Molecular Cell

Article

Progression of the Ribosome Recycling Factorthrough the Ribosome Dissociatesthe Two Ribosomal SubunitsChandana Barat,1,5,6 Partha P. Datta,1,5 V. Samuel Raj,2 Manjuli R. Sharma,1 Hideko Kaji,3 Akira Kaji,2

and Rajendra K. Agrawal1,4,*1 Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany,

NY 12201-0509, USA2 Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA3 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA4 Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12201, USA5 These authors contributed equally to this work.6 Present address: Department of Biotechnology, St. Xavier’s College, 30 Park Street, Kolkata-700016, West Bengal, India.

*Correspondence: agrawal@wadsworth.org

DOI 10.1016/j.molcel.2007.06.005

SUMMARY

After the termination step of translation, theposttermination complex (PoTC), composedof the ribosome, mRNA, and a deacylatedtRNA, is processed by the concerted action ofthe ribosome-recycling factor (RRF), elongationfactor G (EF-G), and GTP to prepare the ribo-some for a fresh round of protein synthesis.However, the sequential steps of dissociationof the ribosomal subunits, and release ofmRNA and deacylated tRNA from the PoTC,are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with un-decagold-labeled RRF, we show that RRF iscapable of spontaneously moving from its ini-tial binding site on the 70S Escherichia coliribosome to a site exclusively on the large 50Sribosomal subunit. This movement leads to dis-ruption of crucial intersubunit bridges andthereby to the dissociation of the two ribosomalsubunits, the central event in ribosome recy-cling. Results of this study allow us to proposea model of ribosome recycling.

INTRODUCTION

The process of translation, in which the genetic informa-

tion encoded in the messenger RNA (mRNA) is converted

into the amino-acid sequence of a protein, occurs on the

ribosome in four steps: initiation, elongation, termination,

and ribosome recycling (Kaji et al., 2001; Ramakrishnan,

2002). After the termination step, the mRNA and a deacyl-

ated transfer RNA (tRNA) remain associated with the ribo-

some. These ligands must be released from the ribosome,

and the ribosome itself must dissociate into its two

250 Molecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier

subunits during the recycling step, to enable entry into a

new round of protein synthesis. Thus, the recycling pro-

cess involving RRF is an essential step in prokaryotic

and eukaryotic organellar translation and involves the dis-

assembly of the posttermination complex (PoTC) (Kaji

et al., 2001; Karimi et al., 1999).

Atomic structures of RRF from six bacterial species

have been determined by X-ray crystallography (Selmer

et al., 1999; Toyoda et al., 2000; Kim et al., 2000; Nakano

et al., 2003; Saikrishnan et al., 2005) and by NMR spec-

troscopy (Yoshida et al., 2001). Each of these structures

shows that the factor is composed of two domains: do-

main I, consisting of three long a-helical bundles, and

the smaller domain II, which is an a/b motif. Differing orien-

tations of domain II seen in these structures (see Figure S1

in the Supplemental Data available with this article online)

have been attributed to interdomain flexibility, which is

thought to be necessary for RRF to function on the ribo-

some (Toyoda et al., 2000; Yoshida et al., 2001). The rev-

elation of an overall similarity, in the dimensions and shape

of RRF, to an L-shaped tRNA molecule prompted the pro-

posal of structural and functional molecular mimicry be-

tween the two molecules (Selmer et al., 1999).

The binding positions of RRF on the 70S ribosome have

been studied by the hydroxyl radical probing method

(Lancaster et al., 2002) and three-dimensional (3D) cryo-

electron microscopy (cryo-EM) (Agrawal et al., 2004;

Gao et al., 2005). The binding position of domain I of

RRF was also determined by X-ray crystallography (Wil-

son et al., 2005); in that study the domain I portion of

RRF was soaked into a preformed crystal of 50S ribo-

somal subunits. Results from all of the studies revealed

essentially the same overall binding position of RRF on

the 70S ribosome. The position of RRF was found to be

such that the long arm would block the two tRNA-binding

sites, namely the aminoacyl (A) and peptidyl (P) sites, of

the ribosome, but in a direction oblique to that of the

long anticodon stem-loop arm of the tRNA (Lancaster

et al., 2002; Agrawal et al., 2004). Previous studies

Inc.

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

(Agrawal et al., 2004; Wilson et al., 2005; Gao et al., 2005)

also revealed that binding of RRF induces a marked con-

formational change in the 70S ribosome, involving one of

the strongest interribosomal subunit bridges, B2a. This

finding suggested a direct role of RRF, by which the factor

could facilitate the disassembly of the PoTC by severing

the intersubunit bridge, on subsequent binding of elonga-

tion factor G (EF-G) (Agrawal et al., 2004). Biochemical

and genetic studies have shown that an interaction be-

tween RRF and EF-G is important in disassembly of the

PoTCs (Rao and Varshney, 2001). Furthermore, initiation

factor 3 (IF3) has been implicated in the release of the de-

acylated tRNA from the dissociated 30S subunit after the

concerted actions of RRF and EF-G on the PoTC (Karimi

et al., 1999). However, this concept is not fully supported,

either by classical (Hirashima and Kaji, 1973) or recent

studies (Peske et al., 2005; Zavialov et al., 2005; for a

detailed discussion, see Hirokawa et al. [2006]).

In the present study, we show that RRF binds in two dif-

ferent binding positions on the ribosome: the first binding

position matches closely with previous determinations

(Agrawal et al., 2004; Gao et al., 2005; Lancaster et al.,

2002; Wilson et al., 2005), while the second binding posi-

tion overlaps with the binding position of tRNA in the ribo-

somal P site. This finding of the second binding position of

RRF allows us to outline the possible sequential steps that

are involved in the disassembly of the PoTC.

RESULTS AND DISCUSSION

To determine the binding positions of RRF on the E. coli

ribosome, we first labeled E. coli RRF with undecagold

(UG), a heavy-metal cluster (core diameter z 8 A; Nano-

probes, Yaphank, NY); the UG-labeled RRF (RRF-UG) was

then bound to the PoTC. A unique cysteine residue pres-

ent at position 16 (Cys16), within domain I of RRF (Ichi-

kawa and Kaji, 1989; Kim et al., 2000; Figure S2), was

labeled using monomaleimido-UG, which forms a covalent

linkage with the SH group of the Cys residue (Hainfeld,

1987; Safer, 1999). After ascertaining that the RRF-UG

was active in disassembling the model PoTC (see Exper-

imental Procedures and Figure S3), we visualized the po-

sition of the UG label by analysis of 3D cryo-EM maps

(e.g., Kikkawa et al. [2000] and Datta et al. [2005]) of the

PoTC�RRF-UG complexes. We were able to capture

RRF on the ribosome in two different conformational

states and in two different binding positions; each position

was marked by a strong density for the UG label. In the fol-

lowing sections, we describe our results and discuss how

these two RRF positions could be related to the process of

ribosome recycling.

Localization of RRF on the Ribosome

We obtained 3D cryo-EM maps (Frank et al., 2000) of the

PoTC�RRF-UG complex, henceforth referred to as the

70S�RRF-UG complex in the rest of the Results and Dis-

cussion section. Application of the supervised classifica-

tion method (Valle et al., 2002) to the 2D cryo-EM data

Mo

set of the 70S�RRF-UG complex revealed a partial disso-

ciation among the 70S ribosome population into ribo-

somal subunits and enabled separation of the images

into more homogeneous subsets, based on individual cor-

relations with several reference maps (see Figures S4 and

S5). Four distinct classes, and subsequently the corre-

sponding 3D cryo-EM maps, were obtained: (1) the empty

70S ribosome; (2) the ratcheted 70S ribosome (Frank and

Agrawal, 2000) with an RRF molecule located in a position

similar to that observed previously (Figures 1A and 2A;

henceforth referred to as ‘‘position 1’’; also see Agrawal

et al. [2004], Gao et al. [2005], and Wilson et al. [2005]);

(3) the empty 50S ribosomal subunit; and (4) the 50S sub-

unit with RRF bound in a completely new position, ‘‘posi-

tion 2’’ (Figures 1B and 2B). The new position, found ex-

clusively on the dissociated 50S subunit, appears to be

a short-lived state that is attained after the movement of

RRF out of position 1 (see below).

The density assigned to RRF in position 1 was isolated

through comparison between the cryo-EM map of the

70S�RRF-UG complex and the map of the empty 70S ri-

bosome. RRF in position 1 is seen as a complex density

mass, with a long cylindrical arm and two short, bifurcat-

ing arms, emerging from the same end of the long arm

but having unequal electron densities. The long arm was

readily identified as domain I of RRF (Agrawal et al.,

2004; Wilson et al., 2005). The two short arms represent

two discrete orientations of domain II of RRF on the ribo-

some. The domain II position represented by the stronger

of the two short-arm masses corresponds to the position

of domain II suggested from the hydroxyl radical probing

study (Lancaster et al., 2002; henceforth referred to as

the IIa configuration of RRF in position 1). The domain II

position represented by the weaker of the two short-arm

masses corresponds to the position of domain II as in-

ferred in the previous cryo-EM study (Agrawal et al.,

2004; henceforth referred to as the IIb configuration of

RRF in position 1; also see Figure S6). The domain IIb con-

figuration has also been proposed in an X-ray crystallo-

graphic study (Wilson et al., 2005). (A similar bifurcated

density feature, corresponding to two possible orienta-

tions of RRF domain II, was observed in the previous

cryo-EM study [Agrawal et al., 2004]; however, the

strengths of the two densities were reversed.) Thus, the

complex mass of density in position 1 represents an en-

semble average of densities, due to two overlapping bind-

ing states of RRF molecules on two separate ribosomes,

with the domain I of RRF in two states lying almost in the

same position.

The density assigned to RRF in position 2 was isolated

through comparison between the cryo-EM map of the

50S�RRF-UG complex and the map of the empty 50S ri-

bosome. This density is located in the ribosomal P site

and has a distinct L shape (Figure 1B). The overall length

of the long arm of the L matches that of the long arms of

both RRF and tRNA and would almost completely overlap

the anticodon stem-loop arm of a tRNA in the ribosomal P

site (Gabashvili et al., 2000; Yusupov et al., 2001; Selmer

lecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier Inc. 251

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 1. Stereo Representations of the RRF-Binding Positions on the 70S Ribosome and on the Dissociated 50S Ribosomal Sub-

unit

(A) RRF (red) in position 1 on the 70S ribosome map (yellow, 30S subunit; blue, 50S subunit); and (B) in position 2 on the dissociated 50S subunit map.

Two orientations of RRF’s domain II within position 1 are indicated. Orientations of the 70S ribosome, shown in the thumbnails to the lower left of each

panel, were chosen to optimally reveal the L-shaped feature of RRF densities in each position. The 30S subunit in the thumbnail for (B) is shown as

a semitransparent yellow mass, to indicate that the position 2 RRF is observed exclusively on the dissociated 50S ribosomal subunit. Landmarks: I

and II, domains I and II, respectively, of RRF. Landmarks of the 30S subunit: hd, head; sp, spur. Landmarks of the 50S subunit: L1, L1 protein; CP,

central protuberance; St, L7/L12 stalk; Sb, stalk base; H38 and H69, 23S rRNA helices 38 and 69, respectively.

et al., 2006). However, the anticodon of the P site tRNA,

which interacts with mRNA on the 30S ribosomal subunit,

would overlap only partially with the outer bend of the el-

bow of the density corresponding to RRF (Figures 3A

and 3B). In addition, there would be no overlap between

the CCA arm of the P site tRNA and the density corre-

sponding to RRF. In fact, the short arm of the L-shaped

density is oriented so as to be diagonally opposed to the

CCA arm of the P site tRNA (Figures 3A, 3B, and 4A; cf.

Selmer et al. [1999]). Therefore, the mass of density that

we see must correspond to RRF in its position 2. This at-

tribution was further verified by presence of a distinct den-

sity feature corresponding to the UG label on RRF.

Comparison of the maps of UG-labeled complexes with

respective unlabeled controls reveals that the overall

shape of the labeled region does not change upon incor-

poration of the UG label. However, the RRF moiety in

each of the two positions showed a unique high-density

252 Molecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier In

spot (within the long arm [domain I] of the L-shaped RRF

density) that persisted after imposition of a very high den-

sity threshold and thus was readily attributable to UG

(Figures 3C and 3D; also see Figure S7). We further vali-

dated the UG positions through calculation of difference

maps for both position 1 (using cryo-EM maps of the

70S�RRF-UG complex and 70S�RRF complex; Agrawal

et al., 2004) and position 2 (using the cryo-EM maps of

the 50S�RRF-UG complex and the 50S subunit, to which

a filtered and resolution-matched X-ray structure of RRF

was added in the matching position). Presence of

a mass of density corresponding to UG on position 2

RRF was also verified by the subtraction of a cryo-EM

map of a complex containing a tRNA bound at the P site

of the 50S ribosomal subunit (isolated by computationally

removing the 30S portion of the cryo-EM map of the

70S�fMet-tRNAfMet complex; Gabashvili et al., 2000)

from the map of the 50S�RRF-UG complex.

c.

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 2. Side-by-Side Comparison of

the Two Binding Positions of RRF on

the Ribosome, and Binding-Associated

Conformational Changes in the Ribo-

somal Bridge Regions

(A) RRF density (red) on the 50S subunit (blue)

portion of the 70S ribosome. A deacylated

tRNA (orange) is present in the P/E state. A

local conformational change, apparently in-

duced via a direct interaction between the tip

of domain II of RRF and the stalk base (Sb) re-

gion of the 50S subunit, is highlighted as a dark

blue mass (#).

(B) RRF density on the dissociated 50S ribo-

somal subunit. A weak mass of density (x)

observed between RRF and L1 protuberance

could be related to an E site tRNA, present in

an extremely small fraction of the dissociated

50S subunit population.

(C and D) The density features (solid blue) cor-

responding to conformational changes in the

50S subunit portion of the 70S�RRF complex

map (C), and in the map of the dissociated

50S�RRF complex (D), are superimposed

onto the respective 50S maps (semitranspar-

ent blue). Locations of intersubunit bridges,

B1a, B1b, B2a, B3, and B5, are marked by

open ovals. A small mass indicated by an as-

terisk (* in [D]) corresponds to a partial shift of

the central protuberance. The 30S ribosomal

subunit has been computationally removed

from the 70S structure shown in (A), to reveal the RRF and tRNA masses, and in (C), to reveal bridge-related conformational changes. The thumbnail

at the bottom, between (C) and (D), depicts the orientation of the ribosome in all four panels. Landmarks: H71, 23S rRNA helix 71; all other landmarks

are the same as in Figure 1.

In order to exclude the possibility that the binding of

RRF to position 2 is a consequence merely of an altered

buffer condition (as absence of reducing agents in our

buffer was a requirement to maintain the Cys16-UG cou-

pling; see Experimental Procedures) and/or the use of

UG labeling, we reanalyzed our earlier data set of the

70S�RRF complex (Agrawal et al., 2004) using the same

scheme of supervised classification (Figure S4). Indeed,

we found partial dissociation of the 70S ribosome in that

data set. A cryo-EM map computed from the further-clas-

sified small fraction of the 50S particle images showed an

RRF signature density in the position 2 region (Figure S8).

These results further support our observation of the posi-

tion 2 binding of RRF, and an RRF-dependent partial and

spontaneous dissociation of the 70S ribosome. Moreover,

our biochemical data (Figure S3B) also show a limited dis-

sociation into ribosomal subunits, upon incubation of 70S

ribosomes with RRF alone.

Conformational Changes of the Ribosome

Due to RRF Binding

Comparisons between the cryo-EM maps of the RRF-

bound and unbound ribosomes, obtained under identical

buffer conditions, revealed conformational changes in the

ribosome that are associated with binding of RRF (Figures

2C and 2D). Most of the conformational changes de-

scribed in the previous cryo-EM study of the 70S�RRF

Mole

complex (Agrawal et al., 2004) have been reproduced in

this study. In addition, we here observe marked conforma-

tional differences between the 50S subunit portion of the

70S ribosome with position 1 RRF, and the dissociated

50S subunit with position 2 RRF. The L7/L12 stalk (St),

which is clearly extended in the 70S�RRF map (position

1), is disordered in the map of the dissociated 50S�RRF

complex (position 2; Figure 2, compare [A] and [C] with

[B] and [D], respectively). However, the functional signifi-

cance of the conformational changes in the stalk region

is not yet understood. For both positions of RRF, confor-

mational changes occurred in the vicinity of 23S rRNA

helices 69 and 71 of the 50S subunit; these helices are

involved in the formation of several of the strongest

bridges (B2a, B3, and B5; Figures 2C and 2D) between

the two ribosomal subunits (Gabashvili et al., 2000; Yusu-

pov et al., 2001; Selmer et al., 2006). We find that a large

conformational change in this region is initiated with bind-

ing of RRF in position 1 on the 70S ribosome (Figure 2C;

also see Agrawal et al. [2004]) and becomes significantly

amplified with RRF in position 2 on the dissociated 50S

subunit (Figure 2D). Furthermore, the conformations of

the 23S rRNA helix 38 and the central protuberance (CP)

of the 50S subunit change upon RRF binding in position

2. Both components shift toward the position 2 RRF.

These two shifts affect the configurations of intersubunit

bridges B1a and B1b and could be partially responsible

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Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 3. Relationship between Position

2 RRF and the P Site tRNA, and Fittings

of the Atomic Structure of RRF into the

Corresponding EM Densities

(A) The resolution-matched cryo-EM densities

of RRF (red) at position 2 and the P site tRNA

(semitransparent green; adapted from

Gabashvili et al. [2000]) are superimposed.

(B) Transparencies of the two cryo-EM densi-

ties have been flipped, to better reveal the rel-

ative positions of the two ligands.

(C and D) Atomic structure of the E. coli RRF

(PDB ID 1EK8; pink ribbons), docked into the

RRF cryo-EM envelope (semitransparent red)

in position 1 (C) and in position 2 (D), shown

in views that reveal the relative positions of

the labeled Cys16 residue (purple balls) and

the UG mass (golden yellow, also see

Figure S7) within domain I. Note that the fitting

shown in (C) corresponds to the position 1 RRF

density, representing the predominant orienta-

tion of domain II (the IIa configuration, rather

than the IIb configuration; see Figures 1A and

2A) in this study. Landmarks: I and II, domains

I and II, respectively, of RRF; AC and CCA,

anticodon and CCA ends, respectively, of the

P site tRNA; landmarks in the thumbnail are

the same as in Figure 1.

for the above-described dissociation of the 70S ribosome

into its two subunits. Because such pronounced confor-

mational changes are not observed in the 50S subunit

that is dissociated from the 70S ribosome upon treatment

with low-concentration Mg2+ (<1 mM) (P.P.D. and R.K.A.,

unpublished data), they must be triggered by the binding

of RRF to position 2.

In an earlier study of RRF binding to a predissociated

50S ribosome in the presence of EF-G (Gao et al., 2005),

it was suggested that domain II of RRF (in a position similar

to our position 1) would sterically interfere with association

of the 30S subunit. However, in that study, domain II of

RRF was found to be sandwiched between EF-G’s do-

main IV (Agrawal et al., 1998) and 23S rRNA helices 69/

71 of the 50S subunit, such that the RRF’s movement to

position 2 and subsequent release from the ribosome

(Kiel et al., 2003) would be precluded. However, that find-

ing corroborated the results of previous study (Kiel et al.,

2003), suggesting that RRF bound to the predissociated

50S subunit cannot be released by EF-G, whereas RRF

bound to the 70S ribosome is readily released in the pres-

ence of EF-G, presumably via position 2 identified in the

present study. Moreover, with the predissociated 50S

subunit, only a weak density related to RRF in position 1

is seen (see Figure S9), unless RRF is trapped by domain

IV of EF-G (e.g., Gao et al. [2005]). This is not surprising,

because the dissociation constant of RRF for the predis-

sociated 50S subunit is significantly higher than that for

the 70S ribosome (Hirokawa et al., 2002a, 2002b). Be-

cause in the present study RRF was allowed to react

with its natural substrate, the PoTC, and because RRF

binding in position 2 could not be detected on a predisso-

254 Molecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier

ciated 50S ribosomal subunit when incubated with satu-

rating quantities of RRF (�60-fold molar excess) (Gao

et al., 2005; also see Figure S9), it is likely that position 2

of RRF is achieved only via the factor’s initial binding to

the 70S ribosome in position 1, rather than representing

a direct binding of RRF to a predissociated 50S subunit.

We believe that the conformational changes, induced by

binding of RRF at position 1, prepare the 50S subunit

within the 70S ribosome for the transition of RRF to posi-

tion 2. The fact that we did not observe position 1 binding

of RRF to 50S subunits that were dissociated upon RRF

action could be related to an unfavorable conformation

of those 50S subunits. However, the possibility that an

extremely small fraction of dissociated 50S subunit

population binds RRF in position 1, but is not classified

as a separate group in our supervised classification

scheme, cannot be ruled out.

Comparison of the Cryo-EM Maps

with the Atomic Structure of RRF

The atomic structure of the E. coli RRF (PDB ID 1EK8) was

docked as two separate domains into the RRF density in

both of its positions, taking into consideration both the

constraints of the envelope of the density and the position

of the UG mass (Figures 3C and 3D). Because configura-

tion IIb of RRF in position 1 is the same as derived in the

previous study (Agrawal et al., 2004), we present here

dockings of the RRF atomic structure into cryo-EM densi-

ties that correspond to the other position 1 configuration,

IIa, and to the position 2. The tight packing of the UG

mass within the helix bundle and the slight broadening of

the density of the RRF domain I at both positions suggest

Inc.

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 4. Comparison between the Posi-

tion 2 Binding of RRF and Positions of the

P/P and P/E Site tRNAs, and Conforma-

tional Changes in RRF on the Ribosome

(A) Stereo view of atomic structure of RRF

(red, domain I; purple, domain II) in position 2

is shown together with mutually exclusive P/P

(green, Gabashvili et al., 2000) and P/E (or-

ange) state tRNAs; the latter was derived by

docking of an atomic structure of tRNAPhe

into the corresponding cryo-EM density (or-

ange mass in Figure 2A). The relative orienta-

tions of the two domains of RRF, as derived

by docking the atomic structure of the E. coli

RRF into the cryo-EM densities corresponding

to RRF in both IIb configuration (position 1) and

in position 2, matched closely with that in the

atomic structure of the Thermotoga maritima

RRF (PDB ID 1DD5). Therefore, we also

docked the T. maritima structure, as a single

rigid body, in the matching positions, and

used it to represent the IIb configuration (posi-

tion 1) and position 2 RRF in this and subse-

quent figures.

(B) Stereo view, showing superimposed struc-

tures of RRF in position 1 (IIa configuration,

golden yellow), and position 2 (red). It should

be noted that the relative orientation of two

RRF domains in its IIb configuration (position

1) is similar to that in position 2.

that RRF undergoes an intradomain conformational rear-

rangement upon binding to the ribosome. However, we

did not introduce any such rearrangement in our dockings.

Additionally, given the two overlapping configurations of

domain II (IIa and IIb; Figures 1A and 2A), it is conceivable

that domain I undergoes a small shift (up to 5 A) and/or

rotation (up to 15� around its long axis) within position 1,

as also inferred from our independent fittings correspond-

ing to both configurations of RRF (Figure 5, compare [A]

and [B]).

Our fittings reveal a large interdomain rearrangement of

RRF on the ribosome. In the IIa configuration, domain II

forms an obtuse angle (�120�) with the long axis of do-

main I, while it is roughly perpendicular (�93�) to that

axis in the IIb configuration (Agrawal et al., 2004). In posi-

tion 2, the domain II of RRF maintains a roughly right angle

(IIb configuration) with the long axis of domain I (Figures

4A and 4B). These observations, and the fact that the den-

sity corresponding to the IIb configuration is significantly

weaker than that in the IIa configuration, lead us to sug-

gest that RRF has moved from position 1 to position 2

via the IIb configuration, maintaining the interdomain an-

gle of �93�. This movement would produce a significant

steric clash with the 30S subunit in the 70S ribosome,

such that the 30S subunit would be separated from the

50S subunit by at least �12 A (Figure 6).

Ribosomal Neighborhood of RRF

in Its Two Positions

In order to determine the binding position of RRF on the ri-

bosome, we docked the X-ray crystallographic structure

Mo

of the E. coli 70S ribosome (Schuwirth et al., 2005) into

our cryo-EM maps of the 70S ribosome and the 50S sub-

unit. Position 2 places RRF in a new biochemical environ-

ment of the ribosome (Figure 5C), thereby satisfying some

of the hydroxyl radical probing (HRP) data (Lancaster

et al., 2002) that are inconsistent with the position 1 siting.

For example, the HRP cleavage of 23S rRNA helix 38 of

the 50S subunit with the probe at amino acid residue 10,

within domain I of RRF, could be easily attributed to the

fraction of the dissociated 50S subunit population having

the RRF bound in position 2. The tip of domain II of the

RRF in the IIa configuration (position 1) would interact

with the base of the 50S subunit stalk, involving the 23S

rRNA helix 43/44 (Figure 5A), as also suggested by HRP

study (Lancaster et al., 2002), while in the IIb configuration

the tip is oriented toward helix 18 of the 16S rRNA and pro-

tein S12 of the 30S subunit (Agrawal et al., 2004).

On the basis of the two binding positions of RRF

observed in this study, we speculate that 23S rRNA helix

80 and ribosomal protein L16 of the 50S subunit (Fig-

ure 5)—the two ribosomal elements that lie in the vicinity

of the tip of domain I (in both positions)—act as anchor-

ing points and perhaps shift along with RRF, during the

factor’s progression from position 1 to position 2. In-

deed, a weak density feature within the difference map

computed between the map of the 50S subunit with

RRF in position 2 and the computationally isolated 50S

subunit portion of the map of the 70S�RRF complex

suggests a conformational change (not shown) near the

helix 80/L16 region, coinciding with the tip of the domain

I of RRF.

lecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier Inc. 255

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 5. The Ribosomal Neighborhood of RRF Binding

Relevant components of the X-ray crystallographic structures of the E. coli (Schuwirth et al., 2005) and T. thermophilus (Yusupov et al., 2001; for the

interpretations of the regions that were disordered in E. coli structure) 70S ribosomes, docked into the cryo-EM map of the RRF-bound ribosome,

together with RRF (red), are shown in stereo views. (A) RRF in position 1 (IIa configuration), (B) RRF in position 1 (IIb configuration), and (C) RRF in

position 2 (IIb configuration). Thumbnail to the lower left depicts the orientation of the ribosome in all three panels. Landmarks: numbers prefixed

‘‘L’’ identify proteins and numbers prefixed ‘‘H’’ identify the 23S rRNA helices of the 50S subunit; P1 and P2, positions 1 and 2 of RRF.

256 Molecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier Inc.

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Relationships of RRF-Binding Positions

with Those of tRNAs and EF-G on the Ribosome

As pointed out in the previous studies (Lancaster et al.,

2002; Agrawal et al., 2004), position 1 of RRF overlaps

the binding positions of both A and P site tRNAs, such

that the long axes of the two ligands are obliquely ori-

ented. These observations indicated that RRF binding,

and tRNA binding to A and P sites, are mutually exclusive,

suggesting that RRF recognizes a ribosome substrate in

which both A and P sites are free of tRNA, as expected

in a PoTC, with the 70S ribosome, mRNA, and deacylated

tRNA in the P/E state (Agrawal et al., 1999b). In position 2,

RRF would partially overlap the anticodon loop of the

deacylated tRNA occupying the ribosomal P/E site (Fig-

ure 4A), the site that is found to be occupied in our

70S�RRF complex (Figure 1A), but not in the 50S�RRF

complex. The implication is that the movement of RRF

to position 2 has displaced the P/E site tRNA.

Because RRF works in concert with EF-G (Hirashima

and Kaji, 1972), knowledge of how the two factors interact

on the ribosome is essential for an understanding of the

Figure 6. Demonstration of the Steric Clash between the

Position 2 RRF and the 30S Subunit(A) RRF in position 1 (IIb configuration) has been superimposed on the

70S ribosome portion of the PoTC�RRF complex map.

(B) RRF in position 2 has been superimposed on the 50S subunit

portion of the 50S�RRF complex. RRF densities (red) were derived

by filtering the docked RRF atomic structures to the resolutions of

the cryo-EM maps. For RRF to attain its position 2, the 30S subunit

must be separated from the 50S subunit by �12 A. All landmarks

and color codes are the same as in Figure 1.

Mo

mechanism of ribosome recycling. Superimposition of

the fitted RRF structure in both of its positions onto the

structure of EF-G (Agrawal et al., 1999a; Valle et al.,

2003) shows that the RRF in its position 1 (in both config-

urations IIa and IIb) would suffer serious steric clashes

with domains III–V of EF-G in both the GTP and GDP

states (see Figure S10). In position 2, RRF does not

show any steric clash with EF-G. Thus, in the presence

of EF-G, RRF can reside in position 2, but not in position

1. In other words, upon EF-G binding to the PoTC contain-

ing RRF, RRF must be displaced from position 1. This ob-

servation is consistent with the finding that RRF can coex-

ist with EF-G on the ribosome, but only with very weak

affinity (Kiel et al., 2003), such that RRF swiftly dissociates

from the ribosome (Seo et al., 2004).

Sequence of Events in the RRF-Mediated

Disassembly of the PoTC

Based on above observations, we propose a model of dis-

assembly of the PoTC (70S ribosome�mRNA�P/E-tRNA

complex) (Figure 7; also see Movie S1). RRF binds to the

PoTC with domain II of RRF facing the stalk base of the

50S subunit (IIa configuration, Figure 7A). In this IIa state,

an interaction of the tip of domain II of RRF with the stalk

base may trigger the subsequent steps. Because domain

II of RRF in this state would be in serious steric conflict

with domain V of the incoming EF-G (see Figure S10),

the initial interaction of EF-G (see Frank and Agrawal

[2001]) might orient the RRF domain II toward the 30S sub-

unit (IIb configuration, Figure 7B). However, our results

suggest that the domain II of RRF is capable of spontane-

ously reorienting to the latter configuration (IIb), given that

density attributable to both possible orientations of do-

main II is observed here and in the previous study (Agrawal

et al., 2004). It is most likely that RRF in this intermediate

state (IIb configuration in position 1) is ultimately pushed

by domain IV of EF-G to position 2 (Figure 7C), without al-

teration to the IIb configuration of the two domains. Due to

disruption of some of the bridges (Figures 2C and 2D), the

two ribosomal subunits dissociate, releasing the 30S sub-

unit, as proposed earlier (Hirokawa et al., 2005; Peske

et al., 2005; Zavialov et al., 2005). The movement of RRF

from position 1 to position 2 (Figures 7B–7D) would ensure

the ejection of the deacylated tRNA from the P/E site, be-

cause the anticodon end of the tRNA would sterically

clash with the elbow region of RRF in position 2 (Figure 4A).

This series of events is consistent with the results of the

kinetic study (Peske et al., 2005) indicating that �63% of

releasable tRNA is released by RRF and EF-G, without ad-

dition of IF3, as has also been indicated in other studies

(Hirashima and Kaji, 1973; Zavialov et al., 2005). Consider-

ing that the separation of the two ribosomal subunits oc-

curs during the RRF progression from position 1 to posi-

tion 2, and that the deacylated P/E tRNA is physically

displaced by the position 2 RRF, the subunit separation

step (Figure 6) would either precede or occur simulta-

neously with the tRNA removal step. It is also likely that

a partial intersubunit separation, induced during RRF

lecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier Inc. 257

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

Figure 7. Proposed Model of the Ribosome Recycling

(A) RRF (red) on the PoTC in position 1, with its domain II oriented (IIa configuration) toward the stalk base (Sb) of the 50S subunit (semitransparent

blue). (B) RRF (still in position 1) with its domain II reoriented (to IIb configuration; see text) toward the 30S subunit (semitransparent yellow). RRF

moves to position 2 (C) and (D) from this intermediate position. Such a movement of RRF, possibly catalyzed by EF-G (solid blue) (C), can also be

induced in vitro, at least transiently, in a significant proportion of the PoTC, by RRF alone, as indicated by the arrow from (B) to (D). The release of

mRNA (green) and the release of P/E tRNA (orange) are depicted from (B)–(D), but the sequence of these two releases could not be inferred from

our study. However, both the P/E tRNA and the 30S subunit must dissociate from the PoTC during RRF occupation of position 2 (see text and Figures

4A and 6B). Due to its low binding affinity at position 2, RRF readily dissociates from the 50S subunit (E).

progression from position 1 to position 2, facilitates the

mRNA removal and allows IF3 to gain access to its binding

site on the 30S subunit (Dallas and Noller, 2001), so as to

promote the actual subunit dissociation (Singh et al., 2005).

The presence of empty 50S subunits suggests (1) that

a significant proportion of the 70S ribosomes with bound

RRF have dissociated into subunits, presumably during

the RRF transition from position 1 to position 2; and (2)

that RRF binding in position 2 is very weak and short-lived,

given that only�6% of the total selected particles showed

RRF in this position (see Experimental Procedures and

Figures S4 and S5). However, even during its short life-

span, the occupancy of position 2 RRF leads to changes

in conformation of three additional intersubunit bridges

(Figure 2D), and it sterically prohibits the 30S subunit

from binding to the 50S subunit (Figure 6). Thus, RRF at

position 2 could act like a transient antiassociation factor

for the dissociated 50S subunit. During the PoTC disas-

sembly, the mRNA dissociates from the 30S subunit,

which is maintained in its isolated form by binding of IF3

(Hirokawa et al., 2005; Peske et al., 2005), until the next

round of translation initiation.

In conclusion, the proposed model of recycling best ac-

counts for all of the binding states observed in this study.

258 Molecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier In

Our findings suggest that in the absence of EF-G, a nonen-

zymatic movement of RRF on the ribosome can take

place, as had been indirectly inferred from previous stud-

ies (Raj et al., 2005; Seo et al., 2004). The transient nature

of RRF/EFG�GTP-driven subunit dissociation has been

recently demonstrated (Hirokawa et al., 2005; Peske

et al., 2005; Zavialov et al., 2005). The partial dissociation

of the 70S ribosome population observed in the present

study could be partly related to the very dilute ribosome

concentration, a requirement to obtain an optimum distri-

bution of ribosome particles on the cryo-EM grid, and to

the presence of large excess of RRF during the grid prep-

aration. Nevertheless, the rapid freezing of the sample on

the cryo-EM grid has enabled us to capture an otherwise

short-lived intermediate. Because only a small population

of the 50S subunits had RRF bound at position 2, it is not

surprising that detection of such a short-lived state had

eluded previous studies. The presence of EF-G would ren-

der such an intermediate even more transient.

EXPERIMENTAL PROCEDURES

Undecagold Labeling of RRF

Purified E. coli RRF was incubated for 2 hr at room temperature with

a 10-fold molar excess of monomaleimido UG (Nanoprobes, Yaphank,

c.

Molecular Cell

Cryo-EM Captures Ribosome Recycling Intermediates

NY), for labeling of its native Cys16 with UG. RRF-UG was chromato-

graphically separated from excess free UG, using a Toyopearl-40

HPLC column in Tris-HCl buffer (pH 6.45). The calculated labeling

efficiency based on the relative absorbances of RRF and RRF-UG

at 280 nm and 420 nm (see Datta et al. [2005]), and as determined

by gel-shift assay (Figure S2), was close to 100%.

Binding and Biological Activity of RRF and RRF-UG

The model PoTC was obtained as described by Hirokawa and co-

workers (Hirokawa et al., 2002b) by treatment of the E. coli polysome

preparation with puromycin in a buffer containing 8.2 mM MgSO4, 80

mM NH4Cl, and 10 mM Tris (pH 7.4), but without DTT. Binding of RRF-

UG to the PoTC was �60% (i.e., 0.6 mol RRF-UG per mol of 70S ribo-

somes), as assessed by quantitative western blot. However, RRF-UG

was efficient in the disassembly of the PoTC (Figure S3A). The PoTC

was subjected to sucrose density gradient centrifugation, and the

70S peak was used for RRF-UG binding and cryo-EM. The

PoTC�RRF-UG complex for the cryo-EM work was prepared by incu-

bation of the 2 mM PoTC with 20 mM RRF-UG at room temperature in

50 ml buffer, containing 50 mM Tris-HCl (pH 7.5), 10 mM Mg(OAc)2,

5.5 mM NH4Cl, and 25 mM KCl. Dissociation of the 70S ribosomes

into subunits was measured by the light scattering method

(Figure S3B) as described previously (Hirokawa et al., 2005).

Cryo-EM and Image Reconstruction

of the PoTC�RRF-UG Complex

Before dilution of the PoTC�RRF-UG complex (to a final concentration

of 32 pmol/ml) for cryo-EM grid preparation (Wagenknecht et al.,

1988), �50-fold molar excess of RRF-UG was added to the buffer

(50 mM Tris-HCl [pH 7.5], 10 mM Mg(OAc)2, 5.5 mM NH4Cl, and 25

mM KCl). EM data were collected on a Philips FEI Tecnai F20 field

emission gun electron microscope, equipped with a low-dose kit

and an Oxford cryotransfer holder, at a magnification of 50,7603.

The majority of the data were collected at close-to-focus settings

(underfocus setting ranging from 0.7 to 1.8 mm), so as to enhance

the signal due to the strongly scattering UG label on the ribonucleo-

protein background (also see Datta et al. [2005]). A total of 109 micro-

graphs for the PoTC�RRF-UG complex were scanned on a Zeiss

flatbed scanner, with a step size of 14 mm, corresponding to 2.76 A

on the object scale. Initially, 136,234 images were picked and sorted

into 18 defocus groups (ranging from 0.68 to 3.10 mm underfocus).

The projection matching procedure (Penczek et al., 1994) of the

SPIDER software (Frank et al., 2000) was used to obtain CTF-

corrected (Penczek et al., 1997) 3D cryo-EM maps.

Supervised Classification of the 2D Cryo-EM Data Set

Initial reconstruction using a manually selected subset of 98,238 im-

ages indicated significant heterogeneity in the data set. Therefore,

the data set was subjected to supervised classification (Valle et al.,

2002). The detailed procedure is provided in the Supplemental Exper-

imental Procedures, where the scheme of supervised classification

and the related data are presented in Figures S4 and S5, respectively.

Through classification, we obtained four distinct classes: (1) empty

70S ribosomes (represented by 18,288 images, �19% of the total se-

lected images), (2) RRF-bound ratcheted 70S ribosome (represented

by 36,439 images, �37% of the total images), (3) empty 50S subunits

(37,803 images, �38% of the total images), and (4) RRF-bound 50S

subunits (5708 images, �6% of the total images). The resolutions of

the 3D maps of the 70S�RRF-UG and 50S�RRF-UG complexes

were 15.3 A and 18.4 A, respectively, according to the FSC criterion

with 0.5 cutoff (see Bottcher et al. [1997] and Malhotra et al. [1998]),

or 11.0 A and 15.0 A, respectively, according to the 3s criterion (Orlova

et al., 1997). We filtered the maps according to the 0.5 cutoff criterion.

Docking of Atomic Coordinates into the Cryo-EM Maps

To optimally incorporate the X-ray crystallographic structure of the

RRF (Kim et al., 2000; PDB ID 1EK8), we docked the RRF structure,

Mol

divided into two domains, which were treated as two separate rigid

bodies, into our cryo-EM maps of ribosome-RRF complexes, using

O (Jones et al., 1991). During our dockings, both the cryo-EM envelope

and the positional constraints imposed by the UG density were taken

into consideration, and the distance between residue Cys16 of RRF

and the position of the UG density was held within 16 A, to accommo-

date the length of the linker and the radius of the organic shell that sur-

rounds the UG core (Safer, 1999). All resulting coordinates of RRF were

energy minimized, to relieve strain from any unfavorable steric interac-

tions. Insight II (Accelrys, USA), with the molecular mechanics/dynam-

ics program DISCOVER, was used to perform energy minimization.

The final distances between the sulfur atom of the target Cys residue

and the center of the mass corresponding to UG density were between

8 and 10 A, slightly shorter than expected (Safer 1999; Datta et al.,

2005).

In order to interpret the ribosomal neighborhood of RRF in both its

positions, we docked the X-ray crystallographic structures of the

E. coli (Schuwirth et al., 2005) and T. thermophilus (Yusupov et al.,

2001) 70S ribosome as a rigid body into the cryo-EM map. The cross-

correlation coefficient (CCC) values between the fitted X-ray coordi-

nates and the corresponding cryo-EM density maps were determined

after conversion of the fitted coordinates to the density map, through

computation of averaged densities within volume elements scale

matched to those of the cryo-EM map (i.e., a pixel size of 2.76 A,

and after filtration of the X-ray map to the resolution of the cryo-EM

density map). The CCC values between the X-ray structures and

cryo-EM densities corresponding to position 1 and position 2 RRFs

were in the 0.74–0.78 range. Visualization of the fitted atomic struc-

tures and the cryo-EM density maps was done with Ribbons (Carson,

1991) and IRIS EXPLORER (Numerical Algorithms group, Downers

Grove, IL), respectively.

Supplemental Data

Supplemental Data include ten figures, one movie, Supplemental

Experimental Procedures, and Supplemental References and can be

found with this article online at http://www.molecule.org/cgi/content/

full/27/2/250/DC1/.

ACKNOWLEDGMENTS

The authors thank T. Booth for help with some of the EM data collec-

tion, M. Breedlove for help with image processing, and Drs. Adriana

Verschoor and Michael Koonce for critical readings of the manuscript.

We thank Dr. Go Hirokawa for critical reading of the manuscript and for

allowing us to include his unpublished light-scattering data on subunit

dissociation as part of the Supplemental Data (Figure S3B). This work

was supported by NIH R01 grants GM61576 (to R.K.A) and GM60429

(to A.K.), and by Creative Biomedical Institute support (to A.K.) and

Nippon Paint grant (to H.K.). The authors also acknowledge the Wads-

worth Center’s Biochemistry Core facility, and NSF grant DBI9871347

for EM infrastructure.

Received: February 12, 2007

Revised: April 18, 2007

Accepted: June 5, 2007

Published: July 19, 2007

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Accession Numbers

The cryo-EM maps of the 70S�RRF complex and the 50S�RRF com-

plex have been deposited in the EM database (http://www.ebi.ac.uk)

under ID codes EMD 1370 and EMD 1369, respectively.

lecular Cell 27, 250–261, July 20, 2007 ª2007 Elsevier Inc. 261