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Ribosom Function

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The Structure and Function of the Eukaryotic


Ribosome

Daniel N. Wilson1,2 and Jamie H. Doudna Cate3,4


1
Center for Integrated Protein Science Munich (CiPSM), 81377 Munich, Germany
2
Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich,
Germany
3
Departments of Molecular and Cell Biology and Chemistry, University of California at Berkeley, Berkeley,
California 94720
4
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Correspondence: wilson@lmb.uni-muenchen.de and jcate@lbl.gov

Structures of the bacterial ribosome have provided a framework for understanding universal
mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is
in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron
microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribo-
somal subunits now provide an unprecedented opportunity to explore mechanisms
of eukaryotic translation and its regulation in atomic detail. This review describes the
X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the
Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstruc-
tions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in
eukaryotes that will require additional structural insights to be resolved are also presented.

ll ribosomes are composed of two subunits, 2000; Harms et al. 2001), and E. coli and
A both of which are built from RNA and pro-
tein (Figs. 1 and 2). Bacterial ribosomes, for
T. thermophilus 70S ribosomes (Yusupov et al.
2001; Schuwirth et al. 2005; Selmer et al. 2006),
example of Escherichia coli, contain a small sub- reveal the complex architecture that derives
unit (SSU) composed of one 16S ribosomal from the network of interactions connecting
RNA (rRNA) and 21 ribosomal proteins (r-pro- the individual r-proteins with each other and
teins) (Figs. 1A and 1B) and a large subunit with the rRNAs (Brodersen et al. 2002; Klein
(LSU) containing 5S and 23S rRNAs and 33 et al. 2004). The 16S rRNA can be divided
r-proteins (Fig. 2A). Crystal structures of pro- into four domains, which together with the r-
karyotic ribosomal particles, namely, the Ther- proteins constitute the structural landmarks of
mus thermophilus SSU (Schluenzen et al. 2000; the SSU (Wimberly et al. 2000) (Fig. 1A): The 50
Wimberly et al. 2000), Haloarcula marismortui and 30 minor (h44) domains with proteins S4,
and Deinococcus radiodurans LSU (Ban et al. S5, S12, S16, S17, and S20 constitute the body

Editors: John W.B. Hershey, Nahum Sonenberg, and Michael B. Mathews


Additional Perspectives on Protein Synthesis and Translational Control available at www.cshperspectives.org
Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011536
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D.N. Wilson and J.H. Doudna Cate

A B C ES9s
S13
h S9
S19 S7
bk
S11

S12 S6p
pt S15
b
S17
ES6s
S20p
sp
h44 rf If
ES12s ES3s

h S9 ES9s
S10
S7
bk S2 S14
S11
pt S18p S3
S5
S6p ES7s
S4
b S15
S8
S17
S16p ES6s
S20p
ES3s

D E
S9
S19e S10
S25e S9 S3
S13 S14
S28e RACK1 S10e
S7
S19 S26e
S31e S11 S31e
S17e S12e
S2
S12e S1e
S21e S5
S8 S30e
S12
S4
S4
S15 S27e

S24e
S24e S17 S7e

S8e
S6e
S6e S4e

Figure 1. The bacterial and eukaryotic small ribosomal subunit. (A,B) Interface (upper) and solvent (lower)
views of the bacterial 30S subunit (Jenner et al. 2010a). (A) 16S rRNA domains and associated r-proteins colored
distinctly: b, body (blue); h, head (red); pt, platform (green); and h44, helix 44 (yellow). (B) 16S rRNA colored
gray and r-proteins colored distinctly and labeled. (C–E) Interface and solvent views of the eukaryotic 40S
subunit (Rabl et al. 2011), with (C) eukaryotic-specific r-proteins (red) and rRNA ( pink) shown relative to
conserved rRNA (gray) and r-proteins (blue), and with (D,E) 18S rRNA colored gray and r-proteins colored
distinctly and labeled.

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Structure and Function of Eukaryotic Ribosome

A cp B cp C cp
St
L1 St ES9L ES7L
L1 St
L1

ES4L

ES31L

ES20L ES41L

cp cp cp
ES12L
ES9L
L1
L7 ES7L ES7L
St L1
L1
St ES5L
ES4L
ES31L
ES19L
ES39L ES26L
ES20L
ES3L
ES41L ES24L

D L5 E
L18 L18
L18 L29e
L4 L21e
L44e L18e
L16 L20e
L30 L15
L13e
L10 L4 L28e
L36e L4
L13e
L8e L40e L15e
L14e L8e
L2 L6
L6e L24
L43e L3 L13 L37e
L33e L29
L34e L14
L3
L32e L23
L27e L24e L22 L38e
L30e L38e L31e L22e L19e
L19e L22e

Figure 2. The bacterial and eukaryotic large ribosomal subunit. (A) Interface (upper) and solvent (lower) views of
the bacterial 50S subunit (Jenner et al. 2010b), with 23S rRNA domains and bacterial-specific (light blue) and
conserved (blue) r-proteins colored distinctly: cp, central protuberance; L1, L1 stalk; and St, L7/L12 stalk (or P-
stalk in archeaa/eukaryotes). (B –E) Interface and solvent views of the eukaryotic 60S subunit (Klinge et al.
2011), with (B) eukaryotic-specific r-proteins (red) and rRNA ( pink) shown relative to conserved rRNA (gray)
and r-proteins (blue), (C) eukaryotic-specific expansion segments (ES) colored distinctly, and (D,E) 28S rRNA
colored gray and r-proteins colored distinctly and labeled.

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D.N. Wilson and J.H. Doudna Cate

(and spur or foot) of the SSU; the 30 major however, only became possible with the im-
domain forms the head, which is protein rich, proved resolution (3.0–3.9 Å) resulting from
containing S2, S3, S7, S9, S10, S13, S14, and S19; the crystal structures of the SSU and LSU from
whereas the central domain makes up the plat- Tetrahymena thermophila (Klinge et al. 2011;
form by interacting with proteins S1, S6, S8, Rabl et al. 2011) and the Saccharomyces cerevi-
S11, S15, and S18 (Fig. 1B). The rRNA of the siae 80S ribosome (Figs. 1D,E and 2D,E) (Ben-
LSU can be divided into seven domains (includ- Shem et al. 2011).
ing the 5S rRNA as domain VII), which—in
contrast to the SSU—are intricately interwoven
RIBOSOMAL RNA OF THE EUKARYOTIC
with the r-proteins as well as each other (Ban
RIBOSOME
et al. 2000; Brodersen et al. 2002) (Fig. 2A).
Structural landmarks on the LSU include the In terms of rRNA, the major differences between
central protuberance (CP) and the flexible L1 bacterial and eukaryotic ribosomes is the pres-
and L7/L12 stalks (Fig. 2A). ence in eukaryotes of five expansion segments
In contrast to their bacterial counterparts, (ES3S, ES6S, ES7S, ES9S, and ES12S, following the
eukaryotic ribosomes are much larger and nomenclature of Gerbi [1996]) and five variable
more complex, containing additional rRNA in regions (VRs) (h6, h16, h17, h33, and h41) on
the form of so-called expansion segments (ES) the SSU, as well as 16 expansion segments (ES3L,
as well as many additional r-proteins and r-pro- ES4L, ES5L, ES7L, ES9L, ES10L, ES12L, ES15L,
tein extensions (Figs. 1C–E and 2C–E). Com- ES19L, ES20L, ES24L, ES26L, ES27L, ES31L,
pared with the 4500 nucleotides of rRNA and ES39L, and ES41L) and two VRs (H16 –18 and
54 r-proteins of the bacterial 70S ribosome, eu- H38) on the LSU (Figs. 1C and 2C) (Cannone
karyotic 80S ribosomes contain .5500 nucleo- et al. 2002). On the LSU most ES are located on
tides of rRNA (SSU, 18S rRNA; LSU, 5S, 5.8S, the back and sides of the particle, leaving the
and 25S rRNA) and 80 (79 in yeast) r-proteins. subunit interface and exit tunnel regions essen-
The first structural models for the eukaryotic tially unaffected (Taylor et al. 2009; Armache
(yeast) ribosome were built using 15-Å cryo– et al. 2010a; Ben-Shem et al. 2010; Klinge et al.
electon microscopy (cryo-EM) maps fitted 2011). The largest concentration of additional
with structures of the bacterial SSU (Wimberly rRNA (40%) on the yeast LSU is positioned
et al. 2000) and archaeal LSU (Ban et al. 2000), behind the P stalk and is formed by ES7L (200
thus identifying the location of a total of 46 nucleotides) and ES39L (150 nucleotides),
eukaryotic r-proteins with bacterial and/or ar- with a second patch (150 nucleotides) located
chaeal homologs as well as many ES (Spahn et al. behind the L1 stalk formed by the clustering
2001a). Subsequent cryo-EM reconstructions of ES19L, ES20L, ES26L, and ES31L (Figs. 2C
led to the localization of additional eukaryotic and 3A). In addition, the highly flexible ES27L
r-proteins, RACK1 (Sengupta et al. 2004) and (150 nucleotides), which was not observed in
S19e (Taylor et al. 2009) on the SSU and L30e the crystal structures (Ben-Shem et al. 2011;
(Halic et al. 2005) on the LSU, as well as more Klinge et al. 2011), adopts two distinct confor-
complete models of the rRNA derived from mations in cryo-EM reconstructions of yeast
cryo-EM maps of canine and fungal 80S ribo- ribosomes (Beckmann et al. 2001; Armache
somes at 9 Å (Chandramouli et al. 2008; Tay- et al. 2010a). On the yeast SSU the majority
lor et al. 2009). Recent cryo-EM reconstructions (75%) of the additional rRNA comprises
of plant and yeast 80S translating ribosomes at ES3S (100 nucleotides) and ES6S (200 nu-
5.5–6.1 Å enabled the correct placement of an cleotides), which interact and cluster together to
additional six and 10 r-proteins on the SSU and form the left foot of the particle (Figs. 1C and
LSU, respectively, as well as the tracing of many 3B) (Armache et al. 2010a; Ben-Shem et al. 2011;
eukaryotic-specific r-protein extensions (Arm- Rabl et al. 2011).
ache et al. 2010a,b). The full assignment of the r- Comparison of rRNA sequences of diverse
proteins in the yeast and fungal 80S ribosomes, organisms, ranging from bacteria to mammals,

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Structure and Function of Eukaryotic Ribosome

A B
L30 ES7Lc ES7La ES6sa
L4 ES6sb
ES7Lb

L14e S7e S4e


L4
ES6se
L33e
S6e
L13 ES3sb
L32e L28e
ES39L S8e
ES3sa
cp
L1 h cp h
L7 bk L1 bk

L7 pt
pt b
b

C D E
S19 S13 S25e L5 L5

S7
S31e
S11

L16 L16
S30e
S1e

A site P site E site


L27
P-tRNA P-tRNA

S12
F G
RACK1 S3
S28e S17e S3

S5 S4
S5
mRNA

3′end 18S S28e


S30e
S26e

S26e

Figure 3. Structural and functional aspects of the eukaryotic ribosome. Interweaving of rRNA and r-proteins
on the (A) LSU near ES7L and ES39L (Klinge et al. 2011), and (B) SSU near ES3 and ES6 (Rabl et al. 2011).
Extension of r-proteins at the tRNA-binding sites on the (C) SSU (Armache et al. 2010b; Rabl et al. 2011), LSU
of the (D) bacterial (Jenner et al. 2010b), and (E) eukaryotic (Armache et al. 2010b) peptidyltransferase centers.
R-proteins located at the mRNA (F) exit, and (G) entry sites (Klinge et al. 2011).

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D.N. Wilson and J.H. Doudna Cate

reveals that the major differences in ES are re- r-protein extensions form an intricate layer of
stricted to four sites on the LSU, namely, ES7L, additional RNA–protein mass that locates pre-
ES15L, ES27L, and ES39L. These ES are signifi- dominantly to the solvent surfaces of the ribo-
cantly longer (850, 180, 700, and 220 some (Figs. 1C and 2B). More than half of the
nucleotides) in human 80S ribosomes than conserved r-proteins contain extensions, which in
in yeast (200, 20, 150, and 120 nucle- some cases, such as S5, L4, L7, and L30, establish
otides, respectively) (Cannone et al. 2002). long-distance interactions far (50–100 Å) from
Moreover, cryo-EM reconstructions of mam- the globular core of the protein. Interaction of
malian ribosomes (Dube et al. 1998; Morgan eukaryotic-specific extensions with conserved
et al. 2000; Spahn et al. 2004b; Chandramouli core proteins using interprotein shared b-sheets
et al. 2008; Budkevich et al. 2011) reveal little to has been noted, for example, between L14e and
no density for the longer ES in mammalian ri- L6 (Ben-Shem et al. 2011) as well as L21e and
bosomes, indicating that they are highly mobile L30 (Klinge et al. 2011).
elements. In Tetrahymena, deletion of ES27L is The eukaryotic LSU contains 1 MDa of
lethal (Sweeney et al. 1994), suggesting a func- additional protein: 200 kDa of eukaryotic-spe-
tionally important role for this ES. Despite the cific domains or extensions and 800 kDa of
high variability in length of ES27L, ranging r-proteins that are absent in bacteria. Most of
from 150 nucleotides in yeast to 700 nucle- this additional protein mass is located in a ring
otides in mammals (Cannone et al. 2002), dele- around the back and sides of the LSU, where
tion of ES27L can be complemented with a cor- it interacts with ES (Fig. 2B). Two large con-
responding ES27L from other species (Sweeney centrations of additional RNA– protein mass
et al. 1994). ES27L has been suggested to play a exemplify the intertwined and coevolving na-
role in coordinating the access of nonribosomal ture of the ribosome (Yokoyama and Suzuki
proteins to the tunnel exit (Beckmann et al. 2008). One cluster on the LSU comprises
2001), but this remains to be shown. The role ES7L, ES39L, five eukaryotic r-proteins (L6e,
of other ES remains unclear. Their presence in L14e, L28e, L32e, and L33e), as well as eukary-
eukaryotic ribosomes may reflect the increased otic-specific extensions of conserved r-proteins
complexity of translation regulation in eukary- (L4, L13, and L30) (Fig. 3A). In this cluster yeast
otic cells, as evident for assembly, translation ES7L comprises three helices, ES7La – c, whereas
initiation, and development, as well as the phe- wheat germ ( plant) ES7L has five helices,
nomenon of localized translation (Sonenberg ES7La– e, including a three-way junction ex-
and Hinnebusch 2009; Freed et al. 2010; Wang tending from ES7Lc (Armache et al. 2010b).
et al. 2010). Curiously, the extension of L6e is longer in
wheat germ as compared with yeast and appears
to wrap around ES7L and insert through the
RIBOSOMAL PROTEINS OF THE
three-way junction of ES7La – c (Armache et al.
EUKARYOTIC RIBOSOME
2010b). ES7La is stabilized by L28e in wheat
The yeast 80S ribosome contains 79 r-proteins germ and Tetrahymena, whereas this helix is
(SSU, 33; LSU, 46), 35 of which (SSU, 15; LSU, more flexible in baker’s yeast lacking L28e.
20) have bacterial/archaeal homologs, whereas Stabilization of ES by eukaryotic r-proteins is
32 (SSU, 12; LSU, 20) have only archaeal homo- also evident for ES27L, with the two different
logs (Lecompte et al. 2002). Thus, 12 (SSU, 6; yeast conformations being stabilized by interac-
LSU, 6) r-proteins of the yeast 80S are specific tion with either L38e or L27e (Armache et al.
for eukaryotes. Cytoplasmic 80S ribosomes of 2010b). The second major ES cluster comprises
Tetrahymena and higher eukaryotes, such as ES19L, ES20L, ES26L, and ES31L, which are
humans, contain an additional LSU r-protein, intimately associated with eukaryotic-specific
L28e, and thus have 13 eukaryotic-specific r- r-proteins L27e, L30e, L34e, L43e, and the
proteins and 80 (SSU, 33; LSU, 47) in total. To- carboxy-terminal extension of L8e (Fig. 2C –
gether with the ES, the additional r-proteins/ E) (Ben-Shem et al. 2011). A single-stranded

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Structure and Function of Eukaryotic Ribosome

loop region of ES31L provides an interaction et al. 2001b; Schuler et al. 2006; Muhs et al.
platform for many of these r-proteins, notably 2011). S30e replaces part of S4 at the mRNA
the carboxy-terminal helix of L34e. Similarly, entry site of the eukaryotic SSU and has con-
ES39L also has many single-stranded loop re- served lysine residues that extend into the
gions that provide interaction sites for r-pro- mRNA channel (Fig. 3G), suggesting that S30e,
teins, such as L20e and L14e. together with S3, plays a role in unwinding
The protein-to-RNA ratio of bacterial SSU mRNA secondary structure (Rabl et al. 2011).
is 1:2, whereas the dramatic increase in r-pro- S3 has a long carboxy-terminal extension that
tein mass for the eukaryotic SSU results in an spans across S17e and interacts with RACK1
almost 1:1 ratio. The SSU structures reveal that (Fig. 3G) (Rabl et al. 2011). RACK1 is a scaffold
most of the additional eukaryotic-specific r- protein that binds to several signaling proteins,
proteins and extensions cover the back of the therefore connecting signaling transduction
SSU particle, forming a web of interactions pathways with translation (Nilsson et al. 2004).
with each other as well as with conserved r-pro- Thus, in addition to stabilization of rRNA ES
teins and rRNA (Fig. 1C – E) (Ben-Shem et al. architecture of the ribosome, eukaryotic-specif-
2011; Rabl et al. 2011). The beak of the eukary- ic r-proteins and extensions appear to be im-
otic SSU has acquired three r-proteins, S10e, portant for binding of eukaryotic-specific reg-
S12e, and S31e, which appear to compensate ulatory factors, particularly factors that interact
for the reduced h33 compared with the bacterial with the SSU to regulate translation initiation of
SSU rRNA (Rabl et al. 2011). R-proteins are also specific mRNAs.
seen to interact with the expansion segments
ES3S and ES6S, via r-proteins S4e, S6e, S7e,
THE tRNA-BINDING SITES ON THE
and S8e (Fig. 3B). S6e has a long carboxy-ter-
EUKARYOTIC RIBOSOME
minal helix that stretches from the left to right
foot, and that is phosphorylated in most eu- The binding sites for the aminoacyl-transfer
karyotes (Meyuhas 2008). Based on the periph- RNA (tRNA) (A site), peptidyl-tRNA (P site),
eral position of S6e, any regulation of transla- and deacylated tRNA (exit or E site) on the
tion via S6e phosphorylation is likely to be via bacterial ribosome are composed predominant-
indirect recruitment of specific regulatory fac- ly of rRNA (Yusupov et al. 2001; Selmer et al.
tors (Rabl et al. 2011). The mRNA exit site on 2006). This rRNA is conserved in archaeal and
the eukaryotic SSU also differs from the bacte- eukaryotic ribosomes, suggesting that the basic
rial one because of the presence of S26e and mechanism by which the ribosome distinguish-
S28e surrounding the 30 end of the 18S rRNA es the cognate tRNA from the near- or noncog-
(Fig. 3F) (Armache et al. 2010a; Rabl et al. nate tRNAs at the A site during decoding (Ogle
2011). S26e overlaps the binding position of and Ramakrishnan 2005; Schmeing et al. 2011)
the E. coli r-protein S21p (Schuwirth et al. is also likely to be conserved. Nevertheless, many
2005), whereas S28e has a similar fold to the r-proteins encroach on the tRNA-binding sites
bacterial RNA-binding domain of r-protein and appear to play important roles in decoding,
S1p (Rabl et al. 2011). Such differences may accommodation, and stabilization of tRNAs
reflect the distinct elements found in the 50 un- (Fig. 3C) (Yusupov et al. 2001; Selmer et al.
translated regions of eukaryotic mRNAs, as well 2006; Jenner et al. 2010b). These r-proteins
as the divergence in the translation initiation may be responsible for the slightly different po-
phase from bacteria (Sonenberg and Hinne- sitioning of tRNAs on the eukaryotic ribosome
busch 2009). Indeed, eIF3, which is absent in compared with the bacterial ribosome (Budke-
bacteria, interacts with this general region of the vich et al. 2011). On the SSU a conserved loop of
SSU (Bommer et al. 1991; Srivastava et al. 1992; S12 participates in monitoring of the second
Siridechadilok et al. 2005), as do internal ribo- and third positions of the mRNA– tRNA co-
some entry site (IRES) elements present in the don –anticodon duplex (Ogle and Ramakrish-
50 untranslated region of viral mRNAs (Spahn nan 2005). Additionally, the carboxy-terminal

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D.N. Wilson and J.H. Doudna Cate

extensions of r-proteins S19 and S9/S13 stretch to the PTC of bacterial versus eukaryotic LSU
from globular domains located on the head of indicates that subtle differences do in fact exist
the SSU to interact with anticodon stem-loop (Wilson 2011). In addition to differences in
(ASL) regions of A- and P-tRNA, respectively, the conformation of rRNA nucleotides, one of
whereas S7, and to a lesser extent S11, interacts the major differences between the bacterial and
with the ASL of E-tRNA (Fig. 3C) (Yusupov eukaryotic PTC is related to r-proteins. Eukary-
et al. 2001; Selmer et al. 2006; Jenner et al. otic L16 contains a highly conserved loop that
2010b). Although these tRNA interactions are reaches into the PTC and contacts the CCA end
likely to be maintained in eukaryotic 80S ribo- of the P-tRNA (Fig. 3D) (Armache et al. 2010b;
somes, additional interactions are probable on Bhushan et al. 2010b). This loop is absent in
the SSU because of the presence of extensions of bacteria, and instead the space is occupied by
four eukaryotic r-proteins that approach the the amino-terminal extension of bacterial-spe-
tRNA-binding sites, namely, the amino-termi- cific r-protein L27p (Fig. 3E) (Voorhees et al.
nal extensions of S30e and S31e that reach into 2009). The binding site of the CCA end of the
the A site; S25e, which is positioned between the E-tRNA on the eukaryotic LSU resembles the
P and E sites; and S1e at the E site (Fig. 3C) archaeal, rather than the bacterial, context.
(Armache et al. 2010b; Ben-Shem et al. 2011; Whereas bacterial-specific r-protein L28p con-
Rabl et al. 2011). S31e is expressed with an ami- tributes to the E site of the bacterial LSU
no-terminal ubiquitin fusion, suggesting that (Selmer et al. 2006), the archaeal and eukaryotic
the lethality from lack of cleavage (Lacombe r-protein L44e contains an internal loop region
et al. 2009) arises because of the inability of (Fig. 2D) through which the CCA end of the E-
tRNA and/or initiation factors to bind to the tRNA inserts (Schmeing et al. 2003). Moreover,
SSU (Rabl et al. 2011). the carboxyl terminus of L44e is longer in eu-
Additional stabilization of tRNA binding is karyotes, such as yeast, than in archaea, provid-
observed via interaction between LSU r-pro- ing the potential for additional interactions with
teins with the elbow regions of tRNAs, namely, the P- and/or E-tRNA. Nevertheless, the E site
the A- and P-tRNA, through contact with con- restricts binding of only deacylated tRNAs via a
served r-proteins L16 and L5, respectively, as direct interaction between the 20 OH of A76 and
well as the E-tRNA with the L1 stalk (Yusupov the base of C2394 (E. coli 23S rRNA numbering)
et al. 2001; Selmer et al. 2006; Jenner et al. (Schmeing et al. 2003; Selmer et al. 2006). The
2010b). The carboxyl terminus of the bacte- base equivalent to C2394 is conserved across all
rial-specific r-protein L25p also interacts with kingdoms (Cannone et al. 2002), suggesting a
the elbow region of A-tRNA (Jenner et al. universal mechanism of deacylated-tRNA dis-
2010b). This r-protein is absent in archaeal crimination at the E site on the LSU.
and eukaryotic ribosomes. At the peptidyltrans-
ferase center (PTC) of the LSU, the CCA ends of
BINDING SITES OF INITIATION FACTORS
the A- and P-tRNAs are stabilized through in-
ON THE RIBOSOME
teraction with the conserved A- and P-loops of
the 23S rRNA, thus positioning the a-amino In bacteria, translation initiation is driven in
group of the A-tRNA for nucleophilic attack large part by base pairing between the mRNA
on the carbonyl carbon of the peptidyl-tRNA just 50 of the start codon and the 30 end of
(Leung et al. 2011). The high sequence and 16S rRNA—the Shine–Dalgarno interaction—
structural conservation of the PTC and of the which defines the ribosome binding site (Geiss-
tRNA substrates suggests that the insights into mann et al. 2009; Simonetti et al. 2009). Three
the mechanism of peptide bond formation proteins contribute to bacterial initiation,
gained from studying archaeal and bacterial ri- termed initiation factors 1, 2, and 3 (IF1, IF2,
bosomes (Simonovic and Steitz 2009) are trans- and IF3), and help to load initiator tRNA into
ferable to eukaryotic ribosomes. Nevertheless, the small-subunit P site at the correct start
the varying specificity for binding of antibiotics codon (Simonetti et al. 2009). In eukaryotes,

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Structure and Function of Eukaryotic Ribosome

translation initiation generally requires a scan- et al. 2009; Chiu et al. 2010; Kouba et al. 2011).
ning mechanism that starts at the 50 -7-methyl- With the determination of the recent X-ray crys-
guanosine (50 -m7G) cap and proceeds to the tal structures of the T. thermophila 40S and 60S
appropriate AUG start codon, often the first subunits, in complexes with eIF1 and eIF6, re-
AUG codon encountered by the initiation ma- spectively (Klinge et al. 2011; Rabl et al. 2011),
chinery (Jackson et al. 2010). To accomplish our understanding of the structural basis for
scanning, a whole suite of eukaryotic transla- translation initiation in eukaryotes has in-
tion initiation factors (eIFs) is involved, with creased greatly, but still lags behind our struc-
names from eIF1 through eIF6, as described in tural knowledge of bacterial translation initia-
more detail by Lorsch et al. (2012). Only two tion (Simonetti et al. 2009).
of the three bacterial proteins, IF1 and IF2, are Initiation factor eIF1 promotes binding of
conserved in eukaryotes, as counterparts of initiator tRNA, in the form of a ternary complex
eIF1A and eIF5B, respectively (Benelli and Lon- of eIF2 – GTP– Met– tRNAMet i , to preinitiation
dei 2009). However, eIF1A and eIF5B have aug- complexes of the SSU. It also serves to prevent
mented or divergent roles to play in eukaryotic initiation at non – start codons, likely by pro-
translation initiation (Jackson et al. 2010). IF3 is moting an “open” state of the SSU (Jackson
not conserved in eukaryotes, but seems to have a et al. 2010; Hinnebusch 2011). Consistent with
functional counterpart in eIF1 (Lomakin et al. this model, a cryo-EM reconstruction of the
2003, 2006). Similar to what is observed for r- yeast 40S subunit in complex with eIF1 and
proteins in eukaryotes, eIF1 and eIF1A have ex- eIF1A revealed that these two proteins induce
tensions or “tails” that are important for their an opening of the mRNA- and tRNA-binding
function (Olsen et al. 2003; Fekete et al. 2005, groove in the 40S subunit that may contribute
2007; Cheung et al. 2007; Reibarkh et al. 2008; to scanning and correct start codon selection
Saini et al. 2010). Most of the interactions be- (Passmore et al. 2007). Release of eIF1 when
tween the 40S subunit and eukaryotic transla- the start codon is recognized is proposed to re-
tion initiation factors are only known from ge- sult in the closing of this groove, thereby locking
netic, biochemical, and low-resolution cryo-EM the mRNA and initiator tRNA in place (Nanda
reconstructions and models of partial initiation et al. 2009). In the structure of the 40S subunit,
complexes (Lomakin et al. 2003; Valasek et al. eIF1 is bound adjacent to the SSU P site, in such a
2003; Fraser et al. 2004, 2007; Unbehaun et al. way that it would prevent full docking of the
2004; Siridechadilok et al. 2005; Passmore et al. initiator tRNA ASL in the P-site cleft (Fig. 4A).
2007; Szamecz et al. 2008; Shin et al. 2009; Yu Notably, the position of eIF1 is more compatible

Head Head Clashes


A Clashes B

PE tRNA

elF1 elF1
B2a B2a
PP tRNA
Platform Head Platform

Figure 4. Positioning of eIF1 near the SSU P site. (A) Steric clash between eIF1 and P-site tRNA in the canonical
P/P configuration. Structure of the 40S subunit–eIF1 complex superimposed with the unrotated state of the
ribosome in Dunkle et al. (2011). (B) Binding of eIF1 is more compatible with tRNA in the P/E configuration.
Structure of the 40S subunit–eIF1 complex superimposed with the rotated state of the ribosome in Dunkle et al.
(2011). Nucleotides in 18S rRNA that would contribute to contacts with the LSU in bridge B2a are colored red.

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D.N. Wilson and J.H. Doudna Cate

with tRNA docked in a hybrid configuration LSU assembly (Senger et al. 2001; Menne et al.
seen in the bacterial ribosome, in which the 2007; Finch et al. 2011), and also how it might
tRNA is bound in the SSU P site and LSU E site be used to regulate the availability of 60S sub-
(P/E-tRNA) (Fig. 4B) (Dunkle et al. 2011). As units as a means to control cell growth and pro-
part of start codon selection, dissociation of liferation (Gandin et al. 2008).
eIF1 may allow initiator tRNA to adopt an in-
termediate P/I orientation, observed in bacte-
THE RIBOSOMAL TUNNEL OF
rial initiation complexes with IF2 (Allen et al.
EUKARYOTIC RIBOSOMES
2005; Julian et al. 2011), or the P/P configura-
tion, in which it could access the LSU P site upon As the nascent polypeptide chain (NC) is being
subunit association (Jackson et al. 2010). synthesized, it passes through a tunnel within
The binding site for eIF1 would also block the LSU and emerges at the solvent side, where
the premature binding of the 60S subunit, be- protein folding occurs. Cryo-EM reconstruc-
cause it is situated right where a critical contact tions and X-ray crystallography structures of
(“bridge” B2a) forms between the two ribosom- bacterial, archaeal, and eukaryotic cytoplasmic
al subunits (Fig. 4) (Rabl et al. 2011). Part of eIF1 ribosomes have revealed the universality of the
also extends into the mRNA-binding groove, dimensions of the ribosomal tunnel (Frank et al.
adjacent to where the P-site codon would be 1995; Beckmann et al. 1997; Ban et al. 2000;
situated. From biochemical and genetic experi- Ben-Shem et al. 2011; Klinge et al. 2011). The
ments, the amino-terminal tail of eIF1 plays an ribosomal tunnel is 80 Å long, 10 –20 Å wide,
important role in recruiting the eIF2– GTP– and predominantly composed of core rRNA
Met – tRNAMet i ternary complex to preinitiation (Nissen et al. 2000), consistent with an overall
complexes (Cheung et al. 2007). However, the electronegative potential (Lu et al. 2007). The
structure of the eIF1 – 40S complex provides extensions of the r-proteins L4 and L22 contrib-
only the first structural hints into how the ter- ute to formation of the tunnel wall, forming a
nary complex is recruited and how start codons so-called constriction where the tunnel narrows
are selected. Future structures with more of the (Nissen et al. 2000). Near the tunnel exit the
translation initiation factors, as well as with ini- ribosomal protein L39e is present in eukaryotic
tiator tRNA, will be needed to unravel the mo- and archaeal ribosomes (Nissen et al. 2000),
lecular basis for start codon selection. whereas a bacterial-specific extension of L23
The role in initiation of translation initia- occupies an overlapping position in bacteria
tion factor eIF6 is not as clearly defined. It has (Harms et al. 2001).
been proposed to be an antiassociation factor For many years the ribosomal tunnel was
that prevents premature association of the two thought of only as a passive conduit for the
ribosomal subunits, and it also acts in late stages NC. However, growing evidence indicates that
of pre-60S assembly (Brina et al. 2011). In the the tunnel plays a more active role in regulating
recent X-ray crystal structure of the 60S subunit the rate of translation, in providing an environ-
(Klinge et al. 2011), and as previously observed ment for early protein folding events, and in
(Gartmann et al. 2010), eIF6 binds to the recruiting translation factors to the tunnel exit
GTPase center, the region of the LSU where GT- site (Wilson and Beckmann 2011). At the sim-
Pases such as those responsible for mRNA de- plest level, long stretches of positively charged
coding (eukaryotic elongation factor 1 [eEF1]) residues, such as arginine or lysine, in an NC can
and mRNA and tRNA translocation (eEF2) in- reduce or halt translation, most likely through
teract with the ribosome. The location of eIF6 interaction with the negatively charged rRNA in
would sterically prevent SSU interactions with the tunnel (Lu and Deutsch 2008). More specif-
the LSU, helping to explain its antiassociative ic regulatory systems also exist in bacteria and
activity. Its position near the GTPase center is eukaryotes, in which stalling during translation
also highly suggestive of how it might be re- of upstream open reading frames (uORFs of
leased in a GTPase-dependent manner during the cytomegalovirus [CMV] gp48 and arginine

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Structure and Function of Eukaryotic Ribosome

attenuator peptide [AAP] CPA1 genes) or lead- the tunnel may have implications for not only
er peptides (TnaC, SecM) leads to modulation protein folding, but also downstream events,
of expression of downstream genes (Tenson and such as recruitment of chaperones or targeting
Ehrenberg 2002). Interestingly, the translational machinery (Bornemann et al. 2008; Berndt et al.
stalling events depend critically on the sequence 2009; Pool 2009).
of the NC and the interaction of the NC with the
ribosomal tunnel. Cryo-EM reconstructions of
INTERACTIONS BETWEEN THE
bacterial TnaC- and SecM-stalled 70S ribo-
RIBOSOMAL SUBUNITS
somes (Seidelt et al. 2009; Bhushan et al. 2011)
and eukaryotic CMV- and AAP-stalled 80S ri- During translation the ribosome undergoes
bosomes (Bhushan et al. 2010b) reveal the dis- global conformational rearrangements that are
tinct pathways and conformations of the NCs in required for mRNA decoding, mRNA and tRNA
the tunnel as well as the interactions between the translocation, termination, and ribosome recy-
NCs and tunnel wall components. Compared cling. These changes involve intersubunit rota-
with bacteria, eukaryotic r-protein L4 has an tion, as well as swiveling of the head domain
insertion that establishes additional contacts of the SSU (Fig. 5A). The interactions between
with the CMV- and AAP-NCs (Bhushan et al. the ribosomal subunits, or “bridges,” change
2010b), whereas the bacterial stalling sequences with each of these rearrangements, and are
interact predominantly with L22 (Seidelt et al. therefore dynamic in composition. The inter-
2009; Bhushan et al. 2011). The dimensions of subunit bridges were originally mapped in bac-
the ribosomal tunnel preclude the folding of teria by modeling high-resolution SSU and LSU
domains as large as an IgG domain (17 kDa) structures into cryo-EM reconstructions and
(Voss et al. 2006), whereas a-helix formation low-resolution X-ray crystal structures (Gabash-
has been demonstrated biochemically (Deutsch vili et al. 2000; Yusupov et al. 2001; Valle et al.
2003; Woolhead et al. 2004) and visualized 2003), and in more recent high-resolution struc-
structurally within distinct regions of the tunnel tures of the intact bacterial ribosome (Schuwirth
(Bhushan et al. 2010a). Folding of NCs within et al. 2005; Dunkle et al. 2011). The bridges in

A B
Head (P proteins) 60S
40S

(L1 arm) GTPase L24e


center eB13
L19e
eB12
* Body
Platform C
Head
h45
h44
40S 60S
L41e
60S eB14
h27
40S Body

Figure 5. Intersubunit rotation required for translation. (A) Key conformational rearrangements in the ribo-
some. Rotation of the SSU body, head domain, and opening of the mRNA- and tRNA-binding groove during
mRNA and tRNA translocation (asterisk) are indicated by arrows. Closing of the SSU body toward the LSU
during mRNA decoding is also indicated by an arrow. Dynamic regions of the LSU (L1 arm, P proteins, and
GTPase center) are labeled. (B) Bridges eB12 and eB13 in the yeast ribosome at the periphery of the subunits.
LSU proteins contributing to the bridges are marked. The view is indicated to the left. (C ) Bridge eB14 in the
yeast ribosome, near the pivot point of intersubunit rotation. LSU protein L41e and 18S rRNA helices in the SSU
contributing to the bridge (gold) are indicated.

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D.N. Wilson and J.H. Doudna Cate

eukaryotic ribosomes have been mapped using and tRNA translocation, translation termina-
similar approaches. The high-resolution struc- tion, and ribosome recycling differ in significant
tures of the yeast 80S ribosome now provide an ways from those in bacteria (Triana-Alonso et al.
atomic-resolution view of the bridges for rotated 1995; Andersen et al. 2000; Gaucher et al. 2002;
states of the ribosome (Ben-Shem et al. 2011), Jorgensen et al. 2003; Alkalaeva et al. 2006;
and cryo-EM reconstructions of translating ri- Khoshnevis et al. 2010; Pisarev et al. 2010). The
bosomes at 5- to 6-Å resolution reveal the in- recent breakthroughs in the structural biology of
tersubunit bridges in the unrotated state of the the eukaryotic ribosome provide a structural
ribosome (Armache et al. 2010a,b). framework to unravel these differences. The large
Whereas the bacterial ribosome preferen- number of approximately nanometer or sub-
tially adopts the unrotated state of the two sub- nanometer cryo-EM reconstructions of eukary-
units, the eukaryotic ribosome seems to adopt otic ribosomes in different functional states
rotated states more readily (Spahn et al. 2004a; (Halic et al. 2004, 2005, 2006a,b; Spahn et al.
Chandramouli et al. 2008; Ben-Shem et al. 2004a; Gao et al. 2005; Andersen et al. 2006;
2011; Budkevich et al. 2011). A possible reason Schuleret al. 2006; Tayloret al. 2007, 2009; Chan-
for this difference in behavior is the fact that the dramouli et al. 2008; Sengupta et al. 2008; Becker
interaction surface between the two ribosomal et al. 2009, 2011, 2012; Armache et al. 2010a,b;
subunits has nearly doubled in eukaryotes com- Bhushan et al. 2010a,b; Gartmann et al. 2010;
pared with bacteria, primarily because of the Budkevich et al. 2011) now can be interpreted
appearance of numerous additional bridges at using high-resolution structures of the ribosome
the periphery of the subunit interface. These (Jarasch et al. 2011) in combination with X-ray
new bridges are composed mainly of protein – crystal structures of the individual factors (No-
protein and protein –rRNA contacts, some of ble and Song 2008; Chen et al. 2010).
the more notable involving long extensions Although there are many differences in the
from the LSU to contact the body and platform translation elongation and termination factors
of the SSU, bridges eB12 and eB13 (Fig. 5B) between bacteria and eukaryotes, these factors
(Ben-Shem et al. 2011). One striking exception seem to exploit common features of the ribo-
to this general trend is one new bridge right at some conserved in all domains of life. One no-
the center of the subunit interface, near the piv- table example is the mechanism for GTPase ac-
ot point of intersubunit rotation (Ben-Shem tivation in mRNA decoding, in which the
et al. 2011). This bridge, termed eB14, is com- sarcin – ricin loop was shown to reorganize the
posed of a single short a-helical peptide, desig- catalytic center in bacterial EF-Tu (eukaryotic
nated L41e, that is nearly entirely buried in a ortholog of eEF1A) during mRNA decoding
pocket composed of 18S rRNA in the SSU. Re- (Voorhees et al. 2010). A second example is
markably, this pocket is highly conserved in eu- the convergent evolution of a motif in release
karyotes and in bacteria (Fig. 5C) (Schluenzen factors that is responsible for stimulating the
et al. 2000; Wimberly et al. 2000; Cannone et al. hydrolysis of completed proteins from pep-
2002; Ben-Shem et al. 2011), but no corre- tidyl-tRNA during termination. Bacterial and
sponding peptide in bacteria has been identi- eukaryotic release factors (RF1 and RF2 in bacte-
fied. The importance of this peptide in eukary- ria, eRF1 in eukaryotes) are composed of entirely
otic ribosome function remains unknown. different protein topologies (Song et al. 2000; Ves-
tergaard et al. 2001; Shin et al. 2004). Furthermore,
eukaryotic RF1 requires the GTPase eRF3 and
MECHANISMS OF mRNA DECODING,
ATPase ABCE1 to stimulate termination and ribo-
TRANSLOCATION, TERMINATION,
some recycling (Khoshnevis et al. 2010; Pisarev
AND RIBOSOME RECYCLING
et al. 2010; Becker et al. 2012), whereas bacterial
Remarkably for processes that are functionally termination and ribosome recycling use different
conserved in all domains of life, the mechanisms factors (Zavialov et al. 2001; Savelsbergh et al.
used by eukaryotes for mRNA decoding, mRNA 2009). Strikingly, given these differences, the key

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Structure and Function of Eukaryotic Ribosome

residues in RFs that insert into the PTC to pro- otide exchange and competition with tRNA in the yeast
elongation factor complex eEF1A:eEF1Ba. Mol Cell 6:
mote peptidyl-tRNA hydrolysis, a GGQ motif, 1261–1266.
are universally conserved. A second example Andersen CB, Becker T, Blau M, Anand M, Halic M, Balar B,
occurs with the GTPases involved in elongation. Mielke T, Boesen T, Pedersen JS, Spahn CM, et al. 2006.
Bacteria rely on the GTPases EF-Tu and EF-G, Structure of eEF3 and the mechanism of transfer RNA
release from the E-site. Nature 443: 663–668.
whereas eukaryotes use the GTPases eEF1A and
Armache JP, Jarasch A, Anger AM, Villa E, Becker T,
eEF2. Eukaryotic eEF2 cannot function on the Bhushan S, Jossinet F, Habeck M, Dindar G, Francken-
bacterial ribosome, unless the bacterial L10 and berg S, et al. 2010a. Cryo-EM structure and rRNA model
L12 proteins in the LSU are replaced by the of a translating eukaryotic 80S ribosome at 5.5-Å resolu-
tion. Proc Natl Acad Sci 107: 19748–19753.
eukaryotic acidic proteins P0 and P1/P2 Armache JP, Jarasch A, Anger AM, Villa E, Becker T,
(Uchiumi et al. 1999, 2002). Notably, this pro- Bhushan S, Jossinet F, Habeck M, Dindar G, Francken-
tein-swapping experiment also illustrates how berg S, et al. 2010b. Localization of eukaryote-specific
ribosomal proteins in a 5.5-Å cryo-EM map of the 80S
the underlying rRNA functions are probably eukaryotic ribosome. Proc Natl Acad Sci 107: 19754–
universal. 19759.
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CONCLUSIONS complete atomic structure of the large ribosomal subunit
at 2.4 Å resolution. Science 289: 905 –920.
The last few years have witnessed a surge of new Becker T, Bhushan S, Jarasch A, Armache JP, Funes S, Jossi-
net F, Gumbart J, Mielke T, Berninghausen O, Schulten K,
structures of the bacterial and eukaryotic ribo- et al. 2009. Structure of monomeric yeast and mamma-
some in different steps of the translation cycle. lian Sec61 complexes interacting with the translating ri-
The recent X-ray crystal structures of the T. ther- bosome. Science 326: 1369– 1373.
mophila 40S and 60S ribosomal subunits and Becker T, Armache JP, Jarasch A, Anger AM, Villa E, Sieber
H, Motaal BA, Mielke T, Berninghausen O, Beckmann R.
yeast 80S ribosome now provide an unprece- 2011. Structure of the no-go mRNA decay complex
dented framework for interpreting the many Dom34–Hbs1 bound to a stalled 80S ribosome. Nat
cryo-EM reconstructions of the eukaryotic ri- Struct Mol Biol 18: 715– 720.
bosome and biochemical insights into the eu- Becker T, Franckenberg S, Wickles S, Shoemaker CJ, Anger
AM, Armache JP, Sieber H, Ungewickell C, Berninghau-
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it is not hard to imagine that many of the steps conserved ribosome recycling in eukaryotes and archaea.
in eukaryotic translation will be understood in Nature 482: 501 –506.
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ACKNOWLEDGMENTS Beckmann R, Spahn CM, Eswar N, Helmers J, Penczek PA,
Sali A, Frank J, Blobel G. 2001. Architecture of the pro-
This work is supported by the EMBO Young In- tein-conducting channel associated with the translating
80S ribosome. Cell 107: 361–372.
vestigator program (to D.N.W.) and by the Na- Ben-Shem A, Jenner L, Yusupova G, Yusupov M. 2010. Crys-
tional Institutes of Health grant R56-AI095687 tal structure of the eukaryotic ribosome. Science 330:
(to J.H.D.C). 1203–1209.
Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L,
Yusupova G, Yusupov M. 2011. The structure of the eu-
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The Structure and Function of the Eukaryotic Ribosome


Daniel N. Wilson and Jamie H. Doudna Cate

Cold Spring Harb Perspect Biol 2012; doi: 10.1101/cshperspect.a011536

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