNA context
duplicate. fortheirabilitytorecognizeGNRAtetraloops(Figure2).
Whereas variations in binding affinity could be observed
RESULTS among them, these variants had the same GNRA selec-
tivityprofile.Thisobservationpromptedustohypothesize
NaturalL39/H89interactionsdonotpromoteefficientself- that receptors with similar structural features might share
assembly similar selectivity profiles. GNRA/helix interactions have
In ribosomes from all kingdoms of life, the L39/H89 in- acommonAminorstemmodule(CCC:GGGtriplet)that
teraction is structurally similar to GNRA/receptor in- is best recognized by GURA and GCRA tetraloops, with
teractions (Figure 1C and D). L39 is either a pen- GGRAtetraloopsbindingtheleast(Figure2B,left).Inthe
taloop (GUAAG in Archaea, Eukaryote) or a hexaloop context of L39/H89, changing the natural A minor stem
(GMUAAV (with M = A, C and V = G, A, C in Bacte- modulesofH89aandH89BintotheCCC:GGGbptriplet
ria)thatfoldslikeaGNRAtetraloop.InArchaeaandEu- resultedinreceptorsH89A2andH89B2with∼20-to100- D
karyotes, the pentaloop L39 has the first and fourth posi- folddecreaseinKdforGUAAincomparisontothenatural ow
tionsinaG:AtransShallow-groove/Hoogsteen(S:H)base H89receptors(H89A2,570nM;H89B2,2073nM;H89A, nlo
pair (bp), with the fifth position in bulge. In bacteria, the 55000nM;H89B;40000nM)(Figure2B,left;Supplemen- ad
e
Gsit:iAontsraonfstSh:eHhbexpailsofooprmLe3d9,bwetiwtheetnhethtehifirrdstaannddsfiixftthhpnuo-- ttiavriytyFpigroufireleSs4fAoraHnd89TAab2leanSd3)H.I8n9tBer2eswtienrgelyn,eGarNlyRidAensetilceac-l d from
cleotidepositionsinbulge.Bothpenta-andhexa-loopsL39 tothoseforGYRA/helixinteractions(Figure2B,leftcol- h
have two conserved adenines that form a type I/IIT A- umn)suggestingthatH89A2andH89B2behavedashelical ttps
minorinteractionwiththesecondandthirdWatson–Crick receptorsratherthan2x bulgemodules. ://a
(W:W) bps of H89 (Figure 1C). Additionally, the recep- Inordertotestwhetherthepresenceofa2x bulgemod- ca
d
tor H89, which folds as a ‘S8-like’ 2bp 2x bulge module ule at the level of H89 could enhance binding affinity of em
(8,12), forms a type 2a platform and a recognition mod- GNRAloops,thesequenceofH89wassubstitutedwiththe ic
ule for stabilizing the docking of L39 (Figure 1C; Supple- sequence of the receptor binding site for protein S8 from .ou
p
mentaryFiguresS2and3).NaturalL39/H89interactions theE.coli(S8a1,S8a2)andThermusthermophilus(S8Tta) .co
fromArchaeal(e.g.Haloarculamarismortui)andBacterial 16SrRNAs.H89receptorsandS8receptorsbelongtothe m
(e.g.Escherichiacoli)ribosomeswerebothtestedwithinthe ‘S8-like’ family of 2x bulge modules (Supplementary Fig- /na
ncoonttperxotmofottheeatneyctsoigRnNifiAcadnitmseerlfs-yasstseemmb(Flyigautr1e51mB)ManMddg2id+ usqreuaSr3eAd)e.vTiahteioynaoref1st.4ruA˚ct(uSruapllpyliedmenentitcaarly,wFiigthuraerSo3oCt)m.Teahne r/article
and10◦C(GUAAG/H89A,50000nM;GAUAAG/H89B, central bp doublets of the 2x bulges from S8 involve clas- -a
2su0b0s0t0it0utniMng;thSeupppenletma-eanntadrhyeTxaab-lloeospSs3foarnmdo4r)e.sMtabolreeoclvaesr-, sniacmAi:cUallaynmd/oorersCta:GbleWthaatsnotnh–oCserifcrkombpHs8a9ndreacerepttohresr,mwohdicyh- bstrac
sic GNRA tetraloops (excepted GAAA) only resulted in contain U:U and U:G Watson–Crick bps. As shown by t/47
breasrpeelyctntootioctehaebrleteimtrpalrooovpems.eAnstsnfootreGdUprRevAiotuestrlyal(o8o),ptshwisitihs RtuNreAofmGoUdeAliAng/S(F8i(gtyupree12Da)(iisii)starnudctu(irva)l)l,ytheqeuteivratilaernyttsotruthce- /1/480
likely due to the inability of natural L39/H89 sequences one of L39/H89 despite differing in sequence (7 positions /51
to fold and assemble into their native structures outside outof17inbacterialL39/H89). 73
thecontext of theribosome. For instance,H89 natural se- Like in L39/H89, the type 2a platform of the 2x bulge 664
quences have suboptimal A-minor submotifs and rather modulecanserveasa‘ribose’stackingplatformforthesec- by
than folding as 2x bulge modules, they can adopt alterna- ond nucleotide of the GNRA (or GNRA-like loop) (Sup- gu
tivestructuresthatarenotconducivetoassembly. plementaryFiguresS2and3).Moreover,thesecondposi- es
AccordingtothestructuralsyntaxofRNAmodulesand tion of the GNRA can form additional contacts with the t on
submodules from stable RNAs, the metastable sequences X3:X11 W:W bp of the A-minor stem module. In the tec- 05
of H89 can potentially be substituted by sequences from toRNA dimer context, S8a2 and S8Tta, with a C:G bp A
p
morestableandstructurallyequivalentsubmodules.Several for X3:X11, demonstrated dramatic decreases in Kds for ril 2
natural2x bulgesadoptthesametertiarystructureasH89 GUAAwithrespectofnaturalH89receptors,inthesame 0
1
(‘S8-like’ 2bp 2x bulges) (Figure 1C ; Supplementary Fig- orderofmagnitudeasH89A2(Figure2andSupplementary 9
ure S3A and C). Additionally, other 2x bulges (‘P12-like’ FigureS4).However,theirGNRAselectivityprofileswere
2bp 2x bulges) have different platforms that could poten- markedlydifferentfromGNRA/helixinteractions(Figure
tiallybefunctionallyequivalenttothosefromH89(8). 2B). S8a2 and S8Tta also had decreased Kds for all other
GNRAloops.Forinstance,S8a2wasabletoassemblewith
GGAA (290 nM) and GGGA (961 nM) almost as well
L39/H89 can be repaired with structurally equivalent mod-
as with GUAA (220 nM) and GUGA (500 nM), demon-
ules
strating that the 2x bulge was likely to contribute to this
Wefirstattemptedto‘repair’L39/H89byoptimizingthese- change of selectivity. Remarkably, the presence of a U:G
quenceoftheA-minorstemmoduleX1X2X3:X11X12X13 inX3:X11dramaticallyincreasesdiscriminationforGUAA
(Figure 2A). The X1:X13 W:W bp is usually not crucial versus the other loops. S8a1, with a U:G bp for X3:X11,
for A-minor module assembly. However, the optimal se- improved binding affinity by 75–100 fold with respect to
quence for X2X3:X11X12 is CC:GG in GYRA/helix in- R89A1andR89B1.Italsodisplayedamuchgreaterselec-
teractions(Figure1C)(8,19).Severalsequencevariantsof tivityforGUAAversusanyotherloops:itsKdforGUAA
484 NucleicAcidsResearch,2019,Vol.47,No.1
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
a
r/a
rtic
le
-a
b
s
tra
c
t/4
7
/1
/4
8
0
Figure2. Re-engineeringtheL39/H89interactionintomorestablevariantswithS8-likeandP12-likereceptors.(A)2Ddiagramsofthedifferentreceptor /5
1
constructstested.Thetypeofproximalplatformfromeachreceptorisindicated.Sub-modulesinthereceptorincludetheAminorstem(withAminor 7
3
G:Cinviolet)anddifferentplatforms(type1a(orange),2a(paleyellow),3a(palebrown)andtWH(palegreen)).(B)SelectivityprofilesofvariantsofH89, 6
S8-likeandP12-likeinteractionstestedagainstGNRA(exceptGAAA).Colorsareindicativeoftherelativestabilityofeachloop/receptorinteractionat 64
10◦Cinpresenceof15mMMg(OAc)2,with0kcal/molcorrespondingtoaKdof4000nM(see‘MaterialsandMethods’section).Y3,C(redsquare);Y3, by
U(bluediamond). g
u
e
s
t o
(358nM)wasapproximatelyninetimeslowerthantheone receptors are not structurally equivalent to H89 but could n
0
for GGAA and about 55 to 110 times lower than the Kds likewiseprovidefunctionalstackingplatformsfordocking 5
fortheotherGNRAloops. RNAloops.Type3aplatformsmimictype2aplatformsand Ap
provide‘ribose’stackingfordockingGNRAtetraloopsin ril 2
NovelL39/H89interactionscanbeengineeredwithfunction- a way that resembles most H89/L39 interactions (Figure 01
9
allyequivalentnucleotideplatformmodules 1D (vi) and Supplementary Figure S3). In the context of
thetectoRNAdimer,P12a2andH68a2,withaC:Gbpfor
Type 3a platforms as docking sites for GNRA. P12-like X3:X11, were both able to assemble to GNRA loops but
2x bulgereceptors,suchas‘P12’fromRnasePRNAsand withdifferentpatternsofGNRAselectivityandnotaswell
‘H68’ from the large RNA ribosomal subunits, have nu- asS8-likereceptors(Figure2andSupplementaryTableS3).
cleotideplatformsthatarestructurallydifferentfromthose Moreover, P12a1 and H68a1, with a U:G bp for X3:X11,
observed in S8-like 2x bulge modules (Figure 1; Supple- did not performed as well as S8a1 (Figure 2B) but never-
mentary Figures S2 and 3). As exemplified by S8Tt and theless increased selectivity for GUAA versus most of the
P12 receptors, sequence variations in no more than 2 nt other GNRA loops. These data suggest that type 3a plat-
positionsinthe2x-bulgemodulecanspecifyeitherforthe formsarenotfullyequivalenttotype2aplatforms,andem-
type 2a and type 1a platforms of S8-like receptors, or the phasizethatthestructuraldifferencesbetweenS8-likeand
type 3a and tWH platforms of P12-like receptors (Figure P12-likereceptorscanhavesignificanteffectsontheability
1C; Supplementary Figure S3A and B). As such, P12-like torecognizeGNRAloops.
NucleicAcidsResearch,2019,Vol.47,No.1 485
Type1aandtWHplatformsasdockingsitesforGNRA. In significantlyhigherthanthoseobtainedfromtype1aS8re-
bacterial 16S rRNAs, it is not the type 2a but the type 1a ceptors.P12boundallloopswithinthesamerangeofK s
d
platform of the S8 receptor that specifically interacts with (191–367nM),withaslightpreferenceforGGGA.H68as-
protein S8 (Figure 1). This platform is very similar to the sembledbestwithGGAA(487nM),GUAA(688nM)and
type1aadenosineplatformusedintheGAAA/11ntinter- GGGA(732nM)butdidnotdiscriminatetheotherloops
action(Figure1)(37).IntheP12andH68receptors,itisthe bymorethan8-fold.
tWH platform that is used for stacking U-turn containing
terminalloops(Figure1;SupplementaryFiguresS2and3).
TheY3:G11bpcontributestoGUAAselectivity
Bothtype1aandtWHplatformsallowbase-basestacking
between the loop and the nucleotide platform rather than AccordingtothestructureofL39/H89fromArchaeaand
ribose-basestackinglikeinH89/L39(SupplementaryFig- Eukaryotes,U3:G11bpisresponsibleforpreferentiallyrec-
ureS2).Moreover,bothS8-likeandP12-likereceptorshave ognizing the uracil at the second position of the GUAAG
an internal two-fold pseudo-symmetry that suggests that loopinL39/H89(SupplementaryFigureS5).Thisislikely
D
theirplatforms could potentially be swapped (Supplemen- favoredbytheformationofaspecificH-bondbetweenatom ow
taryFiguresS2and3).Therefore,wehypothesizethattype O4oftheuracilofL39andG11ofH89andmightbeen- nlo
1aandtWHplatformsofS8-likeandP12-likereceptors,re- hancedbychelationofanion(possiblyapotassium)(Sup- ad
e
spectively,couldpotentiallybeusedtopromotedockingto plementaryFigureS5)(15,57).Liketype2areceptors,most d
GNRA tetraloops (Figure 1). According to the 3D model of the type 1a, type 3a and tWH receptor variants that fro
m
of the GNRA/S8 (type 1a) interaction, type 1a platforms were tested with a U:G bp instead of a C:G bp for posi- h
canprovideoptimalstackingforGNRAloopsmeanwhile tion Y3:G11, displayed a significant increase of selectivity ttp
s
preservingtheH-bondcontactsoccurringbetweenL39and for GUAA versus the other loops (Figure 2 and Supple- ://a
H89(Figure1D(v)andSupplementaryFigureS2).Bycon- mentaryFigureS6).S8.11andS8Tt6significantlyimproved ca
d
trast,thetWHplatformofP12-likemodules,whileoptimal the receptor selectivity for GUAA but with slightly higher e
m
ftoimradlofcokrindgocUk-itnugrnGcNonRtAainteintrgatleorompsin:afolrloionpstsa,niscen,otthaesporpe-- SKudpspthleamnetnhteairryCF:GigucroeusnSte4r,p6aartnsd(ST8abalnedSS3)8.TAt)m(oFnigguarlel2thBe, ic.ou
cisespacingoftheloop/receptorinteractionwithP12-like receptors with increased selectivity for GUAA, S8.11 had p.c
o
modulesisslightlydifferentfromtheonewithS8-likemod- thelowestK forGUAA(21nM):itfavoredGUAAversus m
ulesasthetWHplatformshiftsthestackingnucleotideby GCAAby∼d19-foldandGUAAversusalltheotherGNRA /na
approximatelyhalfabasepair,leadingtoapotentialsteric loopsby65-to85-fold.S8Tt6followedasimilartrend.In- r/a
clashwhenGNRAloopsdocktoP12-likereceptors(Sup- terestingly, H68.9, a P12-like receptor with a U3:G11 bp, rtic
le
plementaryFigureS2).Becauseoftheirdifferentbase-pair also had a significant increase of selectivity for GUAA in -a
b
patterns, P12-like receptors might not be as easily swap- comparison to H68 (with a C3:G11 bp). Despite the fact s
phaobwleevaesrSp8o-slsikibeleretcheapttothrse(flSeuxpibplleemspernintagr-ylikFeignuarteurSe3)o.fIhteis- trhesapteHct68o.f9Hh6a8d(a6n8i8ncnrMea)s,eHd6K8d.9forrecGoUgnAizAed(2b0e5t4tenrMG)UwAitAh tract/4
7
licalstemsmightprovidelocaladjustmentsfortheefficient than GGAA whereas the opposite trend was observed for /1
formation of GNRA/P12-like receptor within the context H68. As a general trend, these data suggest that the U:G /48
0
oftectoRNAself-assemblydimers. bpatpositionX3:X11hasastructuralroleforthespecific /5
1
As anticipated, S8 (type 1a) constructs had remark- recognitionofGUAAversusotherGNRAloops.Theim- 73
6
able self-assembly properties within the context of tec- portanceofthisbasepairforselectivityisalsocorroborated 6
4
toRNA dimers. S8 and S8Tt had K s for GUAA of 19 by the fact that S8.3, with a C:C mismatch bp in X3:X11, b
d y
and 5 nM, respectively. They assembled to GUAA with hadasignificantlossofbindingaffinityforGUAAbutonly g
u
11- to 150-fold improvements in binding affinity in regard moderatelossfortheotherloopsincomparisontoS8(Sup- es
to their respective constructs with proximal type 2a plat- plementaryFigureS4andTableS3).Similarly,inbothP12 t o
n
form(S8a2andS8Tta)(SupplementaryFigureS4E).More- andH68contexts,variantswithaC:CbpinX3:X11(P12b 0
5
over, binding improvements of S8 and S8Tt versus H89 andH68.2)hadmoresignificantlossofbindingaffinityfor A
fvoalrdianfotsr (GHU8A9AA2aanndd ∼H7809-B2to) r∼an50g0e-fforoldmfo3r0-GtGoA∼A40,0a- GYRAloopsthanforGRRAloops. pril 20
1
tetraloop that is poorly recognized by H89A2 and H89B2 9
The length of the A-minor stem is important for optimal
(Supplementary Figure S4E). Both S8 and S8Tt favored
stackingwiththeplatform
GUAA versus all the other loops in the following or-
der GUGA>GGAA>GAGA>GGGA>GCAA>GCGA In the tectoRNA dimer system, the optimal length of the
folds (Figure 2; Supplementary Figure S4 and Table S3). stemsseparatingthetwointeractingGNRA/receptormod-
This data demonstrates that the type 1a platform from S8 ulesis11bp(24,30,31).FortheS8andP12sequences,short-
modules can be used as a docking site for GNRA loops eningorincreasingthestemlengthby1bpledtoasignifi-
leadingtointeractionswithaffinitycomparabletotheone cantlossofbindingaffinitythatwasassociatedtochanges
of the GAAA/11 nt interaction (24,31), albeit with lower of selectivity profiles, corroborating our previous data ob-
selectivity. tainedfromthistectoRNAsystem(24,30,31)(Supplemen-
P12 and H68 receptors with tWH platforms performed tary Figure S7). Shortening the stem length proved to be
better than their type 3a counterparts (P12a2 and H68a2) much more detrimental in the P12 context than in the S8
byoneorderofmagnitude(Figure2B).However,P12dis- context (compare S8.24 and P12a in Supplementary Fig-
playedpoorGNRAselectivityanditsKdsforGNRAwere ureS7B).Thissubstantiatesthemodelwhereshorteningthe
486 NucleicAcidsResearch,2019,Vol.47,No.1
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
a
r/a
rtic
le
-a
b
s
tra
tFairgguertes3a.ndMroedceuplattoironcoonftebxintsdwinigthainffiwnihtyichantdhelosoeqpuseenleccetiovfittyhethprorouxgihmpaloipnltatmfourtmatiwoansscinhaSn8g-elidk:e(aa)ndS8P;1(2b-)liSk8eTntu/cPl1eo2;ti(dce)pHla6t8f.o(rBm)s.B(iAnd)iLngistafofifnGityNaRnAd ct/47
loopselectivityofvariousplatformssequencesintheS8structuralcontext.(C)Variationofbindingaffinityandloopselectivityisbothdependentonthe /1
sequenceidentityoftheproximalanddistalplatforms.DifferentreceptorswiththesameplatformcanhavedifferentGNRAselectivityprofiles.Alldata /48
wereobtainedat15mMMg2+and10◦Casdescribedin‘MaterialsandMethods’section. 0/5
1
7
3
6
stemintheP12contextwouldfurthercreateastericclash formofS8(S8.19)maintainedtheselectivityprofileofS8, 6
4
between the second position of the GNRA tetraloop and withlowerKdsforallloops(SupplementaryFigureS7B). b
y
thetWHplatform.However,increasingthestemlengthin Infact,S8.19turnedouttobethebestGUAAbinderwith g
u
bothS8andP12contextsledtoGNRAselectivityprofiles aK ofapproximately1nM.Thisclearlydemonstratesthat e
d s
resemblingtheoneforGYRA/helixinteractions(S8.23and thedistalplatforminfluencesbindingattheproximalplat- t o
n
P12d,SupplementaryFigureS7),suggestingthatthedock- form. Similarly, removal of the distal type 3a platform of 0
5
ingoftheGNRAtothereceptormightrelyonlooserstack- H68 (S8.28) and P12 (P12c) maintained the GNRA selec- A
p
inginteractionswithitsplatform. tKivdisty(SpurpopfilleemseonftHar6y8FaingdurPe1S28,Are,srpigehctticvoelluy,mbnu,tSwuipthplhemighener- ril 201
tary Table S3). In good agreement with the atomic struc- 9
Sequence variations within nucleotide platforms modulate
turesof2x bulges(SupplementaryFigureS3AandB),this
bindingaffinityandGNRAselectivity
datasuggeststhattype3aandtypetWHplatformsareless
In order to demonstrate the role of the platforms in the independent from one another than type 1a and type 2a
engineered versions of L39/H89, we tested several vari- platforms.
ants within the bulging nucleotides involved in the proxi- Interestingly, some S8-like and P12-like receptors differ
mal and/or distal platforms of S8-like and P12-like recep- by no more than two to four nucleotides positions, sug-
tors(Figure3;SupplementaryFiguresS7and8). gestingthatonlyminorsequencechangesarenecessaryfor
The removal of the bulging nucleotides (N4–5 and N8) controlling the folding of 2x bulge motifs into S8-like or
from S8 led to hGYAA with a typical GYRA/helix selec- P12-like receptors (e.g. compare P12 and S8Tt and H68
tivityprofilesimilartotheoneofH89A2(Figure2andSup- andS8Figure2A).AccordingtoknownX-raystructures,
plementary Figure S7). Deletion of the proximal type 1a G:U, A:A, U:A, U:C and A:C cis Hoogsteen edge (H):
platform of S8 also had a typical GYRA/helix selectivity Shallow-groove edge (S) bps are the most abundant base
profile(S8.20).However,deletionofthedistaltype2aplat- pairsthatcontributetotheformationoftype1aplatforms
NucleicAcidsResearch,2019,Vol.47,No.1 487
(58)(SupplementaryFigureS8).Withinthecontextofthe a more independent fashion in S8-like receptors, our data
S8receptor,proximalAA,ACandUAandUCplatforms supporttheideathatS8-likereceptorsaresomewhatmore
atpositionsN4–5hadKdsforGUAA,rangingbetween7 robusttomutationsthanP12–likereceptors.Moreover,asit
and38nM,buttheycouldalsodisplaystrikingchangesin isthecaseforS8Tt-P12receptors,ourdataalsoemphasize
loopselectivity(Figure3B).Forinstance,theUCplatform thatverysmallvariationsinsequencesleadtothepossibil-
(S8.16)hadadramaticincreaseofselectivityforGUAAver- itytoswitchfromoneclassofreceptortoanother.
sus GGRA and GCGA tetraloops in comparison to AA,
AC and UA platforms (Figure 3). By contrast, other plat-
S8-likereceptorsandP12-likereceptorsarerelatedthrough
forms(CA,GU,AG,AUandUG)displayedweakenedloop
sequence/structurespace
specificitywithhigherKds.Interestingly,theCAplatform
(S8.2)wastheonlyplatformthatalteredthespecificityfrom S8, S8Tt, P12 and H68 are linked through their
GUAA to GGAA within the context of S8 (Figure 3B). sequence/structure space (Figure 4), suggesting that
S8.2boundGGGAandGUAAequallywell.Thispattern receptors with S8-like conformation, such as S8 or H89,
D
of GNRA selectivity was the same as the one of H68, the could have transiently adopted P12-like conformation ow
naturalP12-likereceptorwithaCAtWHplatform(Figure through evolution. Remarkably, P12 and S8Tt, which are nlo
3C). These data strongly suggest that the sequence of the onlytwomutationsawayfromoneanother,displayedvery ad
e
proximalplatformcansignificantlyalterGNRAselectivity distinct selectivity profiles, which are each characteristic d
and can possibly affect the local structure of the 2x bulge of the class of receptor to which they belong (Figure 4B). fro
m
towardeitherS8-likeorP12-likeconformations. S8, which is three mutations away from S8Tt, shares with h
Wefurtherexploredwhetherthe2x bulgestructuralcon- S8Ttthesamecharacteristicselectivityprofile(Figures2B ttp
s
text can affect binding affinity and selectivity profiles of and 4B). S8Tt5, the sequence intermediate between S8Tt ://a
variantswithproximalplatformsofidenticalsequence(Fig- and P12, equally favored GUAA and GGGA, the pre- ca
d
ure 3C). UA platform in the S8, S8Tt-P12 and H68 con- ferredtetraloopsrecognizedbyS8TtandP12,respectively. e
m
textsledtoreceptorswithamarkedpreferenceforGUAA. Moreover,S8Tt5didnotbindGUGAaswellasS8Ttand ic
While S8 and S8Tt had almost identical GNRA selectiv- S8. This suggests that this receptor might eventually have .ou
p
ity patterns, H68.1 preferred GGRA versus GUGA and a propensity to switch from one class to another upon .c
o
GAGA (Figure 3C). Changing the UA platform into the docking of a particular GNRA loop. Walking through m
AA platform had little effect on the loop selectivity in the thesequencespacefromS8toH68indicatesthattheshift /na
contextofS8(S8.14).However,thesamechangeinthecon- in specificity toward GGAA essentially resulted from the r/a
textofS8Tt-P12(S8Tt5)andH68(H68.5)dramaticallyre- change in platform sequence from UA to CA (Figures rtic
le
duced selectivity for GUAA versus the other loops: S8Tt5 3and4).S8,S8.5andH68.1,withaUAproximalplatform, -a
b
boundGUAAandGGGAloopswiththesameKd(37nM) favored GUAA whereas H68, S8.4 and S8.2, with a CA s
whereas H68.5 displayed a marked preference for GGAA platform, slightly favored GGAA versus GUAA and trac
followed by GGGA, with its GNRA selectivity profile re- GGGA (Figure 4B). S8.4, with the same distal platform t/4
7
sembling the one of H68 (Figure 3). Furthermore, S8.15, as S8 but the same proximal platform as H68, had a /1
/4
with an AC platform in the S8 context, had increased se- very similar profile to H68 but with improved K s for all 8
d 0
lectivity for GUAA versus GGRA in comparison to S8 loops(Figure4).Thisindicatesthattheadditionalbulging /5
1
whereasP12.14,withanACplatformintheS8Ttcontext, adenine in the distal platform of H68 is somewhat desta- 73
hadreducedselectivityforGUAAincomparisontoS8Tt. bilizing the GNRA/receptor interaction. The presence 66
4
The selectivity pattern of H68.6, with an AC platform in of this extra adenine in H68.1 is also likely responsible b
y
theH68context,wasclosertotheoneofH68,albeitwith for H68.1 decreased affinity in comparison to S8. The g
u
improved affinity. The AG platform, which is characteris- change from G10:C6 bp to C10:G6 bp had little effect on es
tic of P12, led to very significant reduction of selectivity the receptor profile and binding affinity (Figure 4B). The t o
n
toward all loops in both S8 and H68 contexts (S8.18 and only noticeable trend was that S8 (like S8Tt) preferred 0
5
H68.8 in Figure 3C), resembling most the profile of P12. GUGA versus GGRA whereas S8.5 and H68.1 had the A
p
Overall, these data illustrate that different 2x bulge con- oppositetrend.Overall,thesedatashowthatthesequence ril 2
texts affect and modulate the selectivity of recognition of space associated to 2x bulges allows possible transitions 0
1
GNRAtetraloopsbyproximalplatformswithidenticaldi- from S8-like receptors to P12-like receptors (and/or vice 9
nucleotidesequence.Theyalsoindicatethatsmallchanges versa) without loosing binding affinity for the GUAA
atreceptorpositionsthatarenotdirectlyincontactwiththe tetraloop (Figure 4). Indeed, despite slightly preferring
loop target can still affect the way the loop is recognized. GGRA tetraloops versus GUAA, P12-like receptors still
This is further supported by the fact that a G:U bp is fa- maintain good binding to GUAA. Therefore, our data
voredoverregularWatsonCrickbpsforthedistalclosing suggest that GNRA/2x bulge interactions are modules
bpofS8-likereceptors(X7.2:X7.1bpSupplementaryFig- with high mutational robustness and sequence/structure
ureS8B).Consideringthatbothclassesof2x bulgerecep- plasticity.
tors have structural contacts between proximal and distal
platforms (Supplementary Figure S3), the distal platform
EngineeredH89receptorsbindtonaturalpenta-loopL39but
sub-module likely affect the way the proximal platform is
recognizehexa-loopL39poorly
folded.ThisisespeciallytrueinthecontextofP12-likere-
ceptors for which tertiary contacts are more pronounced. Like natural H89 receptors, helix receptor hGYAA and
Asthedistalandproximalplatformsub-modulescanactin other S8-like receptors with proximal type 2a platform
488 NucleicAcidsResearch,2019,Vol.47,No.1
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
a
r/a
rtic
le
-a
b
s
tra
c
t/4
7
Figure4. Movingwithinthesequence/structuralspaceofS8-likeandP12-likereceptors.(A)MutationalpathwayslinkingP12,S8Tt,S8andH68.Nu- /1/4
cleotidesinredindicatesequencevariationsfromS8(referencesequence).Doublearrowsindicatesinglepointmutationtransitionsbetweentworeceptors. 8
0
Thenumberofwhitenodesonarrowsindicatesthenumberofintermediatesbetweentworeceptors.Selectivityprofilesareshownascoloredbarcodesfor /5
1
receptorstestedagainstGNRA(exceptGAAA),GUAAGandGAUAAGloops(seelegendFigure2).(B)VariationsofKdsandGNRAselectivityfrom 7
P12toH68viaS8TtandS8.Alldatawereobtainedat15mMMg2+and10◦Casdescribedin‘MaterialsandMethods’section. 36
6
4
b
y
(S8a1, S8a2) did not promote efficient recognition of the GUAA tetraloop (Figure 5C). As such, the natural penta g
u
GUAAGpentaloopandGAUAAGhexaloop(Figure5A). and hexa-loop L39 can be seen as evolved metastable ver- e
s
By contrast, all tested receptors with type 1a platforms sionsofGUAA(Figure5C). t o
n
(S8Tt5, S8.15, S8.11, S8, S8tt2, S8.14, S8Tt, S8.19) and 0
5
H68.5 recognized GUAAG with Kds ranging between 122 A
2x bulgereceptorsaremetastableandpronetofoldintoal- p
aGnUdA5A20GnbMou(nFdigtuoreS85)a.nFdoSr8iTnsttawnitche,Kthdseopfen1t7a0loaonpdL21349 ternativestructures ril 20
1
nM,respectively.ThebestreceptorwasS8.14,withaKdof AccordingtothesequenceofnaturalH89receptors,itisap- 9
122nM.However,mosttestedreceptorsdidnothavesignif- parentthatthesereceptorscanadoptalternativesecondary
icantbindingaffinityforthehexaloopGAUAAG.Theonly structures(Figure6A)(18).Forinstance,ratherthanfold-
exceptionwasS8Tt5thatrecognizedthehexaloopL39with inginto2x bulgemodulesasobservedintheribosomecrys-
aKdof909nM.ThenaturalL39loopswereusuallynotrec- tallographicstructures,naturalH89sequencescanformex-
ognized with an affinity stronger than those observed for tended helical elements and, as shown previously, they do
GNRA tetraloops. Among all the receptors tested, S8.11 not promote efficient assembly with L39 (see above). This
provedtobetheexception.Itdisplayedastrongselectivity suggeststhatthesequenceofH89evolvedtobemetastable.
for the pentaloop L39. After GUAA (Kd 21 nM), the sec- However, the sequence of other 2x bulges like the one of
ond most favored loop recognized by S8.11 was GUAAG S8,B7.8orS8.2canalsopotentiallyformalternativebase
(Kd314nM)(Figure5).Consideringthatthebest2x bulge pairs (Figure 6C). For instance, the B7.8 receptor previ-
receptorstestedinthisstudydisplayedamarkedpreference ously isolated by Costa and Michel (29) was initially pro-
for GUAA versus all the other GNRA (Figure 5B), this posed to fold as a 3 nt internal loop (Figure 6C, left side)
data suggests that the ancestral form of L39 was likely a based on its predicted thermodynamic stability using un-
NucleicAcidsResearch,2019,Vol.47,No.1 489
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
tFaenisgdtuerdGeiA5nU.thABisAinsGtduidn(byg.lua(Aeffi)dnSiiaetymveforoanrldGv)aUarriAaenActoGsmopfpeHanr8tea9dl,oStoo8pathanenddoHnG6eA8fUworeAroAetthGeesrhteeGdxNaaltRo1oA0p◦Ctfeotirnraspelorleeocspteesn.dc(erBeo)cfeR1p5atonmrgsMeaonMfdKgr(daOsnAgoebc)so2ef.rBKveidndsdfifonorgraaallfllfirGneicNteypRftoAorr/G/rGeUcNAepRAtoAGr(iinnrettdeerrsaaqccuttiiaoornness) ar/article
testedinthisstudy(total518).(C)EvolutionofL39fromanancestralGUAAtetraloopbyinsertionofnucleotides:L39fromArchaeaandEukaryoteis -a
ametastablepentaloop.Inyeastpre-60Sribosomes(Eukaryote),L39isnotfoldedasaGNRA-like[PDBID:3jct].Inthenativeribosome,itisfoldedas b
s
aGNRA-likestructureoncedockedtoH89[PDBID:3u5d]. tra
c
t/4
7
afold(54). However, itssequencediffersfrom S8 at only 4 (Figure6C).WethengeneratedS8Tt8,aderivativeofS8Tt /1
ntpositionswithintheAminorsubmodule.Asexpected,it withaCAplatform,totestwhetheritsbehaviormimicked /48
0
canfoldintoa2x bulgemodulethatbehavedlikeS8(Fig- thatofP12h(SupplementaryFigureS9B).Contrastingly,it /5
1
ure6).Inordertogarnermoreinformationaboutthestruc- demonstratedunpredictedbehaviorthatseemedtodestabi- 7
3
tural state adopted by the S8 receptor in presence and ab- lizetheloop/receptorinteractionforallloops(Supplemen- 66
4
senceofitscognateGUAAtarget,DMSchemicalprobing tary Figure S9C). S8Tt8 not only had a preference for the b
y
was performed on S8 in the tectoRNA context in absence GUAAloop,butthebindingaffinityforGGAAwasgreatly g
(monomer) or presence of GUAA probe (dimer) (Figures destabilized,withaK of12.3(cid:2)M.Uponcloserexamina- ue
d s
6BandSupplementaryS9A).InabsenceofGUAA,adenine tion of its primary sequence, S8Tt8 could also potentially t o
n
positionswithinthereceptorwereshownaccessibletoDMS adoptanalternativefoldtogenerateahelicalstemwithan 0
5
indicatingthatthecentralWatson–Crickbpdoubletofthe A:C bp mismatch (Supplementary Figure S9B): the stem A
p
G2xUbAuAlgetamrgoedt,utlheedseidandoentifnoersmb.eBcaymceonptrroatsetc,teindporrepseanrtciealolyf sfoeqrmueanticoenboefloawhtehliexroevceerptthoer porfeSd8icTtte8dl2ikxeblyuplgreomseocotenddathrye ril 2
0
1
protected, indicating that the 2x bulge formed by induce structure.Changingtheprimarysequenceofthelowerstem 9
fitwhenbindingtoGUAA.Therefore,likenaturalH89re- to drive the formation of the 2x bulge structure over the
ceptors,theengineeredS8receptorretainsmetastabilityin helix (S8Tt8.a) not only restored the interaction between
absence of cognate loop. This observation is likely to be all GNRA tetraloops, but had a profile similar to that of
a general feature of 2x bulges. Additional indications for P12,withGUAAandGGAAtetraloopshavingcompara-
themetastabilityof2x bulgeswerealsoprovidedbyunex- bleK ’s(370and529nM,respectively).Interestingly,S8Tt,
d
pectedGNRAselectivityprofilesforseveralvariants(com- which can also adopt the same type of alternative fold as
pare P12h with S8.2 and S8Tt8 with S8Tt). For instance, S8Tt8,favoredthe2x bulgefold.Accordingly,changingthe
P12h, which can form an AC platform like P12.14, was primarysequenceinthelowerstemledtoS8Tt4a,whichbe-
initially expected to behave more like P12.14 (Supplemen- havedlikeS8Tt.Thesedataillustratehowsmallvariations
taryFigureS7B).However,itsGNRAselectivityprofilewas of sequences can dramatically affect the thermodynamic
identicaltoS8.2,withaCAplatform.Thispromptedare- stabilityofalternativebasepairslocalizedinthevicinityof
evaluationofthelocalsecondarystructureofP12h.More 2x bulgesandeventuallydrivetheirstructureintoalterna-
likely,P12hadoptsanalternativefoldwithaCAplatform tivefolds.