S16 throws a conformational switch during assembly of30S 5¢ domainPriya Ramaswamy1,3 & Sarah AWoodson2

Rapid and accurate assembly of new ribosomal subunits is essential for cell growth. Here we show that the ribosomal proteinsmake assembly more cooperative by discriminating against non-native conformations of the Escherichia coli 16S ribosomal RNA.We used hydroxyl radical footprinting to measure how much the proteins stabilize individual ribosomal RNA tertiary interactions,revealing the free-energy landscape for assembly of the 16S 5¢ domain. When ribosomal proteins S4, S17 and S20 bind the5¢ domain RNA, a native and a non-native assembly intermediate are equally populated. The secondary assembly protein S16suppresses the non-native intermediate, smoothing the path to the native complex. In the final step of 5¢ domain assembly, S16drives a conformational switch at helix 3 that stabilizes pseudoknots in the 30S decoding center. Long-range communicationbetween the S16 binding site and the decoding center helps to explain the crucial role of S16 in 30S assembly.

Rapidly dividing cells must produce hundreds of new ribosomes eachminute1,2. Consequently, the process of ribosome assembly must beaccurate, so that each subunit is active, and stringently controlled, sothat the capacity for protein synthesis matches the rate of growth3,4.Large ribosomal RNAs (rRNAs) form metastable structures that canlead to errors in assembly5. How the ribosomal proteins and externalassembly factors remodel these intermediates is important to thefidelity of ribosome assembly.

Reconstitution studies on the E. coli 30S ribosomal subunitshowed that the ribosomal proteins induce large changes in thestructure of the 16S rRNA that underlie the cooperativity andhierarchy of the 30S assembly map6–8 (Fig. 1a,b). Whereas themechanisms by which the central and 3¢ domains of the 16S rRNAare assembled have been addressed9–12, assembly of the 16S5¢ domain, which makes up the body of the 30S subunit13,14

(Fig. 1c), is poorly understood. A 16S fragment containing the5¢ domain forms a stable ribonucleoprotein (RNP) with ribosomalproteins S4, S17, S20 and S16 (ref. 15). Primary assembly proteinsS4, S17 and S20 bind the naked rRNA, whereas binding of S16requires S4 and S20 (ref. 16; Fig. 1b). As the 5¢ domain is the first tobe transcribed in vivo and its proteins make interdomain contacts,rapid formation of its stable rRNA and rRNA-protein interactionsnucleates 30S assembly17,18.

Using hydroxyl radical footprinting, we previously showed that theE. coli 16S 5¢ domain RNA can form all of the backbone interactionspredicted by the structure of the 30S subunit in the absence ofproteins19. However, interactions between helices 15 and 17 requiredmore than 5 mM MgCl2, and some helices were protected lessstrongly in the naked RNA than in native 30S ribosomes. Thus,

the 5¢ domain proteins are needed to stabilize the rRNA tertiarystructure in physiological Mg2+ concentrations.

Moreover, time-resolved footprinting showed that half of the5¢ domain RNA became kinetically trapped in non-native foldingintermediates when refolded in vitro in 20 mM MgCl2

19. Tertiaryinteractions between helix 15 and helix 17 required the longest time toform (B1 min), probably owing to misfolding of the central junctionbetween helices 5, 6 and 6a. These results raised the question ofwhether the proteins also change the pathway of assembly, avoidingunproductive conformations.

To determine whether ribosomal proteins redirect the foldingpathway of the rRNA, we probed the assembly landscape of theE. coli 16S 5¢ domain RNP using quantitative hydroxyl radicalfootprinting. This method detects the solvent accessibility of indivi-dual residues along the RNA backbone, providing a detailed picture ofthe RNA tertiary interactions20. Information about the thermo-dynamic stability of each contact in the presence and absence of theproteins was obtained by probing the complexes over a wide range ofMg2+ concentrations.

The results show that binding of S16 to helices 15 and 17 results in aconformational switch at helix 3, 30 A away, which stabilizes tertiaryinteractions in the 30S decoding site. We also find that S16 increasesthe cooperativity of RNP assembly by preferentially stabilizing thenative configuration of helices in the lower half of the 5¢ domain, whiledisfavoring non-native assembly intermediates. Together, these resultshelp explain the crucial role of S16 in 30S assembly. They alsodemonstrate that discrimination against non-native structures isanother way in which RNA-protein interactions increase the selectivityof molecular self-assembly.

Received 29 September 2008; accepted 9 March 2009; published online 3 April 2009; doi:10.1038/nsmb.1585

1Program in Cell, Molecular and Developmental Biology and Biophysics and 2T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland,USA. 3Present address: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. Correspondence should beaddressed to S.A.W. (swoodson@jhu.edu).

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RESULTSStability of the naked 16S 5¢ domain RNATo determine how ribosomal proteins stabilize the folded 16S5¢ domain RNA, we compared the naked rRNA with RNPs containingthe primary binding proteins S4, S17 and S20, or S4, S17 and S20 plusprotein S16. The structures of the complexes were probed by hydroxylradical in 330 mM KCl and 0–30 mM MgCl2 (see Methods andSupplementary Figs. 1–3 online). The extent of cleavage was quanti-fied at more than 65 independent segments of the rRNA back-bone. In general, the stability of RNA tertiary structure is inverselyrelated to the Mg2+ dependence of the foldingtransitions21–23. Thus, we expect the RNAinteractions to form at lower Mg2+ concentra-tions when more proteins join the complex.The extent of cleavage in hydroxyl radical

correlates with the solvent accessibility of each ribose20, which reflectsthe sum of all folding equilibria that lead to exposure or protection ofthat residue. Therefore, the Mg2+ required to protect each segment ofthe RNA reflects the free energy of specific assembly intermediates.

In the absence of proteins (Fig. 2, Supplementary Table 1 andSupplementary Fig. 3b online), we found that tertiary interactions inthe RNA were heterogeneous and fell into three general categories.Some interactions required little Mg2+ to be stable (pink, Fig. 2b), butthey were not protected to the same extent as were control reactionsrun in parallel on native 30S subunits. Others were not protected,even up to 30 mM Mg2+ (green, Fig. 2b). Still others folded in twodistinct phases, suggesting the presence of folding intermediates(black, Fig. 2b).

Residues that were protected in o2 mM MgCl2 included nucleo-tides in helices 17 and 18 adjacent to the ‘upper’ five helix junctionthat binds with protein S4. A stable core of tertiary structure was alsovisible around helix 6a and the central junction, which aligns theinterface between helix 6/6a and helix 7 (red, Fig. 3a). The lowerjunction between helices 7–10 folded in 2–13 mM MgCl2 (orange andgreen, Fig. 3a).

Many other regions of the 5¢ domain remained exposed to hydroxylradical in 20 mM Mg2+ (blue, Fig. 3a), including helices that form thebinding sites for the primary assembly proteins S4, S17 and S20. Inour previous studies, the naked 5¢ domain RNA was almost completelyfolded in 20 mM MgCl2 and 120 mM NH4Cl19. The lesser stability ofthe rRNA tertiary structure reported here reflects the competitionbetween Mg2+ and 330 mM K+ for access to the RNA and the largersize of the K+ ion relative to NH4

+ (ref. 24). K+ is often used toreconstitute 30S subunits and more closely mimics the intracellularenvironment. Thus, under ‘physiological’ conditions, the ribosomalproteins are needed to fully stabilize the rRNA.

Stable 5¢ domain RNPTo determine whether the 5¢ domain RNA can assemble completelywith the four 5¢ domain proteins, we incubated the rRNA withproteins S4, S16, S17 and S20 in 0–30 mM MgCl2 before hydroxyl

17

16

18

a b

c

S17

S16 S6 S9 S19

S13

S14S10

S11

S5

S21 S3 S2

S12

S18

S20 S4 S8 S15 S7

3

5′

3′

S4

S16

S17

S20

5

15

1413

12

7

810

9

11

66a

4

2

5′ domain 3′ domainCentral

1

Figure 1 Structure of the E. coli 5¢ domain. (a) Secondary structure of the

16S rRNA55 with 5¢ domain nucleotides 21–562 in blue. Helices are

numbered as in refs. 13,56. (b) 30S assembly map16 with four 5¢ domain

proteins used in this study in color. (c) Structure of the 5¢ domain in the

E. coli 30S ribosome (PDB 2AVY)28, which forms the body of the small

subunit. S4, pink; S16, blue; S17, green; S20, yellow.

1

0.8

MgCl2 (mM)a b

c

NT

NT

30S

30S

0.01

0.05

0.1

1 2 3 71.5

12 15 20 25 350.2

0.3

0.5

G A H K K RNA only

388–390469

250–254

388–390

469

250–254

0.6

0.4

0.2427–429

451–452

481–483487–488

495–496

499–500504–505

516–518

521–522

525–527

529–531

533–536

513

498

469,471

0

0.01 0.1

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[MgCl2] (mM)1 10 100

Y

1

0.8

0.6

0.4

0.2

0.01 0.1

[MgCl2] (mM)

1 10 100

0

Y

Figure 2 Hydroxyl radical footprinting of the

5¢ domain in the presence and absence of

proteins. (a) The 16S 5¢ domain (nucleotides

21–562) was folded for 30–40 min at 37 1C in

0–35 mM MgCl2 before Fe(II)-EDTA footprinting

and primer extension (see Methods). Lanes: NT,

no treatment; H, RNA only in 80 mM K+-HEPES;

K, RNA only in 80 mM K+-HEPES plus 330 mM

KCl; G A, sequence ladder; 30S, native 30S

ribosomes. Protections due to RNA-RNA contacts

predicted by the structure of the 30S ribosome

are indicated on the right. (b) Folding transitions

for individual RNA-RNA contacts in the absence

of proteins. The relative extent of protectionY was normalized to controls on native 30S

ribosomes (squares) and fit to two- or four-state

models (see Methods). Green, nucleotides

250–254; black, nucleotides 388–390; pink,

nucleotide 469. (c) Fits for the same nucleotides

as in b, but in the presence of proteins S4, S16,

S17 and S20. Symbols as in b.

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radical footprinting with Fe(II)-EDTA. As expected, binding of thefour 5¢ domain proteins strongly stabilized the tertiary interactions inthe 16S 5¢ domain (Fig. 2c and Supplementary Fig. 3a). In 5 mMMgCl2, many tertiary interactions that were undetectable in the RNAalone were formed in the 5¢ domain RNP to the same degree as in thenative 30S subunit (Fig. 3). Moreover, the pattern of hydroxyl radicalprotection and the changes in chemical base modification wereconsistent with previous footprinting studies of 5¢ domain pro-teins25–27 (Supplementary Fig. 1 and 2) and with the backbonecontacts predicted by crystal structures of the 30S ribosome13,28 (seeMethods). Thus, the 5¢ domain RNP assembles completely underphysiological conditions.

Primary binding proteinsWe next asked to what extent the three primary assembly proteinswithout S16 could stabilize the three-dimensional structure of therRNA. When we incubated the 5¢ domain RNA with a mixture of S4,S17 and S20, most of the expected rRNA tertiary contacts formed in2.3 mM MgCl2, (Fig. 3b and Supplementary Fig. 3c). Only a fewinteractions between helices 8 and 6, 10 and 17, at the tip of helix 12and in helix 18, required more than 2.3 mM MgCl2 to formcompletely. Thus, S4, S17 and S20 together stabilize nearly all thenative tertiary contacts in the 5¢ domain, except those near the helix 18pseudoknot and a few positions in the core of the domain.

The primary binding proteins also perturb the ensemble of initialRNA structures. When the naked 5¢ domain RNA was incubated in330 mM KCl without Mg2+, most of the RNA backbone wasmoderately cleaved (Fig. 2a), suggesting that the entire domain isdynamic or disordered, adopting many conformations. By contrast,certain nucleotides were cleaved much more strongly in low Mg2+

concentrations in the presence of S4, S17 and S20 than in the nakedRNA (Fig. 4a,b). The exposed nucleotides are located in helix 5(nucleotides 50–60, 352, 355, 365), helix 7 (118), helix 8 (176–177),helix 12 (314), helix 13 (328) and helix 15 (370, 372, 392, 396), where

they participate in tertiary interactions with adjacent helices in themature 30S subunit13,14 (Fig. 4c,d).

Notably, the residues exposed at low Mg2+ concentration by bindingof S4, S17 and S20 overlap the binding site for protein S16(refs. 25,27; Fig. 4d). As the S4–S17–S20 complexes were titratedwith Mg2+, the exposed residues became protected from hydroxylradical cleavage, consistent with formation of additional tertiarystructure (Fig. 4a,b and Supplementary Fig. 4 online). Thus, theprimary assembly proteins not only stabilize the overall tertiarystructure of the 5¢ domain RNA, they also specifically pre-organizethe S16 binding site. Moreover, S4, S17 and S20 in combination narrowthe ensemble of assembly intermediates, favoring rRNA conformationsthat are prepared to make the desired tertiary interactions. These resultshelp explain the cooperative interactions between S16 and S4 or S20 inthe 30S assembly map16.

RNAa b c

0

S4–S17–S20

≤20 mM Mg2+

S4–S17–S20–S16

Figure 3 Global stabilization of rRNA tertiary structure by ribosomal proteins. Individual residues in the 5¢ domain RNA were clustered according to the

[Mg2+]1/2 of the folding transition: red, 0–2.3 mM; orange, 2.3–4.9 mM; green, 4.9–13.4 mM; blue, 413 mM. Residues protected in two transitions were

ranked according to the midpoint of the second transition (further details in Supplementary Table 1). Two-dimensional schematics and three-dimensional

ribbons prepared as in Figure 1. (a) 5¢ domain RNA only; (b) RNA plus S4, S17 and S20; (c) RNA plus S4, S17, S20 and S16.

GANNHKK

RNA onlya c

db

35 mM

30 mM

314

H17

H4

H16

H15

H13

H17

H4

H15

30S

H5

H13

H8

H10 H8

H5

328

352355

365370372

314

328

352355

365370372

392396

392396

S4–S17–S20

0.01

GANNHKKM 0.01

Figure 4 Primary assembly proteins pre-organize the S16 binding site.

Enhanced hydroxyl radical cleavage of specific nucleotides in low Mg2+

concentrations when proteins S4, S17 and S20 are bound, relative to the

naked RNA. These residues become protected in 20 mM MgCl2. Lanes arelabeled as in Figure 2. (a) RNA only. (b) RNA plus S4, S17 and S20. See

Supplementary Figure 4 for additional data. (c,d) Exposed residues in low

Mg2+ concentrations (red) overlap the S16 binding site (nucleotides 51 and

362–364 on both sides of helix 5, and nucleotides 120, 315, 324 and

390–393)25,27,38.

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S16 binds the native 5¢ domain coreProtein S16 is essential for viability29, and its absence strongly affectsthe kinetics of 30S reconstitution in vitro30. When bound to the 16SrRNA, S16 induces large changes in the conformation of 5¢ and centraldomains25,27, consistent with its important role in 30S assembly. Tounderstand the function of S16 in assembly, we deduced the specificeffects of S16 on the stability of the 5¢ domain by comparing Mg2+-titrations of S4–S17–S20 complexes with titrations of complexescontaining four proteins (Fig. 3b,c).

In the 30S subunit, S16 straddles a C-loop motif in helix 15 thatstacks against bases in a sharp kink in helix 17, and Tyr17 in S16donates a hydrogen bond to the 2¢ OH of A374 in the C-loop13.Addition of S16 stabilized this important tertiary contact in the rRNA,reducing the midpoint for protection of nucleotides 481–483 at thekink in helix 17 from 4.9 mM to 2.4 mM MgCl2 (SupplementaryTable 1). More unexpectedly, we found that S16 also stabilized manybackbone contacts with helix 6/6a in the core of the 5¢ domain,including contacts with helix 8 (nucleotides 151–153), helix 13 andS20 (nucleotides 107–108), helix 10 and the tip of helix 17 (nucleo-tides 203–204). Helix 6 forms a spur that juts into the solvent,emerging from a bundle of helices at the base of the 30S subunit13,14.Thus, S16 binding directly stabilizes the interaction between helices 15and 17 and indirectly improves helix packing around the 30S spur.

S16 changes the structure of helix 12S16 was previously shown to induce a shift in the secondary structureof helix 12 and to decrease the accessibility of G31 in helix 3 and basesin the central pseudoknot (helix 2)27. We observed that S16 stabilizedtertiary interactions between helix 3, 12 and 18, which form part of theinterface between the body of the 30S subunit and the platform. Theseinclude a contact between the tip of helix 12 (nucleotides 295–297)and the minor groove of helix 3, as well as an interaction betweennucleotide 301 and a flexible loop in S17 (Fig. 3c).

S16 stabilizes the decoding site pseudoknotIn addition to its interactions with helix 15 and the domain core, theMg2+ titrations showed that protein S16 stabilizes the pseudoknotformed by base pairs 505–507 and 524–526 in helix 18 at physiologicalMg2+ concentrations (Fig. 3c and Supplementary Table 1). The helix18 pseudoknot positions the universally conserved G530 in the 30Sdecoding site and is essential for protein synthesis31. In the 30Ssubunit, nucleotides 505–507 are buried by a kink in the RNAbackbone created by the pseudoknot and by contact with theN-terminal domain of protein S4. These residues were only partiallyprotected in complexes with S4 alone (P.R. and S.A.W., unpublisheddata), indicating that interactions elsewhere in the rRNA are impor-tant for the stability of the helix 18 pseudoknot. Addition of S16lowered the [Mg2+]1/2 for protection of nucleotides 505–506 to2.3 mM (versus 5.6 mM with S4–S17–S20) and increased the maxi-mum protection to 80% of the value obtained for 30S controls. Thus,in our experiments, the helix 18 pseudoknot forms in physiologicalMg2+ only when S16 is added to the complex.

Reorganization of intermediate complexesAlthough many 5¢ domain interactions become more stable asproteins join the RNP, segments of the rRNA backbone showed amore complex change in solvent accessibility that revealed the reorga-nization of intermediate RNPs during the assembly process (Fig. 5).When the 5¢ domain RNA was incubated with the three primarybinding proteins, certain residues were protected in two transitions,indicating the presence of one or more intermediates in the pathway.For example, in the S4–S17–S20 complex, nucleotides 379–380 inhelix 15 fold in two stages. These nucleotides are partially protected in0 mM Mg2+ and fully protected in 10 mM Mg2+ (Fig. 5a). A secondgroup of residues, such as those in helix 12, become lightly protectedin 0.1 mM Mg2+ and fully protected in 10 mM Mg2+ (Fig. 5b).

Notably, a third group of residues in helix 18 were partiallyprotected in 0–1 mM Mg2+, exposed to hydroxyl radical cleavagebetween 1 mM Mg2+ and 5 mM Mg2+ and reprotected in 10–20 mMMg2+ (Fig. 5c). This oscillation in the solvent accessibility of the RNAbackbone is best explained by the remodeling of tertiary interactionsduring assembly. We observed the same behaviors for other residues inthese helices, consistent with a structural change affecting the entirehelix (Supplementary Figs. 5 and 6 online). Single-protein complexesshow similar multistage folding (P.R. and S.A.W., unpublisheddata), suggesting that these structural changes are intrinsic to the 5¢domain RNA.

S16 suppresses non-native assembly intermediatesAddition of S16 eliminated this multistage folding, causing tertiaryinteractions with helices 12, 15 and 18 to form cooperatively over anarrow range of Mg2+ concentrations (Fig. 5 and SupplementaryFig. 6). Not only did the final folding transition occur at a lower Mg2+

concentration when S16 was added, but the first folding transition

1

0.8

a

b

c

Helix 15

+1°

+1°

+1°

1° +S16

1° +S16

RNA

RNA

1° +S16

Helix 12

Helix 18

RNA

0.6

0.4

0.2

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0.8

0.6

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0.2

0

0.8

0.6

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0.2

0

0.01 0.1 1 10 100

[MgCl2] (mM)

Y

Y

Y

Figure 5 S16 discriminates against non-native assembly intermediates.

(a,b) Formation of tertiary interactions in helice 15, 12 and 18. The extent

of protection (Y ) relative to native 30S ribosomes was as above (see

Methods). Representative titrations are shown for (a) nucleotides 379–380

(helix 15); (b) nucleotide 315 (helix 12); (c) nucleotide 501–502 (helix 18).

Open circles, RNA only; filled squares, S4–S17–S20 (11); filled circles,

S4–S16–S17–S20. The lower baseline varies for residues in helix 18 in

titrations with four proteins, owing to heavy cleavage of unfolded RNA

controls in these particular experiments. Further data are shown in

Supplementary Figure 5.

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moved to a higher Mg2+ concentration or disappeared. Thus, bindingof S16 stabilizes certain assembly intermediates while antagonizingothers. We observed the conversion of multistage transitions in theabsence of S16 to two-state transitions in the presence of S16 for manyresidues in the 5¢ domain, suggesting that fewer assembly intermedi-ates are populated when S16 binds the rRNA.

Conformational switch at helix 3The structures of candidate assembly intermediates were deduced byclustering residues that followed similar folding transitions whenbound by S4–S17–S20. Residues folding in two stages were locatedon both faces of helix 15, the inward face of helix 17 and the tips ofhelices 11 and 18 (blue, Fig. 6). The second cluster of residues map tothe base of helix 12, the interface between helices 6a and 10, and helix16 where it contacts helix 18 (orange, Fig. 6). In the 30S subunit, helix15 lies between helix 17 and helix 6a, whereas the other side of helix 6apacks against helices 8 and 10 (refs. 13,14). Thus, these two clustersrepresent a set of helix packing interactions in the lower half ofthe domain.

Because these residues are protected in two stages, we deducethat the core of the 5¢ domain adopts at least two folded con-formations during assembly: a native-like conformation (IN) that issimilar to the 30S structure, and a non-native conformation (InC)that leaves certain residues exposed to hydroxyl radical. In themature subunit, helices 6, 6a and 12 are arranged co-axially and arelinked to each other and to helix 5 through the central junction.Residues in these helices fold together (orange), suggesting that thedifference between the native and non-native intermediates may bedue to alternative base-pairing of the central junction, which wehave observed previously19.

Residues that are exposed in moderate Mg2+ concentrations andreprotected in high Mg2+ concentrations cluster in helix 18 and in thetip of helix 12 (nucleotides 295–297) (pink, Fig. 6, and Supplemen-tary Fig. 3c). The backbone of these residues is protected by helix 3,which lies between helix 18 and the tip of helix 12 in the maturesubunit. Consequently, the transient exposure of helices 12 and 18 tosolvent at moderate Mg2+ concentration can be explained by a switchin the relative orientation of helix 3.

When we compared folding transitions in different parts of the5¢ domain (Fig. 6a), it was apparent that helix 18 (pink) becomesexposed over the same Mg2+ concentration range in which helix 15and the base of helix 12 (blue and orange) become fully protected

(Fig. 6a). Therefore, we infer that interactions between helices 3 and18 break when the native-like IN intermediate is populated. Thecorrelation between the orientation of helix 3 and the IN state iscorroborated by changes in the solvent accessibility of all the residuesin these helices and in the core of the 5¢ domain (SupplementaryFig. 6). Core residues that are protected in a single transition centeredat 1–2 mM Mg2+ (nucleotides 151–153, 469, 370–374) probably reflectinteractions that exist in the native-like core but not in the non-nativecore. Other residues (nucleotides 203–204, 301, 481–483, nucleotides505–506) require 5 mM or 10 mM Mg2+ to become protected in theabsence of S16, representing contacts that appear in the native RNP. Asdiscussed below, the self-consistency of the footprinting results allowsus to propose a structural model for assembly of the 16S 5¢ domain.

DISCUSSIONMany RNA binding proteins preferentially bind and stabilize thenative three-dimensional structure of their RNA target. In multi-protein RNPs such as the signal recognition particle (SRP) and the 16Scentral domain, the resulting protein-induced changes in the structureof the RNA favor the addition of subsequent proteins, drivingcooperative assembly of the entire complex32–34. Our studies on the16S 5¢ domain show that S16 not only stabilizes the native conforma-tion of the rRNA, it also destabilizes certain rRNA interactions at earlystages of assembly. These results reveal a second important role ofRNA binding proteins in RNP assembly, which is to suppressunproductive intermediates. We propose that the suppression ofmetastable intermediates is an important factor in the cooperativityof RNP assembly.

Assembly energy landscapeThe actions of the 5¢ domain proteins can be understood in terms ofan energy landscape for assembly (Fig. 7), as previously proposedbased on the kinetics of 30S assembly35. As each protein binds therRNA, the relative free energies of the rRNA structures are changed,resulting in a new distribution of assembly intermediates. Together,the three primary assembly proteins S4, S17 and S20 stabilize nearly allthe RNA tertiary interactions in the 16S 5¢ domain. However, theseform at different Mg2+ concentrations, suggesting that assembly passesthrough at least two intermediates, IN and InC (see below). S16 lowersthe free energy of IN but not InC, preventing the accumulation of InC

complexes. Consequently, when S16 is present, assembly proceedsdirectly from IN to N under physiological conditions (2–5 mM Mg2+).

Evidence for competing I states comes from the multistage changesin backbone accessibility as the complexes are stabilized by Mg2+. Onepotential explanation for the plateau in the protection of helix 15 isthat assembly passes through an intermediate in which helix 15 is onlypartially buried (scheme I). However, if ribosomal proteins such asS16 bind the native rRNA more strongly than non-native or unfoldedrRNA, then, according to the thermodynamic cycle, they must

1

0.8

a b

c

H15

H12 H180.6

0.4

0.2

0.01

H16

H12

H12

H11 H7H17

H17

H16

H15

H4

H3

H18

H15

H6

H6

H10

H10

H18

H3

0.1[MgCl2] (mM)

10 10010

Y

Figure 6 RNA conformational changes during assembly. Residues protected

in two or more steps during assembly in the presence of S4, S17 and S20

form three clusters: blue, 20–60% protected in KCl and fully protected in

1 mM MgCl2; orange, 20–30% protected in 0.1 mM MgCl2 and fully

protected in 1 mM MgCl2; pink, partially protected in KCl, exposed in

1 mM MgCl2 and reprotected in 10 mM MgCl2. (a) Sample titration curves

for H15 nucleotides 379–380 (blue), H12 nucleotide 312 (orange) and

H18 nucleotide 495–496 (pink). Curves for other residues are shown in

Supplementary Figure 6. (b,c) Residues in each cluster are projected on the

two-dimensional and three-dimensional structures of the 30S 5¢ domain.

Stereo ribbon of the three-dimensional structure as in Figure 1c, with proteins

shown as semitransparent surfaces: S4, pink; S17, green; S20, yellow.

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stabilize native RNA interactions more than they do non-nativeinteractions. If the partially protected intermediate in scheme Icontains only native RNA interactions, then both the intermediateand the native state should be stabilized when S16 binds, and bothtransitions should occur at lower Mg2+ concentrations. However,we observe that the initial folding transition is disfavored whenS16 is added to the reaction (Fig. 5a and Supplementary Fig. 6).Moreover, the transient exposure of helix 18 during assembly in theabsence of S16 strongly argues for a second intermediate with adifferent conformation.

Scheme I:

U

Cleaved 50% protected Protected

NIN

An alternative model that is consistent with all of our data is thatassembly involves at least two intermediates with different structures(scheme II): IN in which helix 12 is buried and helix 18 is exposed, andInC in which helix 12 is exposed and helix 18 is buried. We proposethat some residues in helix 15 are protected in both IN and InC. If InC

forms at low Mg2+ concentrations, this would explain why suppres-sion of InC by S16 increases cleavage of the helix 15 backbone. In this‘energy landscape’ model, the extent of protection plateaus when IN

and InC have similar free energies, and thus neither intermediatedominates the population (Fig. 7a).

Scheme II:

UCleaved

Cleaved

Protected

ProtectedN

InC

IN

Protein S16 smooths the pathway of assembly considerably bystabilizing IN more than InC, such that only IN is populated(Fig. 7a). This could occur by selective binding to IN. However, S16could also raise the free energy of InC. In either case, once there is asubstantial energy gap between the two intermediate states, only themost stable state will be populated at equilibrium.

The four-state model in scheme II can account for all of themultistage changes in backbone accessibility. By contrast, the oscillat-ing protection of helix 18 cannot be explained by the three-statemodel in scheme I (Supplementary Fig. 7 online). Thus, at least twointermediates are needed to explain assembly of the 5¢ domain.Additional intermediates containing only one or two proteins arealso likely. Therefore, the free-energy landscape for assembly is almostcertainly more complex than depicted in Figure 7.

Mechanism of 5¢ domain assemblyFrom the footprinting results presented here, we propose the followingminimal mechanism for 5¢ domain assembly (Fig. 7b,c): at low Mg2+

concentrations, the 5¢ domain RNA forms an ensemble of partly foldedstates with a minimal set of stable tertiary interactions (IC). Thisensemble contains both native-like and non-native configurations ofcore helices 6, 6a, 7 and 11 that are bound and further stabilized byprimary assembly proteins S4, S17 and S20. In IN, we propose thathelix 3 is oriented away from the upper five-helix junction, leaving thetip of helix 12 and the stem of helix 18 exposed to solvent (Fig. 7b).Although we do not know the exact orientation of helix 3 in IN, aswitch in the topology of the right angle junction between helices 3 and4 could swing helix 3 away from the rest of the 5¢ domain36. In InC,helix 3 is aligned correctly with helix 18, but the base of helix 12 isunprotected owing to the perturbed conformation of the core helicesand the central junction between helices 5, 6a and 12.

When S16 binds (or when the Mg2+ concentration reaches 3 mM),IN becomes more stable than InC, resulting in full protection of helix 15and exposure of helix 18 and the tip of helix 12 (Fig. 6a). Helix 18 and12 are finally reprotected as helix 3 swings back into its native orien-tation (N). Under equilibrium conditions, we cannot distinguishwhether InC proceeds to N directly in the absence of S16, or whethernon-native complexes must first transform to IN before reaching thenative RNP (dashed arrows, Fig. 7c). In the presence of S16, we pro-pose that most of the complexes assemble along the path from IN to N.

The fact that ribosome assembly can proceed by more than onepathway was previously shown by the kinetics of 30S ribosomeassembly, as measured by time-resolved footprinting of the RNAbackbone37 and the rates of protein addition measured by MS35. Theequilibrium and kinetic assembly intermediates of the 5¢ domain arequalitatively similar, even though the time-resolved experiments werecarried out on the entire 16S rRNA and total 30S proteins in 20 mMMgCl2. The most stable interactions in the core of the 5¢ domainformed within 50 ms. By contrast, the folding kinetics of the upperjunction were slower (2 s) and multiphasic37, consistent with slowerconformational changes in this region of the rRNA. One discrepancy isthat the tip of helix 12 (nucleotides 295–297) is rapidly protected in the

RNA

a

c

b

∆G

+S16

Nativecore

H1,H2

N

H18

H18

H18

S16

S16

H4 H12

H12

H12

H17

H15

H3

H3 in

H3 out

Icore

IC

IN

InC

InCNon-nativecore

IN

N

+1°

Figure 7 Model for assembly of the 30S 5¢ domain. (a) Free-energy diagram

illustrating how selective stabilization of a native-like intermediate by S16

depopulates competing Is and results in more cooperative assembly; 11,

S4–S17–S20. (b) Helices that switch conformation when bound by S16

(blue surface) are highlighted in a structure of the 30S ribosome (PDB

2AVY). (c) An ensemble of partly folded RNA (IC) containing different

configurations of core helices are bound and further stabilized by primary

assembly proteins S4, S17 and S20 (pale pink, green and yellow). In the

absence of S16, a native-like (IN) and a non-native intermediate (InC) have

similar free energies and are both formed. S16 (light blue) preferentially

stabilizes IN, resulting in depopulation of InC and a smoother transition to

the native RNP (N). Long-distance communication between the binding site

of S16 in H17 (cyan) and H15 (blue) and helix 3 (purple) stabilizes the

helix 18 pseudoknot (pink) in the 30S decoding center. Equilibrium data

do not distinguish paths from InC to N (dashed arrows).

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16S rRNA, possibly because of the presence of S16 and the high Mg2+

concentration, or the presence of other 16S sequences.

Protein S16 and 30S assemblyThe crucial role of S16 in the assembly of 30S subunits25,30 can beexplained by the conformational switch in helix 3 that comes about bythe preferential stabilization of IN. S16 was previously shown tochange the secondary structure of helix 12 and stabilize the centralpseudoknot27. We hypothesize that tight binding of S16 to helices 15and 17 is communicated via helix 4 to helix 3, and via helix 17 to thestem of helix 18. We note that the kink in helix 17 and the helix 18pseudoknot both become protected in 2–5 mM MgCl2, during thetransition from IN to the native RNP (Fig. 7). Tertiary interactionsbetween helices 3, 18 and the tip of helix 12 are expected to stabilizethe helix 18 pseudoknot. In the 30S subunit, helix 18 is presumablyfurther stabilized by its interactions with protein S12, which is the nextprotein to join the complex in the 30S assembly map38.

By changing the orientation of helix 3, binding of S16 influencesthe connections between the head, body and platform of the smallsubunit that are crucial for forming the decoding site and for finalmaturation of the 30S subunit39,40. First, helix 18 is itself anessential component of the decoding site31,41. Second, in the 30Sribosome, helix 3 stacks with helix 1, which in turn contributes halfof the central pseudoknot (helix 2). Reorientation of helix 3 duringthe transition from IN to the native RNP may help to ensure thathelix 3 is not locked in place until helix 12 is correctly aligned withhelix 6a in the core of the 5¢ domain.

In the cell, ribosome assembly is coupled to transcription42,allowing secondary and tertiary structures to form at the 5¢ end ofthe 16S rRNA before the 3¢ end has been synthesized. When foldingis sequential, local structure is favored over long-distance interac-tions such as helix 3 (ref. 43). Wagner and co-workers havesuggested that the rRNA leader serves as a scaffold for assemblyof the 5¢ domain44,45, by forming metastable interactions that holdthe place of downstream partners until the rest of the 16S rRNAis transcribed. They found that the leader interacts with helix 6 and is cross-linked to nucleotides in helices 3, 4, 5, 7 and 11a,precisely those regions of the 5¢ domain that can form alternativestructures19. The cold-sensitive mutation C23U in helix 1 inhibits5¢ processing of the 16S rRNA46 and produces 30S particlesresembling reconstitution intermediates (RI) formed in vitro atlow temperature6. Thus, an interesting possibility is that theconformational switch we have identified in helix 3 is coupled toprocessing of the pre-rRNA and later steps of 30S subunit assembly.

METHODSPurification of recombinant ribosomal proteins. We overexpressed E. coli

ribosomal proteins S16, S17 and S20 and purified them by ion-exchange

chromatography as described47. Plasmids for overexpression of S16, S17 and

S20 were a gift from G. Culver. We overexpressed S4 as described48 (gift from

D. Draper) and purified it as above. Protein footprints were verified using

DMS footprinting49,50.

Purification of 5¢ domain. We transcribed the 542-nt 5¢ domain RNA in vitro

with T7 RNA polymerase from pRNA1 (ref. 19) using standard methods and

purified it by denaturing 4% PAGE. The RNA concentration was determined by

UV absorption at 260 nm and e260 ¼ 5.4 � 106 M–1 cm–1.

Assembly of ribosomal protein complexes. For hydroxyl radical footprinting

reactions, 12 pmol 5¢ domain RNA was folded 30–40 min at 37 1C in 42 ml

reconstitution buffer (80 mM K+-HEPES, pH 7.6, 330 mM KCl, 20 mM MgCl2,

0.01% (v/v) Nikkol detergent, 6 mM b-mercaptoethanol) before treatment

with Fe(II)-EDTA. Where stated, we varied the MgCl2 concentration from

0 mM to 30 mM. The 5¢ domain RNA was previously shown to fold in less than

5 min19. We prepared protein-RNA complexes by pre-incubating 12 pmol

RNA in reconstitution buffer containing the desired MgCl2 concentration for

15–20 min at 37 1C, before addition of 5-molar equivalents of S16, S17 or S20

and 4-molar equivalents of S4 in a total volume of 56 ml. The RNA was

incubated with the proteins for an additional 45 min at 37 1C.

We determined the optimum protein:RNA ratios by titrating 50 pmol

5¢ domain RNA with individual ribosomal proteins in reconstitution buffer

at RNA: protein ratios ranging from 1:1 to 1:10 in a 42 ml volume. Protein

contacts were saturated with 5-molar equivalents of S16, S17 or S20 and

4-molar equivalents of S4. The protein-RNA co-incubation time required to

achieve saturation was determined by verifying the integrity of RNA-protein

complexes on a native 8% polyacrylamide gel between 0–1.5 h; 40 min was

sufficient for maximal complex formation, and longer incubation produced no

change in the extent of hydroxyl radical protection.

DMS modification of protein complexes. We folded 50 pmol of 5¢ domain

RNA alone, with the individual proteins or all four proteins in 50 ml of

reconstitution buffer as described above, with RNA:protein ratios of 1:0.25,

1:0.5, 1:1, 1:2, 1:3 and 1:5 at 42 1C for 1 h. Samples were cooled on ice for

10 min and then treated with DMS as described50. The RNA was extracted once

with equal volumes of phenol and 1:25 isoamyl:chloroform and precipitated

before primer extension.

Fe(II)-EDTA reactions. Following folding of the 5¢ domain RNA or assembly

of 5¢ domain RNPs, we removed 14 ml containing 3 pmol of 5¢ domain RNA

from each reaction and treated it with Fenton reagents as described51. We

carried out control reactions in parallel with each titration on native E. coli 30S

ribosomal subunits, 5¢ domain RNA in 80 mM K+-HEPES, 80 mM K+-HEPES

plus 330 mM KCl, or reconstitution buffer. 30S subunits (10 pmol) in 14 ml

reconstitution buffer were heat activated for 30 min at 42 1C, placed on ice

for 10 min and treated on ice with 2 ml 10 mM Fe(II)-EDTA for 1 min as

described above. Samples were analyzed by primer extension and AMV reverse

transcriptase as described19.

Data analysis. We compared protected regions of the RNA backbone with the

solvent-accessible surface area for each C4¢ atom in the 5¢ domain of the 16S

rRNA using coordinates from the structure of the E. coli 30S ribosome28 and

the program Calc-surf52. The extent of cleavage at each position in the

5¢ domain RNA was quantified with a Molecular Dynamics Phosphorimager

and normalized to a reference nucleotide whose intensity did not change over

the course of the experiment53. We obtained the fractional saturation (Y) by

normalizing the relative cleavage to the amount of cleavage in 80 mM HEPES

(Y ¼ 0) and buffer plus 20 mM MgCl2 or native 30S subunits (Y ¼ 1),

whichever was greater.

Assuming that the extent of hydroxyl radical cleavage reflects the equili-

brium between an ‘open’ and ‘closed’ state at each nucleotide, we fit the

fractional saturation (Y) of each backbone protection versus Mg2+ concentra-

tion (C) to an isotherm for a cooperative two-state equilibrium,�Y ¼ �Y0 + ðC=CmÞn=½1 + ðC=CmÞn�, in which n is the Hill coefficient and Y0 is

the extent of protection in 330 mM KCl and no MgCl2. Multiphasic transitions

were fit to a four-state model in which the statistical weight of each term was

taken from the equilibrium constant for the open and closed state,

Ki ¼ ðC=Cm;iÞn;i (refs. 22,54):

�Y ¼ ðC=Cm;2Þn;2 + ðC=Cm;N Þn;N

1 + ðC=Cm;1Þn;1 + ðC=Cm;2Þn;2 + ðC=Cm;NÞn;Nð1Þ

The initial state (Icore) and one intermediate are assumed to be completely

exposed, whereas the second intermediate and the final state (N) are fully

protected. In many cases, the data could be described equally well by two

sequential transitions,

�Y ¼ A1ðC=Cm;1Þn;1

1 + ðC=Cm;1Þn;1+

A2ðC=Cm;2Þn;2

1 + ðC=Cm;2Þn;2ð2Þ

in which A1 and A2 are the magnitude of change in protection with each step.

Parameters from equation (2) were used for the purposes of clustering the

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data. Reproducibility in the fit parameters between experimental trials was

typically ±20% for Cm or ±50% for n, but no greater than than ±50% (Cm)

or ±100% (n).

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSThe authors thank G. Culver (Univ. Rochester) and D. Draper, R. Moss andT. Adilakshmi (Johns Hopkins Univ. (JHU)) for gifts of plasmids andT. Adilakshmi, A. Cukras, J. Brunelle (JHU) and R. Green (JHU andHoward Hughes Medical Institute) for their help and advice. This work wassupported by a grant from the US National Institutes of Health (GM60819).

AUTHOR CONTRIBUTIONSP.R. performed experiments, analyzed and interpreted data and wrote the paper;S.A.W. conceived the project, interpreted the data and wrote the paper.

Published online at http://www.nature.com/nsmb/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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