A Threefold RNA Protein Interface in the Signal ... · the unlabeled wild-type SRP19 protein (see Materi-als and Methods). Site-directed cleavage experi-ments also demonstrated that

doi:10.1016/j.jmb.2007.03.032 J. Mol. Biol. (2007) 369, 512–524

A Threefold RNA–Protein Interface in the SignalRecognition Particle Gates Native Complex Assembly

Tuhin Subhra Maity and Kevin M. Weeks⁎

Department of Chemistry,University of North Carolina,Chapel Hill, NC 27599-3290,USA

Abbreviations used: SRP, signal rRNP, ribonucleoprotein; FRET, fluorenergy transfer.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2007 E

Intermediate states play well-established roles in the folding andmisfoldingreactions of individual RNA and protein molecules. In contrast, the roles oftransient structural intermediates in multi-component ribonucleoprotein(RNP) assembly processes and their potential for misassembly are largelyunexplored. The SRP19 protein is unstructured but forms a compact coredomain and two extended RNA-binding loops upon binding the signalrecognition particle (SRP) RNA. The SRP54 protein subsequently binds tothe fully assembled SRP19–RNA complex to form an intimate threefoldinterface with both SRP19 and the RNA and without significantly alteringthe structure of SRP19. We show, however, that the presence of SRP54during SRP19–RNA assembly dramatically alters the folding energylandscape to create a non-native folding pathway that leads to an aberrantSRP19–RNA conformation. The misassembled complex arises from thesurprising ability of SRP54 to bind rapidly to an SRP19–RNA assemblyintermediate and to interfere with subsequent folding of one of the RNAbinding loops at the three-way protein–RNA interface. An incorrecttemporal order of assembly thus readily yields a non-native three-component ribonucleoprotein particle. We propose there may exist ageneral requirement to regulate the order of interaction in multi-componentRNP assembly reactions by spatial or temporal compartmentalization ofindividual constituents in the cell.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: ribonucleoprotein assembly and misassembly; compartmentali-zation; energy landscape
*Corresponding author
Introduction

Assembly of most ribonucleoprotein (RNP) com-plexes is accompanied by significant conformationalchanges in both protein and RNA components.1,2

In some cases, these conformational changes mayoccur in a progressive fashion such that earlyinteractions in assembly lead to further conforma-tional rearrangements that uniformly facilitate latersteps. Several structurally linked steps in assemblyof the 30 S ribosomal subunit appear to proceed inthis manner.3–5 However, in view of the multiplicityof RNA–protein and protein–protein interactionsthat can potentially occur during assembly of multi-component RNPs, the extent to which incorrect or

ecognition particle;escence resonance

ng author:

lsevier Ltd. All rights reserve

premature interactions among components mightinterfere with native complex formation remains tobe assessed. The idea that some RNA6,7 andprotein8,9 molecules readily adopt stable, but mis-folded, states is now well established. We haverecently identified a striking example of robust mis-assembly in the mammalian signal recognitionparticle.10 Our work extended the concept of kineti-cally trapped, non-native conformational states tomulti-component RNP complexes. Here we analyzethe kinetic mechanism for this misassembly.The signal recognition particle (SRP) recognizes

nascent membrane or secretory proteins emergingfrom translating ribosomes and directs ribosome–peptide–mRNA complexes to the endoplasmic reti-culum, where peptide synthesis continues and theproteins are dispatched to their final intra– or extra-cellular destinations.11 The mammalian SRP consistsof one RNA molecule of ∼300 nt (SRP RNA) and sixprotein cofactors, SRP9, SRP14, SRP19, SRP54, SRP68and SRP72, (Figure 1(a)). Structurally, the SRP iscomprised of two distinct and functionally separable

d.

mailto:[email protected]

Figure 1. The signal recognition particle (SRP). (a) Architecture of the mammalian SRP. SRP proteins are shown ascolored and gray ovals; the RNA is yellow. (b) “SRP54-late”model for cellular assembly of the mammalian SRP. Five of sixSRP proteins (SRP9, SRP14, SRP19, SRP68, and SRP72) (gray and green ovals) enter the nucleus or nucleolus to assemblewith the SRP RNA. The partially assembled complex is then transported back into the cytoplasm to bind SRP54 (purple)and form the native SRP holocomplex. (c) In vitro scheme for native SRP19–SRP54–SRP RNA ternary complex formationvia the SRP54-late assembly pathway. (d) “SRP54-early” pathway that leads to formation of a non-native ternary complex,termed the non-compartmentalized complex, in which two RNA binding loops in SRP19 do not fold to their nativeconformation.10 (e) Threefold protein–RNA interface in the native SRP ternary complex. SRP19 loop 2 (gray) is positionedin a cleft formed by SRP54 and the RNA. The Figure shows the same complex20 as (c), but has been rotated ∼90° to afforda view from the “top” of the three-component interface.

513Threefold Interface Gates SRP Assembly

subunits.12,13 About half of the RNA and fourproteins (SRP19, SRP54, SRP68 and SRP72) form the‘large’ subunit. The other half of the RNA and SRP9and SRP14 form the “Alu” subunit. In bothmammalian14–16 and yeast17,18 cells, SRP appears to

assemble via an “SRP54-late” pathway in which allSRP proteins except SRP54 are imported into thenucleolus where they bind the SRP RNA (green andgray proteins; Figure 1(b)). This partially assembledcomplex is then exported to the cytoplasm where

Figure 2. SRP19 and the large subunit SRP RNA (LSRNA). (a) SRP19 structural motifs when bound to the SRPRNA. SRP19 core is green and the two RNA binding loopsare gray. Sites of fluorophore attachment are shown asspheres. (b) Alexa 555 derivatized RNA–DNA hybrid.RNA and DNA are shown in black and gray, respectively.(c) LS RNA.

514 Threefold Interface Gates SRP Assembly

SRP54 binds in a distinct step (in purple; Figure 1(b))to form the fully functional SRP holocomplex.Mammalian SRP19 is unstructured in its unbound

state and assembles with the SRP RNA via formationof multiple obligatory intermediate complexes.10,19

Assembly of the SRP19–RNA complex is an exampleof mutually induced fit because both SRP19 and theSRP RNA undergo significant conformational rear-rangements during assembly.10,19–22 A native ternarycomplex forms in vitrowhen SRP54 binds to the stableand preformed SRP19–SRP RNA complex (Figure1(c)) and is the same as the complex visualized incrystallographic studies.10,21 This SRP54-late in vitroassembly pathway mimics the two-step, or compart-mentalized, in vivo assembly pathway.14–18

In contrast, if SRP19 and SRP54 are allowedsimultaneous access to the SRP RNA in vitro, thesethree components interact robustly to form a dis-tinct, non-native, complex (Figure 1(d)).10 In thiscomplex, two RNA binding loops in SRP19 remainmisfolded (black broken lines; Figure 1(d)). We termthis assembly pathway the “SRP54-early” pathwayand the misfolded ternary complex the “non-compartmentalized” complex. Here we show that,beginning with identical sets of protein and RNAcomponents, the non-compartmentalized structurearises from the surprising ability of SRP54 to bindan SRP19–RNA assembly Intermediate. The result-ing cleft-like interface that forms between SRP54and the SRP RNA (Figure 1(e)) is sufficientlynarrow that it disfavors insertion of an RNA-binding loop from SRP19 that normally occurs inthe native complex.

Results

Strategy for monitoring assembly of SRP19 withthe SRP RNA

Free SRP19 is a natively unstructured protein.Upon binding to the SRP RNA, SRP19 spans threestructural motifs, a small globular core domain andtwo RNA binding loops that extend out from thecore (Figure 2(a)).10,20,21,23 We monitored assembly-induced conformational changes in each motif byattaching an environmentally sensitive fluorophoreat four sites in SRP19. Each of the four positionsis solvent accessible in both SRP19–SRP RNAbinary and SRP19–SRP54–SRP RNA ternary com-plexes.10,20,21 Fluorophores were placed individu-ally in each of the two RNA binding loops (positions31 and 72) and in the core domain (positions 93 and106) (Figure 2(a)). Control experiments showed thatRNA binding affinities for each fluorophore-deriva-tized SRP19 were either identical or very similar tothe unlabeled wild-type SRP19 protein (see Materi-als and Methods). Site-directed cleavage experi-ments also demonstrated that derivatizing SRP19 atthese positions with Fe(II)-EDTA groups retains thenative conformation of the complex.10

Conformational changes specific to each positionwere monitored by either of two approaches. We

detected RNA–protein assembly using fluorescenceresonance energy transfer (FRET). Energy transferoccurred between a donor Alexa 488 fluorophoretethered to SRP19 and an acceptor Alexa 555fluorophore tethered to the large subunit of theSRP RNA (LS RNA) via a hybridized DNA oligo(Alexa 555-LS RNA) (Figure 2(b)). Control experi-ments showed that SRP19 binding to the Alexa 555-LS RNA is indistinguishable from that of the nativeLS RNA. In the second approach, SRP19 assemblywith the SRP RNA was monitored in single


fluorophore experiments using the environmentallysensitive fluorophores Alexa 488 or BODIPY-FLtethered to one of the four SRP19 labeling sites.Assembly of SRP54 with the SRP19-RNA complexcould also be monitored as the effect that SRP54binding has on the environmentally sensitive SRP19-tethered fluorophores.

FRET based analysis of SRP assembly

Assembly of the SRP19–RNA binary and theSRP19–SRP54–RNA ternary complexes was initiallymonitored as illustrated in Figure 3(a). In this ex-periment, Alexa 488 functioned as both the FRETdonor and also as an environmentally sensitivefluorophore. Thus, RNA binding and subsequentconformational changes in SRP19 were detectedboth as the initial change in the resonanceenergy transfer efficiency to the RNA-tetheredAlexa 555 acceptor fluorophore and also as thelocal change in donor fluorophore environment(Figure 3(a)).When SRP19, labeled at position 31, was added to

the Alexa 555-LS RNA, we observed a clear burst

Figure 3. FRET-based analysis of SRP assembly. (a) SchemeSRP19 (green) and the Alexa 555-LS RNA (yellow and graySRP19–RNA complex. (b) and (c) Three distinct assembly stternary complex formation. (1) A rapid burst phase quench(green). (2) Well-resolved increase or decrease in fluorescencethe native structure (green). (3) An increase in fluorescence intecomplex (purple). Free Alexa 647 reference fluorophore is gra

phase in which the donor emission intensity waspartially quenched, followed by a well-resolvedslow phase during which the emission intensitydecreased further (phases 1 and 2 in the green tracein Figure 3(b)). This change in fluorescence wasspecific to SRP19–RNA interactions because theemission of a free reference fluorophore (Alexa 647)present in the solution remained unchanged overthe course of the experiment (gray trace in Figure3(b)). Similar overall behavior was observed whenassembly was monitored using SRP19 labeled atposition 72. Fluorescent emission initially decreasedin a rapid burst phase followed by a well resolvedslow phase in which the fluorescence emissionintensity increased (green trace; Figure 3(c)). Thesedata indicate that both RNA-binding loops in SRP19assemble with the LS RNA in at least two well-resolved steps.Assembly of SRP54 with the preformed SRP19–

RNA complex was monitored as the effect thatSRP54 binding has on emission of the fluorophoreattached to SRP19 (Figure 3(a)). Addition of SRP54 toa SRP19–RNA complex, labeled at either SRP19position 31 or 72, yielded a clear increase in

for monitoring SRP assembly using Alexa 488-derivatized). SRP54 (purple) binding is detected by its affect on theeps are detected during native SRP19–SRP54–SRP RNAing of fluorescence during initial SRP19–RNA assemblyemission intensity as the SRP19–RNA complex matures tonsity due to SRP54 binding to the pre-formed SRP19–RNAy.

Figure 4. Burst phase assembly step corresponds toencounter complex formation between SRP19 and theRNA. (a) and (b) SRP19–RNA assembly was monitoredfor the native sequence RNA or an A149U mutant thatcannot form native RNA–RNA interactions (filled andopen symbols, respectively). Arrows indicate burst phasesobserved with both RNAs. (c) Addition of SRP54 has noeffect on the encounter complex formed between SRP19and the A149U RNA.

Figure 5. Conformational rearrangements at distinctSRP19 structural motifs during RNP assembly. (a) Schemefor monitoring SRP19–RNA assembly using single Alexa488 fluorophore experiments. (b) Fluorescence-detectedassembly at the RNA-binding loops (open symbols)proceeds significantly faster than assembly of the SRP19core domain (filled symbols).


fluorescence emission intensity due to the perturba-tion of the local fluorophore environment uponSRP54 binding (purple traces in Figure 3(b) and (c)).

Burst phase of SRP19–RNA assemblycorresponds to encounter complex formation

To assign the binding event that corresponds to therapid burst phase change in fluorescence for SRP19,labeled at either position 31 or 72, we monitored

assembly using an A149Umutant RNA. NucleotidesA149 and A201 form a non-canonical base pair thatlinks helices III and IV in the SRP RNA (Figure2(c)).20,21 SRP19 forms a labile encounter complexwith the A149U mutant RNA but cannot assemblebeyond this step to form the native SRP19–RNAcomplex.19

SRP19 binding to the A149U mutant RNA pro-ceeded via a rapid burst phasewhosemagnitudewascomparable to that observed for the native sequenceRNA as monitored at either positions 31 or 72 (Figure4(a) and (b)). In contrast, neither of the fluorescentlylabeled SRP19proteins showed the second, slow,phasecharacteristic of assembly with the native sequenceRNA (compare open and filled symbols in Figure 4(a)and (b)). We infer that the burst phase corresponds torapid formation of an encounter complex betweenSRP19 and the SRP RNA; whereas, the second, slowerphase (visualized in Figure 3(b) and (c)) corresponds toslow conformational rearrangements in the nascentSRP19–RNA complex that ultimately requires forma-tion of the native RNA tertiary interaction betweennucleotides A149 and A201.We then evaluated whether SRP54 interacts with

the SRP19–RNA encounter complex. A stalledSRP19–RNA encounter complex intermediate wasfirst formed using the A149U mutant RNA andSRP19 derivatized at position 72. As expected, weobserved the burst phase fluorescence change uponformation of the initial SRP19–RNA complex (open


circles; Figure 4(c)). When SRP54 was added tothis complex, no additional fluorescence changeoccurred (filled symbols; Figure 4(c)). These resultscontrast strongly with the ability of SRP54 to induceconformational changes in the native RNA–SRP19complex (Figure 3(c)). We infer that SRP54 does notbind the encounter complex intermediate.

SRP19 loop and core structures bind the SRPRNA via distinct mechanisms

SRP19 is comprised of distinct core and extendedloop motifs (Figure 2(a)). We detected local con-formational changes specific to the two RNAbinding loops (positions 31 and 72) and to theSRP19 core domain (positions 93 and 106) usingsingle fluorophore experiments (Figure 5(a)). RNAbinding caused an increase in Alexa 488 fluores-cence emission efficiency for SRP19 proteins labeledat positions 72, 93 and 106 and a decrease for theprotein labeled at position 31 (Figure 5(b)).Strikingly different time-resolved behavior was

observed depending on where the fluorophore wastethered to SRP19. When the fluorophore was linkedto either of the RNA-binding loops in SRP19, theobserved change in fluorescence intensity was

Figure 6. SRP19 structural motifs assemble with the Rfluorescence intensity as a function of RNA concentration for SRconstants for assembly as detected at SRP19 loop and core sAlexa 488-labeled SRP19 (squares). For the 72Cys and 106CysFL fluorophore (circles).

tenfold faster than when attached to the SRP19core (compare open and filled symbols; Figure 5(b)).We characterized local assembly at each SRP19

motif by following the rate of change in fluorescenceintensity as a function of RNA concentration. Forall four Alexa 488-labeled SRP19 proteins, rates in-creased with RNA concentration (Figure 6). SRP19derivatized at positions 31, 93 and 106 showed goodlinear behavior in rate versus RNA concentrationplots: the slopes of these lines yield the second-orderrate constants for complex formation (squares;Figure 6(a), (c) and (d)).In contrast, SRP19 derivatized with Alexa 488 at

position 72 exhibited a distinctive behavior. At RNAconcentrations below 100 nM, the observed rateincreased with increasing RNA concentration, asexpected for a single rate-limiting binding step(dashed line; Figure 6(b)). However, at RNAconcentrations above 100 nM, the apparent rate ofcomplex formation became independent of concen-tration and leveled off at 0.9 min−1 (continuous line;Figure 6(b)).These distinctive kinetic behaviors are independent

of the nature of the reporter fluorophore. The SRP19variants labeled at positions 72 and 106 were alsomonitored using the BODIPY-FL fluorophore, which

NA via distinct mechanisms. (a)–(d) Rate of change inP19 variants. Slopes (broken lines) yield second-order ratetructural motifs. Most experiments were performed withvariants, assembly was also monitored using the BODIBY-


is chemically and sterically dissimilar to Alexa 488.The distinctive slow and fast time-resolved behaviorswere identical as detected by either fluorophore(compare circles and squares; Figure 6(b) and (d)).These experiments thus illustrate two distinc-

tive features for assembly of the SRP19–RNAcomplex. First, second-order rate constants varyby an order of magnitude and fall into twoclasses. SRP19 derivatized at either of the twoRNA binding loops (positions 31 and 72) havesimilar second-order rate constants (2 × 106

M−1min−1 to 8×106 M−1min−1). SRP19 variantsderivatized in the core (positions 93 and 106)also have similar rate constants (3×105 M−1min−1

to 5×105 M−1min−1) that are an order ofmagnitude slower than those observed at theRNA-binding loops. Second, the SRP19 variantderivatized at position 72 has a distinctivebiphasic kinetic behavior such that a concentra-tion-independent process with rate constant0.9 min−1 limits assembly in the vicinity ofRNA binding loop 2.These experiments support a three-step mechan-

ism for assembly of SRP19 with the SRP RNA. FreeRNA and protein rapidly interact to form theencounter complex. Encounter complex formationis observed directly as the fast phase in FRET-basedassembly experiments (Figure 3). This step isfollowed by two kinetically separable concentrationdependent conformational changes to form a stableintermediate complex. In these steps, the RNA

Figure 7. One-step assembly of SRP54 to the pre-formed SRbinding to the pre-formed Alexa 488-labeled SRP19–RNA comintensity over time to a second-order rate equation. (c) and (d) Aconstants.

binding loops in SRP19 undergo RNA-inducedconformational changes more rapidly than does thecore domain (Figure 5). Finally, regions near loop 2participate in an additional concentration indepen-dent structural rearrangement step that converts thestable complex into the native SRP19–RNA complex(Figure 6(b)).

Assembly of SRP54 with the preformedSRP19–RNA complex

Assembly of SRP54 with the preformedSRP19–LS RNA complex was monitored by theeffect that SRP54 binding had on an environ-mentally sensitive fluorophore attached to SRP19.These experiments were similar to thosedescribed in Figure 3(a), except that onlySRP19 was fluorescently labeled. For SRP19variants derivatized at either position 31 or 72,fluorescence emission intensity increased uponSRP54 binding (Figure 7(a) and (b)). SRP54 bindsto the preformed SRP19–RNA complex with aKd=12 nM10 and, as expected, the net amount ofcomplex formed increased as the SRP54 concen-tration was increased from 15 nM to 200 nM.Over a broad range of SRP54 concentrations,second order rate constants were identical, at1.1×107 M−1 min−1 (Figure 7(c) and (d)), inde-pendent of the site of derivatization in SRP19.SRP54 thus binds in a single kinetically signifi-cant step.

P19–RNA complex. (a) and (b) The rate constant for SRP54plex was obtained by fitting the change in fluorescencell SRP54 concentrations yielded identical second-order rate


Concentration dependence ofnon-compartmentalized ternary complexformation

As described above, if SRP19 and SRP54 assemblewith the RNA simultaneously, these three compo-nents interact to form the non-compartmentalizedcomplex whose structure is significantly differentfrom the native complex (Figure 1(d)). We usedsingle fluorophore experiments to characterize thekinetic step that gates whether SRP54 assembles toform the native or non-compartmentalized complex(Figure 8(a) and (d)).We followed formation of the native complex by

sequential addition of SRP19 and SRP54 (green andpurple traces; Figure 8(b) and (c)). This is the SRP54-late pathway. As monitored using the 31Cys-SRP19protein, fluorescence emission intensity decreased asfree SRP19 bound to the RNA. The rate of bindingincreased as the protein concentration was increasedfrom 50 nM to 200 nM (green traces in Figure 8(b)).Fluorescence intensity then increased upon subse-quent addition of SRP54. The rate of SRP54 bindingalso increased with protein concentration, asexpected (purple traces in Figure 8(b)). These dataare consistent with the SRP19 and SRP54 bindingexperiments outlined in Figures 6(a) and 7(a),respectively.We then monitored assembly of the non-compart-

mentalized ternary complex by adding SRP54 priorto adding SRP19. This is the SRP54-early pathway(black traces in Figure 8(b)). These experiments wereperformed under the identical component concen-trations used to monitor native complex formation.Thus, the experiments represented by the green andpurple versus black traces (Figure 8(a) and (b))differed only the order in which SRP componentsinteract but not in the identity or amounts of SRP19,SRP54 or SRP RNA.Strikingly, when assembly was monitored for

component concentrations of 200 nM, complexformed via the SRP54-early pathway had signifi-cantly higher fluorescence than complex formed viathe SRP54-late pathway (compare the endpoints forpurple and black traces in Figure 8(b)). Thisdifference in fluorescence intensity directly reportsformation of the non-compartmentalized complexvia the SRP54-early pathway. A similar, but smaller,difference in fluorescence was observed at 100 nMprotein concentrations; whereas, the difference dis-appeared when component concentrations werereduced to 50 nM (Figure 8(b)).We performed comparable experiments with the

72-Cys SRP19 variant. As expected, the rate ofSRP19–RNA complex formation was concentration-independent (green traces; Figure 8(c)) because theconformational rearrangement involving loop 2binding plateaus at 0.9 min−1 (recall Figure 6(b)).The rate of SRP54 binding increased with increasingSRP54 concentration, consistent with simple one-step assembly with the preformed SRP19–RNAcomplex (purple traces; Figure 8(c)). Similar toassembly as monitored at position 31, the fluores-

cence of the final 72Cys-complex was again stronglyconcentration dependent and the difference influorescence intensity in the SRP54-late versusSRP54-early complexes was larger at 200 nM com-ponents than at 100 nM or 50 nM concentrations(compare purple and black traces in Figure 8(c)).In summary, as monitored at either of two loca-

tions within SRP19, there exists a kinetic competitionsuch that misassembly is specifically favored athigher component concentrations. As outlinedunder the Discussion, this concentration-dependentbehavior provides strong evidence regarding thephysical step that gates misassembly.

Discussion

Assembly of the SRP19–RNA complex viadistinct folding stages for the loop andcore domains

Our physical model for the alternating interactionsthat lead to the native SRP19–RNA complex isshown in the first four panels of Figure 9(a). BothSRP19 and the SRP RNA are in non-native con-formations prior to forming a specific ribonucleo-protein complex. The SRP RNA forms a relaxed butbase paired state; whereas, free SRP19 exists in anunstructured coil conformation.SRP19 and the RNA initially interact in a rapid,

approximately diffusion limited step to form anencounter complex. This step occurs with similar,very rapid, kinetics for both the native sequenceSRP RNA and a mutant (A149U) RNA that is inca-pable of maturing to the native complex (Figure 4(a)and (b)).We infer that this encounter complex largelyreflects a fast forming heterogeneous electrostaticinteraction between the RNA and SRP19.Further assembly from the encounter complex

involves a series of complex SRP19 folding steps toform the stable intermediate complex (Figure 9(a)).SRP19 loops 1 and 2 undergo, RNA-induced, secondorder conformational changes with rate constantsthat are an order of magnitude faster than folding ofthe SRP19 core domain (Figures 5(b) and 6). Foldingof both the core and loop motifs are concentration-dependent (below 100 nM) even though they followformation of the encounter complex. This observa-tion suggests that the encounter complex establishesa rapid pre-equilibrium with the free RNA andSRP19.An additional SRP19 folding step is revealed by

the concentration-independent folding behavior ofloop 2. Below 100 nM, folding at loop 2 shows thesame simple concentration-dependent behavior asloop 1 (Figure 6(a) and (b)); whereas, at higher con-centrations, the rate of the conformational changeassociated with loop 2 plateaus at 0.9 min−1. Thus, afirst-order conformational rearrangement in the still-immature RNA–protein complex limits overallassembly at SRP19 loop 2 (Figure 9(a)). Completionof the first order rearrangement of loop 2 yields thenative SRP19–RNA complex.

Figure 8. The extent of non-compartmentalized complex formation is concentration dependent. (a) Scheme formonitoring native versus non-compartmentalized assembly using single fluorophore experiments. Native assembly (theSRP54-late pathway) involves ordered binding by SRP19 (green traces) and SRP54 (purple traces). For non-compartmentalized assembly (the SRP54-early pathway), both proteins assemble simultaneously (black traces). (b) and(c) Visualization of native and non-compartmentalized ternary complex formation using single fluorophore experiments.Ratios of SRP19, SRP54 and RNA components were held constant at 1:1:2 in all experiments; protein concentrations aregiven for each panel. Non-compartmentalized complex formation is reported directly as the intensity difference betweenpurple and black traces (see red brackets). (d) Expected concentration dependence for SRP54 binding post-encounterversus post-stable complex formation. k1 is a compound rate constant reflecting formation of the Stable complex via thekinetically linked Encounter complex. Broken arrows show putative, concentration-dependent (k'3 [ ]), steps for SRP54-mediated misassembly.


Figure 9. Mechanisms for native and non-compartmentalized SRP assembly. (a) Native and non-compartmentalizedassembly pathways are gated at the stable complex. (b) and (c) Folding energy landscapes for SRP19 during SRP ternarycomplex formation via SRP54-late and SRP54-early pathways.


The threefold protein–RNA interface involvingSRP19 loop 2 gates native assembly of the SRPternary complex

SRP54 does not bind stably to the free SRPRNA.10,24,25 Therefore, SRP54-mediated misassem-bly via the SRP54-early pathway must involveSRP54 binding to an intermediate SRP19–RNA

complex. The two candidate classes of intermediatecomplexes could be either (i) after formation of theencounter complex or (ii) after formation of thestable intermediate complex and prior to maturationof the loop 2–RNA interaction (illustrated by brokenarrows in Figure 8(d)).Two independent experiments support a model in

which SRP54 binds after formation of the stable


complex. First, direct binding experiments showthat SRP54 does not modulate the fluorescence ofthe complex formed between SRP19 and the A149URNA, which is trapped at the encounter complexstage (Figure 4(c)). Second, formation of the non-compartmentalized complex is strongly dependenton the total concentration of SRP protein and RNAcomponents (Figure 8(b) and (c)). This is exactly theconcentration dependence expected if SRP54-mediated misassembly occurs after stable complexformation but not if SRP54 binds after encountercomplex formation (summarized in Figure 8(d)).The governing kinetic competition involves bothconcentration–independent (k2) and –dependent(k'3[ ]) steps such that the fraction of non-compart-mentalized complex formed (k'3 [ ]/k2) increases athigher SRP54 concentrations, if SRP54 binds afterthe stable complex forms.Folding of loop 2, which occurs after formation of

the stable SRP19–RNA intermediate, has exactly thecorrect slow concentration-independent behaviorrequired to gate native versus non-compartmenta-lized assembly. SRP19 loop 2 is packed in a cleft-likeinterface formed by SRP54 and helix IV of the RNA(Figure 1(e)).21 Thus, loop 2 both exhibits anintrinsically slow local folding behavior and alsolies in a highly constrained threefold interface in thenative particle. These observations support a modelin which formation of the non-compartmentalizedcomplex involves rapid RNA binding by SRP54 to anearly native SRP19–RNA interface but in whichfolding of loop 2 is still incomplete (Figure 9(a)).Early binding by SRP54 then results in a conforma-tion in which loop 2 lies outside the correct threefoldinterface and further insertion of this loop into itsnative interaction cleft is kinetically unfavorable.

SRP54–RNA interaction alters the SRP19 foldingenergy landscape

SRP19 has an almost identical structure in both theSRP19–RNA20 binary and native SRP19–SRP54–RNA21 ternary complexes. SRP54 thus has no rolein the native folding of SRP19. In contrast, SRP54-induced misfolding of SRP19 indicates that interac-tions between SRP54 and the RNA alter the foldingenergy landscape for SRP19. In the absence of SRP54,SRP19 folds to reach its global energy minimum in alandscape whose most important features includeformation of the encounter and native complexesand a single major intermediate, the stable complex(Figure 9(b)). SRP54 binding to the preformedSRP19–RNA complex does not significantly alterthe SRP19–RNA complex but further stabilizes it,creating a deeper well in the landscape (Figure 9(b)).The presence of SRP54 alters the folding landscape

for SRP19 to create a second, deep, low energyminimum corresponding to the non-compartmenta-lized complex (Figure 9(c)). Non-compartmentalizedcomplex formation is concentration dependent,indicating that the alternative pathway is kineticallydriven and leads to a local rather than a new globalminimum relative to the native ternary complex.

Broad implications for multi-component RNPassembly reactions

A simple three-component RNP, derived fromthe mammalian signal recognition particle, hasthe ability to form a stable alternative, non-nativecomplex. The native and non-compartmentalizedcomplexes differ only in the order by which the threecomponents assemble. The structural requirementsfor this order-of-interaction driven misassemblyare very modest. Alternative complex formation re-quires only that the three components communicatestructurally and that a portion of one binary inter-face be modulated by a third component. In theSRP example, structural communication involves adirect threefold interface, but indirect interactionswould suffice, as well.Evidence that assembly of other RNPs requires

spatial or temporal regulation is currently circum-stantial, but strongly suggestive. Like the SRP,several other cellular RNPs have multi-site assemblyphases involving transit through nucleolar and Cajalbody compartments.26,27 Examples in which spatialcontrol potentially facilitates RNP assembly includesnRNPs, spliceosomal RNPs and telomerase. Asecond strategy for regulated RNP assemblywould be to impart structural specificity throughpreferred temporal assembly phases. In the smallsubunit of the bacterial ribosome,28 three proteins,S3, S10 and S14, and the 3' domain of 16S RNAform an intimate fourfold interface that appearsto physically require that proteins S10 and S14assemble with the RNA prior to protein S3.Consistent with this structural view, S10 assembleswith 16 S RNA more rapidly than does S3.3,29 Wespeculate that slow RNA binding by S3 specificallyfunctions to reduce RNP misassembly at thisprotein–RNA interface. Multi-fold interfaces thatmay require early or rapid binding by buriedcomponents also exist in the large subunit of theribosome,28 including those formed by L14, L19 and23 S RNA and by L23, L29 and 23 S RNA, and in thearchaeal H/ACA complex formed by L7ae, Cbf5,Nop10, Gar1 and the H/ACA RNA.30Given that multi-fold interfaces are common in

RNP complexes, we propose that avoiding forma-tion of stable, but misassembled, complexes is offundamental importance for the structural biogen-esis of many RNPs. Mechanisms for regulatingassembly, including preferred early and late assem-bly phases and cellular compartmentalization, mayplay critical regulatory roles in preventing order-of-interaction driven misassembly for multi-compo-nent RNPs in the cell.

Materials and Methods

Preparation of SRP RNA and SRP54

Native and A149U mutant RNAs spanning nt 101–255of the human SRP RNA (LS RNA) were transcribed in vitrofrom plasmid phR31 and purified by denaturing gel


electrophoresis. The 5′-truncated native and A149Umutant LS RNAs were transcribed from PCR-generatedtemplates. All RNAs were refolded by heating to 95 °C(1 min), snap cooling at 0 °C, incubating at 60 °C (10 min)in RNA refolding buffer (300 mM potassium acetate (pH7.6), 5 mM MgCl2, 20 mM Hepes (pH 7.6), 0.01% (v/v)Triton), followed by slow cooling to room temperature(∼40 min). The Alexa 555-labeled LS RNAwas generatedby co-folding of the appropriate 5′-truncated RNA and afluorescently labeled DNA oligonucleotide (Trilink) (seeFigure 2(b)). SRP54 was expressed and purified asdescribed.10

BODIPY-FL and Alexa 488 labeled SRP19 variants

Single cysteine (E31C, W72C, L93C or L106C) SRP19variant proteins were expressed in Escherichia coli strainBL21-CodonPlus(DE3)-RIL (Stratagene) and purified asdescribed,10 except that the final dialysis buffer was pH 7.2and contained 2 mM TCEP as the reducing agent. All foursites involve residues whose amino acid identity isphylogentically variable32 and are solvent accessible inboth the SRP19–RNA binary20 and SRP19–SRP54–RNAternary21 complexes. SRP19-fluorophore conjugates weregenerated by treating each protein (∼20 μM)with a 50–100-foldmolar excess of aAlexa 488 or BODIPY-FL fluorophoremaleimide derivative (22 °C, 2 h; Molecular Probes).Unreacted fluorophore was removed by incubating the(His)6-tagged protein with an Ni2+-NTA slurry (Invitro-gen) and washing extensively with protein dilution buffer(PDB) (300 mMNaCl, 50 mM sodium phosphate (pH 8.0),10 mM 2-mercaptoethanol). Fluorophore-labeled proteinswere eluted in PDB containing 200 mM imidazole,subjected to dialysis to remove imidazole, and stored inPDB supplemented with 50% (v/v) glycerol at −20 °C.Derivatization extent was calculated from UVabsorbanceat 280 (protein) and 500 nm (fluorophore). For Alexa-labeled SRP19 variants, the extent of derivatization was95–100% and for the BODIPY-FL-labeled proteins was∼30%. Equilibrium dissociation constants for SRP19-fluorophore derivatives were determined by filterpartitioning19 using internally labeled [32P]-LS RNA(0.01 nM) or the Alexa 555 fluorophore RNA–DNAhybrid. Equilibrium dissociation constants for all fluor-ophore-containing complexes were either identical orvery similar to the native SRP19–LS RNA complex(native SRP19, 5 nM; ΔCysSRP19, 4 nM; Alexa 488-31CysSRP19, 11 nM; Alexa 488-72CysSRP19, 1 nM;Alexa 488-93CysSRP19, 3 nM; Alexa 488-106CysSRP19,11 nM, BODIPY-FL-72CysSRP19, 4 nM; BODIPY-FL-106CysSRP19, 19 nM).

Monitoring the SRP assembly pathway using FRETand single fluorophore experiments

Fluorescent measurements were performed in a Varian/Cary Eclipse spectrofluorometer. Final reaction mixtures(22 °C, 500 μl) contained RNA refolding buffer supple-mented with 1/5 volume of PDB. Calibration betweendifferent measurements, when required, was performedusing an inert Alexa 647 reference fluorophore. Formationof the encounter complex between SRP19 and the SRPRNA was monitored via fluorescence resonance energytransfer (FRET) between 20 nMAlexa 488-labeled 31Cys or72Cys SRP19 variants and 25 nM Alexa 555-labeled LSRNA. Conformational changes in SRP19 upon RNAbinding were monitored using single-fluorophore experi-

ments using 5 nM or 10 nM protein labeled with Alexa488 or BODIPY-FL and varying concentrations of LSRNA (as indicated in Figure 6). Fluorescence intensitychange over time was fit to a single exponentialequation. Second-order rate constants were calculatedfrom the slope of the line obtained by plotting observedrate versus RNA concentration. For SRP54 binding tothe preformed SRP19–RNA complex, assembly wasmonitored using 20 nM Alexa 488-labeled SRP19-RNAcomplex and the change in fluorescence intensity as afunction of time was fit directly to a second-order rateequation. Observed fluorescence=A [(exp(kc1t)-(exp(kc2t))(c1c2)/(c1exp(kc1t)-c2(exp(kc2t)]+b, where k is thesecond-order rate constant, c1 and c2 are the initialconcentrations of SRP54 and the SRP19-RNA complex,A is the amplitude of the fluorescence change, and b isthe initial fluorescence of the pre-formed SRP19–RNAcomplex. For experiments comparing native versus non-compartmentalized assembly, protein concentrations aregiven in Figure 8(b) and (c) and the RNA concentrationwas twice that of the proteins.

Acknowledgements

We are indebted to Howard Fried for manyhelpful conversations. This work was supportedby the US National Institutes of Health (GM065491to K.M.W.).

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Edited by J. Doudna

(Received 7 December 2006; received in revised form 9 March 2007; accepted 10 March 2007)Available online 20 March 2007

Documents

A Threefold RNA Protein Interface in the Signal ... · the unlabeled wild-type SRP19 protein (see Materi-als and Methods). Site-directed cleavage experi-ments also demonstrated that