insights into protein biosynthesis from structures of bacterial ribosomes

Insights into protein biosynthesis from structures ofbacterial ribosomesVeysel Berk1 and Jamie HD Cate1,2,3

Understanding the structural basis of protein biosynthesis on

the ribosome remains a challenging problem for cryo-electron

microscopy and X-ray crystallography. Recent high-resolution

structures of the Escherichia coli 70S ribosome without ligands,

and of the Thermus thermophilus and E. coli 70S ribosomes

with bound mRNA and tRNAs, reveal many new features of

ribosome dynamics and ribosome–ligand interactions. In

addition, the first high-resolution structures of the L7/L12 stalk

of the ribosome, responsible for translation factor binding and

GTPase activation, reveal the structural basis of the high

degree of flexibility in this region of the ribosome. These

structures provide groundbreaking insights into the mechanism

of protein synthesis at the level of ribosome architecture, ligand

binding and ribosome dynamics.

Addresses1 Department of Molecular and Cell Biology, University of California at

Berkeley, Berkeley, CA 94720, USA2 Department of Chemistry, University of California at Berkeley, Berkeley,

CA 94720, USA3 Physical Biosciences Division, Lawrence Berkeley National Laboratory,

Berkeley, CA 94720, USA

Corresponding author: Cate, Jamie HD ([email protected])

Current Opinion in Structural Biology 2007, 17:302–309

This review comes from a themed issue on

Nucleic acids

Edited by Dinshaw J Patel and Eric Westhof

Available online 15th June 2007

0959-440X/$ – see front matter

# 2007 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.sbi.2007.05.009

IntroductionThe ribosome is composed of two subunits that work

together to carry out mRNA-directed polypeptide syn-

thesis, or translation. Translation involves the highly

dynamic interplay of these two subunits with each other

and numerous accessory factors. Our understanding of

translation is most advanced for that in bacteria, which

contain 70S ribosomes composed of a small (30S) and a

large (50S) subunit [1]. As with DNA and RNA poly-

merases, the activity of the ribosome involves initiation,

elongation and termination steps, and also includes a

recycling step to allow reinitiation (Figure 1). The ribo-

some adopts many different functional states during each

of the above steps. Understanding the mechanistic details

of translation, therefore, will require atomic-resolution


snapshots of each of the functional states of the ribosome,

in addition to extensive biochemical, genetic and

biophysical data. This review focuses on recent high-

resolution X-ray crystal structures of the 70S ribosome,

as well as cryo-electron microscopy (cryo-EM) reconstruc-

tions and NMR experiments, that illuminate many new

aspects of the different states of the ribosome during the

translation cycle.

Interactions between ribosomal subunitsThe first high-resolution crystal structures of the 30S and

50S ribosomal subunits were a major breakthrough in

understanding the architecture and function of the ribo-

some [1]. However, much of protein synthesis requires

the two subunits to work together in the intact 70S

ribosome (Figure 1). A 5.5 A resolution X-ray crystal

structure of the 70S ribosome provided the first structural

model of how the subunits interact [2]. This structure

used the high-resolution crystal structures of the 30S and

50S subunits to define the contacts between the riboso-

mal subunits, termed bridges in prior cryo-EM recon-

structions [2]. These bridges are composed of rRNA–

rRNA contacts, rRNA–protein contacts and protein–

protein contacts. In the past two years, three groups have

determined high-resolution structures of the intact 70S

ribosome that now reveal in atomic detail how the two

ribosomal subunits interact with each other [3��–6��].Structures of the E. coli 70S ribosome at 3.5 A resolution

revealed the bridges at high enough resolution to accu-

rately model many of the molecular contacts, including

structural rearrangements of the long 16S rRNA helix

(h44) that runs the length of the 30S subunit body [3��].One striking aspect of the interface between the riboso-

mal subunits is the high degree of solvation, whereby

RNA elements from both subunits approach but do not

contact each other [3��]. In the higher resolution structure

of the T. thermophilus 70S ribosome, additional magnes-

ium ions were identified at the interface, creating new

bridges [4��]. Without further experiments at physiologi-

cal salt concentrations, however, it is unclear which of

these ions is critical for ribosome function.

Dynamics of the 30S subunit head domainThe most striking finding from the apo-70S ribosome

structures is the swiveling motion of the head domain of

the 30S subunit (Figure 2a). In two independent struc-

tures of the E. coli 70S ribosome (ribosome I and II in

[3��]), the head domain of the 30S subunit of ribosome I is

rotated about 68 as a rigid body around the 16S rRNA

‘neck’ helix (h28, Figure 2b) towards the tRNA exit site

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http://dx.doi.org/10.1016/j.sbi.2007.05.009

Insights into protein biosynthesis from structures of bacterial ribosomes Berk and Cate 303

Figure 1

Overall scheme of the bacterial translation cycle. During initiation, the 30S subunit, mRNA, initiation factors IF1 and IF3, initiator tRNA (fMet-tRNAfMet)

and IF2�GTP form a pre-initiation complex (a), which subsequently recruits the 50S subunit to form a 70S initiation complex containing initiator tRNA in

the peptidyl-tRNA-binding site (P-site) (b) [33]. The 70S initiation complex enters the elongation cycle (c), which begins with mRNA decoding in the

ribosomal aminoacyl-tRNA-binding site (A-site) (d) [1]. In mRNA decoding, aminoacyl-tRNA binds to the A-site via a two-step proofreading mechanism

that involves the GTPase elongation factor Tu (EF-Tu) and two ribosome-binding steps separated by GTP hydrolysis [7]. The aminoacyl-tRNA is

delivered as part of a ternary complex with EF-Tu and GTP (T3 complex). Following GTP hydrolysis by EF-Tu and its dissociation, aminoacyl-tRNA

accommodates into the ribosomal A-site (e) and participates in peptide bond formation in the PTC of the 50S subunit [34]. Peptide bond formation

leaves peptidyl-tRNA in the ribosomal A-site and deacylated tRNA in the P-site, termed the pre-translocation state. With the aid of the GTPase

elongation factor G (EF-G), the ribosome translocates the mRNA and tRNAs from the A- and P-sites to the P- and E-sites, respectively (g), by transiting

a hybrid state of tRNA binding (f) [7,35,36]. The resulting complex, the post-translocation state, contains peptidyl-tRNA in the P-site and deacylated

tRNA in the E-site, and is ready for another elongation cycle [7]. Exposure of a stop codon in the A-site recruits release factors (RFs) that induce

hydrolysis of the nascent polypeptide (h–j) [1]. Finally, ribosome recycling factor (RRF) works with EF-G to dissociate the two ribosomal subunits and

recycle the ribosome for another round of polypeptide synthesis (k) [1]. States of the translation cycle that correspond to structures of particular

interest in this review are marked by double dots (��).

(E-site) of the ribosome, when compared with the T.thermophilus 70S ribosome structures containing mRNA

and tRNA substrates [2,4��,5��]. The 30S head domain of

ribosome II is swiveled even further, about 128 towards

the E-site, relative to the liganded ribosome structures.


The dynamic nature of the 30S head might be critical for

unlocking a steric block to the translocation of tRNAs

from the aminoacyl-tRNA and peptidyl-tRNA sites

(A- and P-sites) to the P- and E-sites, respectively [3��](Figure 2c).


304 Nucleic acids

Figure 2

Conformational changes in the 70S ribosome during mRNA and tRNA translocation. (a) Swiveling of the head of the small subunit. Difference vectors

between phosphorous or Ca atoms are shown for the small subunit (blue). The large subunit is in grey and magenta, with the positions of ribosomal

proteins L1, L5 and L11, and the central protuberance (CP) indicated. Binding of mRNA and a P-site tRNA ASL (green) is sufficient to stabilize the 30S

subunit head in a single conformation. Reproduced with permission from [6��]. (b) Helical distortions in the neck helix of the small subunit are

responsible for swiveling of the head domain. The direction of view is shown to the left. The red and blue helices are taken from the superposition of the

two different ribosome structures in [3��]. Reproduced with permission from [3��]. (c) Five global motions within the ribosome, revealed by cryo-EM

reconstructions and by the 3.5 A structures of the E. coli 70S ribosome [3��,37,38]. The small ribosomal subunit (blue) ratchets counterclockwise by

�78 relative to the large ribosomal subunit (grey and magenta) to form the hybrid state of tRNA binding (Figure 1f). Within the 30S subunit, the head

domain swivels towards the E-site (curved arrow) and opens the tRNA-binding groove (asterisk). The L1 and L11 arms of the large subunit move

towards and away from the center of the ribosome, depending on the substep of translocation (Figure 1e–g). Reproduced with permission from [3��].

Notably, binding of mRNA and the anticodon stem-loop

(ASL) of initiator tRNA to the P-site stabilizes the 30S

head domain of both E. coli 70S ribosomes in the con-

formation seen in the 5.5 A T. thermophilus 70S ribosome

structure [2] and in high-resolution structures of the T.thermophilus 70S ribosome [4��,5��] (Figure 2a). Thus, the

30S head domain is likely to be inherently dynamic in the

absence of bound ligands. Once mRNA and P-site tRNA

bind to the ribosome, the ASL portion of the tRNA

stabilizes the head region in a single conformation. This

stabilization by P-site tRNA may help to maintain the

mRNA reading frame [6��].70S ribosome complexes with mRNA andtRNAThe three new high-resolution structures of the 70S

ribosome complexed with mRNA and tRNA ligands

noted above have provided many new insights into the

translation cycle [4��–6��] (Figure 1). Here, we describe

some highlights from these structures by focusing on the

tRNA-binding sites. The process of mRNA decoding by


cognate tRNAs occurs in the ribosomal A-site and uses a

proofreading mechanism involving the GTPase EF-Tu

[7]. Structures of the 30S subunit with ASL analogs of

tRNA revealed that the ribosomal elements responsible

for mRNA decoding in the A-site interact with the minor

groove of the codon–anticodon helix to discriminate

between cognate, near-cognate and non-cognate tRNAs

[8]. As most of the free energy of tRNA binding comes

from the ASL part of tRNA [9], these structures probably

reveal a significant part of the decoding mechanism.

Consistent with this view, the 2.8 A crystal structure of

the T. thermophilus 70S ribosome complexed with mRNA

and A-, P- and E-site tRNAs [4��] reveals the same minor

groove readout of the A-site codon–anticodon helix

(Figure 3a). In the intact ribosome, this rearrangement

is accompanied by a change in the interface with 23S

rRNA of the large subunit immediately adjacent to the

decoding center [4��] (Figure 3a). Notably, the acceptor

arm of the A-site tRNA is not visible in the 2.8 A structure

of the T. thermophilus 70S ribosome. The inherent



Figure 3

70S ribosome interactions with tRNAs. (a) Interactions in the A-site. Universally conserved residues A1492 and A1493 of 16S rRNA (orange) pack in the

minor groove of the mRNA–tRNA codon–anticodon helix (purple and green, respectively). A1913 of helix H69 of the large subunit (cyan) also

rearranges upon tRNA binding. The structure was determined in the presence of the antibiotic paromomycin (red). Reproduced with permission from

[4��]. (b) Stereo view of ribosome interactions with the P-site mRNA and tRNA ASL. Structures of the E. coli 70S ribosome and the 2.8 A T.

thermophilus 70S ribosome are shown in yellow and orange, respectively [4��,6��]. The structure of the 3.7 A T. thermophilus ribosome is shown in blue

[5��]. (c) Interactions of deacylated 30-terminal A76 of E-site tRNA with the 50S subunit. Nucleotides in 23S rRNA are in cyan and L28 is shown in blue.

Reproduced with permission from [4��]. (d) Comparison of the PTC in structures of the 70S ribosome and 50S subunit. A76 of P-site tRNA in 70S

ribosome structures and the A76 analog in the H. marismortui 50S subunit are shown for comparison. The 2.8 A structure of the 70S ribosome [4��] and

the 50S subunit structure [15] are in gold and orange, respectively. The 3.7 A structure of the 70S ribosome is in blue [5��]. The atoms used for

superposition of the PTC are described in [3��].

dynamics of the acceptor end of A-site tRNA is intriguing

in light of the proposed role of the flexibility of A-site

tRNA in mRNA decoding [10].

A detailed depiction of interactions between the ribo-

some, mRNA and tRNA within the P-site also emerges

from the recent 70S ribosome structures [4��–6��]. In

contrast to the minor groove readout in the ribosomal A-

site, RNA and protein elements in the P-site of the 30S

subunit interact with the mRNA and tRNA anticodon

via backbone contacts and stacking interactions. An

open A-site with dynamic minor groove readout makes

sense for aminoacyl-tRNA discrimination, whereas

extensive interactions in the P-site probably help the

ribosome to maintain the mRNA reading frame. A kink

between the A- and P-site mRNA codons, which may

also help define the mRNA reading frame, is stabilized

by single Mg2+ ion in the 2.8 A structure of the 70S

ribosome [4��]. Differences in the A- and P-site contacts


also help to explain how wobble base pairing is allowed

in the first position of the P-site codon during initiation

at non-canonical start codons (GUG and UUG),

whereas wobble pairing is only allowed in the third

position of the A-site codon. Notably, there are some

differences in the interpretation of how the P-site tRNA

ASL interacts with the 16S rRNA, when comparing the

structure determined in [5��] with those determined by

[4��,6��] (Figure 3b). These differences remain to be

resolved.

Large-scale rearrangements of the ribosome are apparent

in the recent 70S ribosome structures, when compared

with 30S subunit structures. In the 70S ribosome, the

head domain of the 30S subunit adopts a ‘closed’ con-

formation by tilting towards the central protuberance of

the 50S subunit, when compared with structures of the

isolated 30S subunit. This closure reorients P-site tRNA

to avoid tRNA clashes with the central protuberance of


306 Nucleic acids

the 50S subunit [4��–6��]. Additional distortions of P-site

tRNA, relative to tRNA in solution, are observed in the T.thermophilus 70S ribosome structures with intact tRNAs

[4��,5��]. The P-site tRNA is flexed towards the peptidyl

transferase center (PTC) in the large subunit and is also

bent towards the large subunit region of the A-site.

Release of these distortions after peptide bond formation

may play a role in the translocation of tRNA from the

P-site to the E-site [4��,5��].

The E-site plays an important role in mRNA and tRNA

translocation [11,12], and release of E-site tRNA may be

coupled to mRNA decoding in the A-site [13�]. The

tRNA in the E-site forms a large number of contacts

with the ribosome from its anticodon to its acceptor ends,

although some are likely to be transient in nature. In

earlier crystal structures of the intact ribosome complexed

with mRNA and E-site tRNA, the E-site in the 30S

subunit was shown to be composed primarily of proteins

S7 and S11, and backbone elements of 16S rRNA [2]. The

high-resolution structures suggest that there are no base

pairs between the E-site tRNA anticodon and the mRNA

[4��,5��]. However, these structures lack cognate E-site

tRNA. Recent lower resolution structures of the T. ther-mophilus ribosome provide some hints that the E-site

tRNA may in fact base pair with mRNA in the E-site

[14�]. Another set of tRNA contacts with the ribosome

occurs in the tRNA elbow region, which stacks with

rRNA in the L1 stalk [4��,5��]. The L1 stalk, in a closed

conformation in 70S ribosome structures with E-site

tRNA, probably has to flex to an open conformation

Figure 4

Model of the L7/L12 stalk region of the ribosome. (a) Model of the L7/L12 s

interface side of the 50S subunit. Shown are the L10/L11-binding region (be

C-terminal domains (CTDs) of L12 (red). The locations of the flexible connect

[25��]. (b) Regions of the C-terminal domain of E. coli L12 that contact GTP

with permission from [26�].


relative to the central protuberance of the 50S subunit

in order for E-site tRNA to dissociate [2,4��,5��,13�]. A

third set of ribosomal interactions involves the 30 end of

E-site tRNA, a critical element for E-site function [11].

The new 70S ribosome structures provide a structural

explanation for the functional importance of both ribose

hydroxyls of the 30-terminal A76 of E-site tRNA [4��,5��](Figure 3c). Notably, the conformation of the 30-CCA end

of tRNA bound in the E-site may differ in bacteria,

archaea and eukaryotes. In model E-site tRNA acceptor

end complexes with the isolated Haloarcula marismortui50S subunit, the position of tRNA nucleotide C75 differs

from that in the new 70S ribosome structures, possibly

due to phylogenetic differences in how the E-site is

constructed [15].

Peptidyl transferase centerStructures of 50S subunits and the 70S ribosome all

show that the PTC, where peptide bonds are formed, is

composed of RNA [3��–5��,16,17�]. Thus, the ribosome

is a ribozyme. Notably, the recent 70S ribosome struc-

ture determined by Ramakrishnan and colleagues

revealed that the N terminus of protein L27 probably

interacts with the 30 end of P-site tRNA [4��]. However,

it has been shown that the peptidyl transferase activity

of the ribosome does not depend on L27 [18,19]; thus,

its role may be to stabilize substrate binding in the P-

site. This new 70S ribosome structure [4��], together

with many determined of the 50S subunit [17�,20],

indicates that no metal ion is involved in the peptidyl

transfer reaction.

talk based on cryo-EM and X-ray crystal structures, viewed from the

ige), L11 (yellow), L10 (blue), and the N-terminal domains (NTDs) and

ion and hinge regions are also shown. Reproduced with permission from

ase translation factors (red), as determined by NMR. Reproduced



Comparisons of available 50S subunit and 70S ribosome

structures suggest that some of the key elements of the

PTC might adopt different conformations. In the apo-70S

ribosome structures from E. coli, for example, the PTC is

in a slightly more ‘open’ conformation than that seen in

other structures with bound substrates [3��]. This open

structure may play a role in A-site tRNA accommodation

before peptide bond formation [21] (Figure 1), whereas

local rearrangements of nucleotides in the PTC are more

likely to be involved in controlling the peptidyl transfer

reaction itself [17�]. Notably, the 3.7 A structure of

the T. thermophilus 70S ribosome reveals a different

conformation of the PTC to that seen in the other

available structures [3��–6��,16,17�]. The non-canonical

A2450�C2063 base pair in 23S rRNA and A76 of P-site

tRNA are located closer together in this new structure

(Figure 3d). This movement brings the 20-OH of A76,

Figure 5

Newly identified factor- and ligand-binding sites on the ribosome. (a) Bindin

The view is from the perspective of the small (40S) subunit. P-site tRNA is in

‘closed’ conformation (left) to an ‘open’ conformation (right). Reproduced w

bound in the P/I-site (red ribbon and density), which resides in between the

sites. The direction of view in the right panel is shown globally in the left pa

30S subunit head; GAC, GTPase activating center; sp, 30S subunit spur. Re


which has been shown to be essential to catalysis [20,22],

within hydrogen-bonding distance of the A�C base pair.

Future experiments will be required to determine

whether this new conformation plays a role in peptidyl

transfer or in termination, whereby the polypeptide is

hydrolyzed from the P-site tRNA by release factors RF1

or RF2 (Figure 1).

The L7/L12 stalkThe L7/L12 stalk of the ribosome, named for the L7/L12

ribosomal proteins (hereafter termed L12), protrudes

from the crown-like structure of the 50S subunit and is

crucial for recruiting translation factors to the ribosome

and enhancing their GTPase activity [7]. It has therefore

been termed the GTPase-activating region, or GAR. The

L7/L12 stalk contains ribosomal proteins L10, L11 and

L12 and the rRNA to which they bind. Unfortunately, the

g site for the ATPase eEF3 near the E-site of the yeast 80S ribosome.

green, 60S subunit in cyan and eEF3 in red. The L1 arm moves from a

ith permission from [13�]. (b) 70S initiation complex with initiator tRNA

position of tRNA when bound to the P/P- (magenta) and P/E- (green)

nel. Features of the ribosome are labeled CP, central protuberance; h,

produced with permission from [31�].


308 Nucleic acids

L7/L12 stalk is nearly invisible in the available X-ray

crystal structures of isolated 50S subunits and 70S

ribosome complexes, due to its high degree of flexibility

[23,24]. The structure of the stalk has now been revealed

in a tour-de-force effort that combined cryo-EM recon-

structions of the ribosome with new X-ray crystallo-

graphic information [25��] (Figure 4a). It can be

dissected into three subsections that are separated by

highly flexible hinge regions. First, the L10/L11-binding

region of 23S rRNA, protein L11 and the N-terminal

domain of L10 form the base of the stalk. Second, this

base is connected by a flexible region to the second part of

the stalk, the C-terminal a helix of L10, which interacts in

a modular fashion with the N-terminal domains of L12

dimers. Third, the C-terminal domains of L12 are con-

nected to the N-terminal domains by highly flexible

hinges. Overall, the C-terminal domains of L12 can span

a volume with a radius of 45 A. The two hinges give the

stalk a high degree of freedom, which is probably import-

ant for recruiting translation factors and for controlling

different states of the ribosome during translation. For

example, elongation factors EF-Tu and EF-G associate

with the ribosome far more rapidly than would be

expected based on diffusion alone. The flexibility and

extent of the L7/L12 stalk might dramatically increase

the encounter frequency of factors with the ribosome

[25��]. Notably, all of the translation factor GTPases

(IF2, EF-Tu, EF-G and RF3) form weak interactions

with the same surface of the L12 C-terminal domain, as

determined by NMR [26�] (Figure 4b). In addition,

contacts between L12 C-terminal domains and trans-

lation factors contribute to their GTPase activity [27]

and to phosphate release [28]. Although insights into the

contacts between EF-G and the L7/L12 stalk continue to

be gleaned from cryo-EM reconstructions [29], high-

resolution structures will be required to unravel the

molecular mechanisms of GTPase activation and phos-

phate release [7].

ConclusionsCryo-EM reconstructions have so far provided nearly all of

the structural information about how the ribosome inter-

acts with translation factors, from initiation to ribosome

recycling. As has been the case in the past, cryo-EM

reconstructions point to new directions for high-resolution

structural work. One of many intriguing examples is

eukaryotic elongation factor eEF3, which might be the

functional homolog of the ATPase RbbA in bacteria [30]

and binds near the ribosomal E-site, where it may aid E-site

tRNA release [13�] (Figure 5a). A second example comes

from cryo-EM reconstructions of 70S ribosome initiation

complexes, in which initiator tRNA is bound to a novel

tRNA-binding site, termed the P/I site (Figures 1 and 5b)

[31�,32�]. Structural biologists will need to tackle the

difficult problem of taking many atomic-resolution snap-

shots of the ribosome to understand its dynamics,

as revealed in cryo-EM reconstructions. The recent


high-resolution structures of the 70S ribosome provide

encouragement that this effort will pay off.

AcknowledgementsThe authors thank JA Doudna for helpful comments on the manuscript, andR Beckmann, V Ramakrishnan, J Frank, M Wahl and M Akke for allowingus to use figures from their work. This work was supported by funds fromthe National Institutes of Health (grant GM65050) and the Department ofEnergy (grant DEAC03-76SF00098) to JHDC.

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� of special interest�� of outstanding interest

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3.��

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5.��

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The structure of the E. coli ribosome complexed with a short mRNA andthe ASL portion of initiator tRNA bound in the P-site is presented. Bindingof the ASL portion of P-site tRNA is proposed to lock the head of the 30Ssubunit in a single state, similar to that in [4��,5��]. Interactions betweenmRNA and the P-site ASL are similar to those in the 2.8 A structure of theT. thermophilus ribosome [4��].

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13.�

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14.�

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insights into protein biosynthesis from structures of bacterial ribosomes

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