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review
Approaching translation at atomic resolution
Joseph D. Puglisi1, Scott C. Blanchard1 and Rachel Green2
© 2000 Nature America Inc. • http://structbio.nature.com
Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction.
These ribosomal structures show often unusual folds of ribosomal RNAs and proteins, and provide molecular
explanations for fundamental aspects of translation. In the 50S structure, the active site for peptide bond
formation was localized and found to consist of RNA. The ribosome is thus a ribozyme. In the 30S structures,
tRNA binding sites were located, and molecular mechanisms for ribosomal fidelity were proposed. The 30S
subunit particle has three globular domains, and relative movements of these domains may be required for
translocation of the ribosome during protein synthesis. The structures are consistent with and rationalize
decades of biochemical analysis of translation and usher in a molecular age in understanding the ribosome.
The ribosome is the ribonucleoprotein particle that performs
protein synthesis using a messenger RNA template. The ribosome (a 70S particle in prokaryotes) is composed of two subunits. The small subunit (30S) mediates proper pairing between
transfer RNA (tRNA) adaptors and the messenger RNA, whereas
the large subunit (50S) orients the 3′ ends of the aminoacyl (Asite) and peptidyl (P-site) tRNAs and catalyzes peptide bond formation. The ribosome must translocate directionally along
mRNA in 3 nucleotide steps to read the sequential codons. Thus,
the ribosome is both an enzyme and a molecular motor1 (Fig. 1).
The essential features of ribosome function have been dissected during 40 years of genetic, biochemical, and biophysical
analysis. The current view ascribes the primary functions in the
ribosome to its RNA components, the 16S and 23S rRNAs
(reviewed in ref. 2), though definitive proof that the ribosome is
a ribozyme has remained elusive. Electron microscopy (EM) has
delineated much of the global information about ribosome morphology. Recent advances in cryoelectron microscopy and single-particle image analysis have resulted in substantial
improvements in resolution3. Electron mictroscopy has not provided atomic-level information, yet it is a powerful tool for
unraveling the various functional states of the ribosome4.
Since the discovery that well-diffracting crystals of the large
ribosomal subunit could be formed5, crystallographers have
envisioned obtaining the ribosome structure at atomic resolution. A year ago, several groups reported structures of the 30S
and 50S subunits at ∼4.5–5.5 Å resolution6–8. A structure of the
70S ribosome with tRNAs substrates bound was also published
at 7.8 Å resolution9. Now, marking one of the most stunning
achievements of structural biology, atomic resolution structures
of 30S and 50S subunits have been determined (∼3 Å)10–14. These
massive structures (~0.9 and 1.7 MDa, respectively) provide a
sumptuous feast of RNA and protein structures, more than
quintupling the database of known RNA–protein complexes,
and open the door to atomic-level discussions of the mechanism
of translation.
The development of high-intensity synchrotron radiation
sources and improved computational ability were essential to
solving these large structures (discussed in detail in ref. 13).
Harnessing these developments through traditional isomorphous replacement and anomalous scattering methods, the
investigators used good old-fashioned hard work to solve these
Fig. 1 Schematic of the morphology of the 70S ribosomal particle as
revealed by electron microscopy55. The 30S subunit (front) and 50S subunit (back) are shown, as are gross features described in the text. The
ribosome is depicted as bound to mRNA and tRNA ligands. States of
tRNA on the ribosome, based on the hybrid states model of Noller and
colleagues56, are indicated (taken from ref. 56 and modified). The A/T
state refers to tRNA bound to the A site on the 30S subunit and to EF-Tu,
the A/A state is tRNA bound to the A site on both subunits, the P/P state
is tRNA bound to the P site on both subunits. Following peptide bond
formation, deacylated P-site tRNA moves spontaneously to the P/E state
where the anticodon interacts in the 30S subunit P site and the 3′ end of
the tRNA in the 50S subunit E site.
breakthrough structures. The structure of the Haloarcula marismortui 50S subunit was solved by Steitz, Moore and coworkers to
a resolution of 2.4 Å10,11, and the structure of the Thermus thermophilus 30S subunit was solved by Ramakrishnan and coworkers13,14 and Yonath, Franceschi and coworkers12 to resolutions of
3.0 Å and 3.3 Å, respectively. (Figs 2–5) Each of these structures
allows almost complete tracing of the RNA and peptide chains.
The large subunit
The 50S subunit of H. marismortui contains two RNA chains, the
23S rRNA (2,923 nts) and 5S rRNA (122 nts), and 31 proteins. As
a testimony to the quality of the structural data, the investigators
had to perform protein sequence analysis using their electron
density, since only 28 proteins had previously been sequenced.
Though H. marismortui is an archaebacterium and a halophile,
the similarities between this ribosome and that of Escherichia coli
are extensive, readily allowing functional and structural linkage
to the vast E. coli literature. Steitz, Moore and coworkers identify
1Department of Structural Biology, Stanford University School of Medicine, Stanford, California 21205, 94305-5126, USA. 2Howard Hughes Medical Institute and
Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Correspondence should be addressed to J.D.P. email: [email protected] and R.G. email: [email protected]
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Fig. 2 Three-dimensional structures of
the H. marismortui 50S subunit (top) and
the T. thermophilus 30S subunits (bottom). The RNA chains are shown in tan
for both subunits; 50S subunit proteins
are depicted in orange and 30S subunit
proteins in blue. Rendering of proteins in
the 50S subunit was achieved by back calculating the positions of amide and carboxyl atoms based on the Cα coordinates
(PDB code 1FFK). This procedure was performed by Michael Levitt. (Top) Two
views of the 50S subunit showing the
face that interacts with the small subunit
(left) and the solvent-accessible face
(right). The positions of the missing L1
and L7/L12 stalks are indicated. On the
subunit face, the long RNA ridge in
domain IV of 23S rRNA that forms the lip
of the active site cleft is indicated.
(Bottom) Two views of the 30S subunit,
showing the face that interacts with the
large subunit (left) and the solvent-accessible face (right). On the subunit face, the
long RNA helix 44 is indicated. In both
subunits, the interface region is composed mainly of RNA, whereas proteins
uniformly distribute over the solvent
exposed surface of both subunits.
most of the rRNA (2,711 of 2,923 nucleotides in 23S RNA and all
of 5S RNA) and 27 proteins in their electron density. No clear
density was observed for proteins L1, L10, L11 and L12 at 2.4 Å
resolution or for the rRNA regions that interact with these proteins. In the traditional EM-defined ‘crown’ view of the 50S particle (Fig. 1), seen from the surface that interacts with the 30S
subunit, it is known that protein L1 constitutes the left hand
point of the crown and L10, L11 and L12 form the majority of
the right-hand point of the crown (Fig. 2). These proteins have
been crystallized in isolation and were visible in lower resolution
structures6,9. Either the loss of these proteins during purification/crystallization or residual dynamics of these domains has
left these functionally interesting regions of the subunit disordered.
The 50S subunit is ~250 Å in diameter and is roughly isometric in mass distribution. The face that interacts with the 30S subunit is somewhat flattened, except for a deep cleft that lies behind
an rRNA ridge that runs longitudinally across the equator of the
50S subunit. This cleft is of sufficient size to accommodate the 3′
acceptor arms of three tRNA molecules (the aminoacyl, peptidyl
and exit) and contains the active site for peptide bond formation
(Fig. 3). Exiting downward from the center of this cleft is a tunnel roughly 100 Å in length and 15 Å in diameter through which
the polypeptide products of translation are released.
A striking feature of the 50S subunit is the relative distribution
of proteins and nucleic acid within the particle. The 23S rRNA
represents a majority (∼75%) of the 50S subunit mass and its
globular fold within the particle forms the bulk of its shape and
856
overall architecture. Although secondary structure maps of 23S
rRNA divide the rRNA chain into six domains (Domains I–VI)
that emanate from a central loop, the structure of the subunit is
remarkably uniform (Fig. 2). Ribosomal proteins are largely
found on the edges of the subunit–subunit face, and on the backside, which is solvent exposed in the 70S particle. Indeed, the proteins seem to distribute evenly along the periphery of the particle,
forming a protein lattice on which the rRNA structure forms (Fig.
2). With the exception of proteins L1, L10, L11 and L12 that form
the tips of the 50S subunit’s protuberances, the proteins do not
extend significantly beyond the particle envelope defined by
rRNA.
The surface that interacts with the 30S subunit and the active
site cleft where tRNAs bind are conspicuously devoid of proteins.
The authors take note of this and point out that these regions of
the 50S subunit also contain the most highly conserved
sequences of rRNA. Thus, the structure of the 50S subunit lends
the most substantial evidence to date that rRNA plays the dominant role in the function of the ribosome. Domain IV of 23S
rRNA forms the ridge of the cleft (Figs 2, 3) and the bulk of the
subunit interface as previously predicted from biochemical
experiments15–17. Domain V of 23S rRNA forms the active site
cleft (see below) below and behind the domain IV ridge, also in
agreement with a sea of biochemical data (reviewed in ref. 2).
Although RNA structure forms the core of the active site cleft,
this region of the ribosome does not appear to fold independently of ribosomal proteins. Throughout the particle, proteins
appear intimately intertwined with the rRNA. More than a third
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Fig. 3 Side views of the 50S and 30S
subunits. The subunit interfaces of
both particles are flat. A tRNA molecule, drawn to scale, is positioned with
its 3′ CCA-end pointed towards the
active site cleft of the 50S subunit and
its anticodon stem-loop directed
towards the decoding site of the 30S
subunit. Landmark features of each
particle are indicated.
of the nucleotides in 23S rRNA
make van der Waals contact with a
ribosomal protein.
The 50S subunit represents a
true collaboration between RNA
and proteins; the physical properties of the components create
something that neither could
achieve on its own. All large subunit proteins except L12 interact
directly with RNA. Most (23/29)
RNA-binding proteins interact
with more than one domain of 23S rRNA, helping to create the
compact structure of the 50S subunit. The protein structures
observed in both the 50S and 30S subunits are perhaps the most
novel feature of the ribosome structures. Nearly half (13/30) of
the large subunit proteins have unusual, extended structural
domains that snake through regions of compact rRNA structure
to stabilize interhelical contacts between RNA domains. Buried
extensions of proteins L2 and L3 reach deep into the compact
rRNA core (Fig. 4). These protein moieties are among those that
most closely approach the active site and thus have the greatest
potential to stabilize its structure. These data are remarkably
consistent with previous protein extraction and reconstitution
experiments18–20. The extended protein structures beautifully
highlight the interdependence of the rRNA and proteins in this
ribonucleoprotein assembly.
The ribosome structure explains how 23S rRNA, whose secondary structure had been predicted by phylogenetic comparisons21, folds within the 50S subunit. Phylogenetically conserved
residues either stabilize tertiary folds of rRNA, mediate sequencespecific contacts to proteins or reside on surface regions of the
ribosome for direct interaction with tRNA ligands or the 30S subunit. The global architecture of the rRNA is mediated by the now
standard array of tertiary interactions, including pseudoknots,
base triples, tetraloop–receptor interactions, and ribose zippers.
Adenosine-mediated interactions, first observed in the P4-P6
domain crystal structure of the group I intron22, are essential for
close packing of the RNA elements. The disproportionate number of adenosines in bulge and loop regions of rRNA likely reflects
the unusual contribution of this nucleotide to helix packing.
The structure of the 50S subunit will also be valuable to protein folding and protein secretion enthusiasts. Beyond just synthesizing proteins, the ribosome plays a role in maturing
peptides as they are made. The peptide exit tunnel of the 50S
subunit is lined with both rRNA and protein (with few
hydrophobic patches), and as many as 50 of the newly synthesized amino acids of a polypeptide are contained in the tunnel
before they become solvent and chaperone accessible. The
polypeptide tunnel with conspicuous constrictions, bends, and
irregularities may play an intimate role in the protein folding
process. The structure also provides a physical map of the specifnature structural biology • volume 7 number 10 • october 2000
ic protein and RNA components of the ribosome that may be
involved in protein folding and secretion which may be compared with existing biochemical data23, 24.
In the end, the finding of Steitz, Moore and coworkers that will
reverberate through all areas of molecular biology far into the
future is the demonstration that the active site for peptide bond
formation by the 50S subunit is composed solely of RNA (Figs 4,
5). While the notion that the ribosome is a ribozyme has been
increasing in popularity for many years, Steitz, Moore and
coworkers prove this point beyond reasonable doubt by presenting two supporting crystal structures of the 50S subunit with
bound tRNA substrate analogs. One analog consists of a short
helix (minihelix) that mimics the acceptor stem of tRNA with a
terminal puromycin residue instead of adenosine. Puromycin is
an aminoacyl tRNA analog equivalent to the terminal adenosine
(A76) of tRNA linked through a 3′ amide linkage to a methoxytyrosine residue. A second analog is a transition-state analog of
peptide bond formation first developed by Yarus and coworkers25.
This inhibitor contains 3′-terminal CCdA (representing P-site
tRNA) covalently attached to the amino group of the puromycin
(mimicking A-site tRNA) amino acid through a tetravalent phosphate linkage. The phosphoramide group mimics the tetrahedral
geometry and the developing negative charge on the carbonyl of
the presumed transition state for peptidyl transfer. The inhibitor
binds with increased affinity to 50S subunits relative to the individual substrate components, consistent with it representing at
least certain chemical properties of the transition state, though
the affinity is lower than that predicted for the transition state,
suggesting limitations to the mimicry25.
The substrate analogs soaked into the 50S crystal lattice interact with residues of domain V within the active site cleft, near the
entrance to the peptide exit tunnel. Neither substrate dramatically perturbs the structure of the 50S particle, except that
nucleotide A2637 (A2602 in E. coli) acquires interpretable density as a result of binding the Yarus analog. The puromycin residue
of the transition state analog superimposes on that of the minihelix bound to the A site, allowing the authors to create a model
depicting the interaction of the 3′ ends of both tRNAs with the
ribosome simultaneously. The active site cleft consists entirely of
domain V of 23S rRNA. None of the 30 large subunit proteins
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Fig. 4 Global and close-up views of the A and P sites on the 50S subunit.
The four proteins of the large subunit L2, L3, L4 and L10e, which contain
long extended regions that penetrate closest to the active site of the particle, are indicated in red. Nucleotides of the large subunit present in the
structure that are protected from chemical probes by the terminal
residues of A- and P-site tRNA are indicated in green and pink, respectively26. In the upper panel, A and P loop structures are rendered with
color as well for purpose of clarity28,29. A, P and E sites spatially distribute
from right to left as indicated; tRNAs move sequentially through A,
P and E sites in the translation elongation cycle.
vent inaccessible phosphate on A2485 (A2450 in E. coli) might be
sufficient to shift the pKa of the N3 of this residue from a normal
pKa of about 1.5. Shifted pKa values for adenosine N1 and cytosine
N3 have been observed in other RNA structures, and are thought
to result from coupling of favorable electrostatic and other noncovalent interactions upon protonation30–32. Such a proposed
mechanism for perturbing the pKa of A2486 has similarity to the
buried carboxylate of Asp102 in chymotrypsin that enhances the
nucleophilicity of the active site serine.
In an accompanying study, Strobel and coworkers independently demonstrate that the pKa of A2451 in E. coli (A2486 in
H. marismortui) is substantially perturbed33. Using dimethylsulfate (DMS) as a protonation-sensitive probe, A2451 was found to
be the only accessible adenosine in the 50S subunit with a perturbed pKa. Their results indicate that some group on A2451 (N1
or N3) titrates at pH 7.6, far from the measured pKa values of 3.5
and 1.5.
How then might such a nucleotide with a perturbed pKa and
the other active site residues conspire in catalysis of peptide bond
formation? The authors propose a mechanism whereby A2486
come within 18 Å of the phosphate group representing the tetrahedral intermediate and the catalytic center of the 50S subunit.
The structure clearly shows that the ribosome is an RNA enzyme
with proteins stabilizing the RNA structure of the active site.
Both tRNA analogs interact with nucleotides biochemically
identified as P-site or A-site binding determinants26,27. The C74
and C75 residues of P-site tRNA of the analog developed by
Yarus form Watson-Crick base pairs with two guanosines, G2285
and G2284 (G2252 and G2251 in E. coli) in the P-loop whereas
C75 of A-site tRNA forms a Watson-Crick pair with G2588
(G2553 in E. coli) in the A-loop (Fig. 6). These interactions with
the P-loop and A-loop had been predicted based on biochemical
and in vitro complementation analysis28,29. Interaction with the
two recognition loops positions the tetrahedral phosphoramide
within a complex RNA structure formed by the central loop of
domain V RNA and its peripheral helices. Thus, nucleotides
from the A-loop, the P-loop, the 2600 helix (E. coli) and the central ring of domain V are juxtaposed together above the opening
of the peptide exit channel — these nucleotides define the active
site (Figs 4, 6).
The resulting structure places the phosphoramide oxygen of the
tetravalent phosphate of the transition state analog within hydrogen bond distance of the N3 of A2486 (A2451 in E. coli). To form
this hydrogen bond in the crystals at pH 5.8, one of these atoms
must be protonated. The authors propose that an extensive network of hydrogen bonding interactions of A2486 coupled to a solFig. 5 Global and close-up views of the A and P sites on the 30S subunit13.
Protein S12, functionally important to tRNA selection at the
A site, is rendered in blue. Nucleotides of the small subunit present in the
structure that are protected from chemical probes by A and P site tRNA
are indicated in green and pink, respectively57. In the global view, the
mRNA-like oligonucleotide seen in the crystal structure reported by
Ramakrishnan et al. is rendered in P-site color as well.
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Fig. 6 Schematics of the active sites for a, decoding on the 30S subunit
and b, peptide bond formation on the 50S subunit showing molecular
interactions known to occur during these processes. a, A1492 and A1493
in helix 44 of 16S rRNA read the shape of the codon–anticodon helix
(blue) formed by the A-site tRNA–mRNA complex (taken from ref. 54).
b, The peptidyl transferase center of the 50S subunit showing the modeled interactions with the Yarus transition state analog. P-site tRNA
residues C74 and C75 (red) form base pairs with G2251 and G2252 (E. coli)
in the P-loop, and A-site tRNA residue C75 forms a base pair with G2553
(E. coli) of the A-loop. The line traces the phosphodiester backbone of
the modeled transition state analog that links P-site and A-site tRNA; the
tetrahedral phosphoramide group is shown as a red sphere. A2451 (E.
coli) is in closest proximity to this charged group and may directly participate in the catalysis of peptide bond formation. A2602 wedges between
the P and A site substrates.
(A2451 in E. coli) acts as a general base, abstracting a proton
from the amino group that attacks the aminoacyl ester bond of
the P-site tRNA. No bound divalent ions, critical for many
ribozymes that perform phosphodiester chemistry34, are
observed in the active site. However, a potassium ion is found
near the active site interacting with G2102 (G2061 in E.coli) and
G2482 (G2447 in E.coli); both of these residues directly contact
A2486 (A2451). Both groups assert that the collective environment surrounding A2486 (A2451 in E. coli), which may include
charge relay mechanisms and tautomeric shifting of bases, ultimately gives rise to a nucleotide empowered to catalyze the peptide bond forming reaction in the 50S subunit.
The atomic resolution structure and biochemical experiments
are compelling. The interactions of the CCA-moieties of the
tRNA substrate analogs with the P- and A-loops were predicted
by biochemical and genetic studies28, 29 and thus serve to validate
the placement of the analog. These constraints in turn limit
potential error in positioning of the phosphoramide linkage and
the surrounding active site nucleotides. The phenomenal overlap
of nucleotides identified by chemical modification analysis as
being protected by bound tRNA26, 35 and the nucleotides found in
the active site by crystallography cannot be coincidental. The
structure must be relevant.
On a more conservative note, several issues should be considered. The crystals may not be found in a catalytically active conformation and molecular movement (subtle or less subtle) may
be required for peptide bond formation to occur. As the reaction
is considerably less energetically demanding than proteolysis,
orientation of the activated substrates of this reaction may be
sufficient to account for the observed rates of translation. Or,
deprotonation by A2486 (H. marismortui) of the protonated
form of the amino acid (NH3+), as observed bound to EF-Tu36,
may well be sufficient to promote nucleophilic attack.
Determination of the precise mechanism of peptidyl transferase
and the relative contributions made by various active site components to the reaction energetics will require rigorous enzymology coupled with further structural analyses.
The small subunit
The genetic code is deciphered on the 30S subunit where tRNA
anticodons pair with mRNA codons. Two groups, Yonath and
coworkers12 and Ramakrishnan and coworkers13,14 present the
structure of the Thermus thermophilus 30S subunit. Although
the Ramakrishnan group has data at a slightly better resolution,
the structures are globally similar. The small ribosomal subunit
contains a single RNA chain of 1,518 nucleotides (16S rRNA)
and 20 proteins. The 30S particle has a distinctly different global
structure than the 50S subunit (Figs 2, 3). Whereas the 50S particle is thick and monolithic, the 30S particle is thin and flexible.
nature structural biology • volume 7 number 10 • october 2000
a
b
The particle is divided into three domains which each contain
one of the principle domains of 16S rRNA observed in the secondary structure. The head, body and platform of the 30S subunit, terms that originate from EM reconstructions of 30S
particles, are composed of the 3′ major, 5′ and central domains of
16S rRNA secondary structure, respectively. The 3′ minor
domain of 16SrRNA forms an extended helix (H44 in the E. coli
helix numbering system) that runs down the long axis of the 30S
subunit surface that interacts with the 50S subunit. All four
domains of the 30S particle join at a narrow neck region (also
EM derived nomenclature).
The general organization of RNA and protein within the 30S
subunit is similar to that observed in the 50S subunit. Small subunit proteins cluster on the solvent-exposed surface of the particle and are largely absent from the surface that interacts with the
50S particle (Fig. 2). The exception to the rule is protein S12,
which is found at the subunit interface bound to several functionally important components of the 30S particle. In contrast to
the 50S structure, the proteins in the more globular, flexible 30S
structure do not penetrate as deeply into 16S rRNA. While many
of the small subunit proteins do contain extended domains that
interact intimately with the rRNA, the more flexible nature of the
30S structure eliminates the need for proteins that would compact the rRNA domains into a monolithic sphere. The small subunit also contains a short 26 residue peptide called Thx, which
helps to stabilize the structure of the head domain. Similar to the
interactions seen in the 50S structure, RNA tertiary structure is
often stabilized by protein interactions, as well as by the standard
RNA–RNA interactions discussed above. Indeed, Ramakrishnan
and coworkers13,14 take note of the prevalence of adenosinemediated helix packing interactions within the 30S subunit and
make an effort to catalog them.
A major function of the 30S subunit is decoding of mRNA, a
process by which a tRNA is properly matched to the codon within
the A site of the ribosome. Although neither 30S structure contains tRNAs or mRNA ligands, they provide insights into how
tRNA–mRNA complexes are recognized by the 30S subunit.
Ramakrishnan’s group reports that intersubunit interactions are
observed in the crystal lattice that place the ‘spur’, an element that
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EF-Tu
ternary complex
Aminoglycosides
aa
GTP
A. a
E
cC.
b
B.
P A
aa
GTP
aa
GTP
50S
30S
P/P
P/P
T
tetracyclines
D.
E.
d
e
aa
Chloramphenicol,
(macrolides)
Streptogramin A
Sparsomycin
Puromycin
F.
f
aa
GDP
Pi
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OH
peptidyl
transfer
GDP
P/P A/T
P/P A/T
P/P A/A
P/E A/P
Fusidic acid
Fig. 7 The ribosome is a molecular machine. The hybrid
states model for translation elongation56. A, P and E sites
on the 30S and 50S subunits are indicated schematically.
a, Initially the P site is filled with peptidyl tRNA and the A
site is unoccupied. b, Aminoacyl-tRNA binds to the A site
as a ternary complex with EF-Tu–GTP in a codon-independent manner. c, Subsequently, the anticodon of the
ternary complex tRNA interacts with the A-site mRNA
codon on the 30S subunit d, GTP is hydrolyzed and EF-Tu
dissociates from the 3′ CCA-end of tRNA and the ribosome e, allowing the acceptor end to engage in the A
site. f, Peptide bond formation occurs rapidly, transferring the peptide chain to the A-site tRNA and the acceptor end of P-site tRNA spontaneously moves to the E-site
(P/E state) and the A-site tRNA moves to the P-site (A/P*
state). g, Elongation factor G (EF-G) binds to the ribosome
as a complex with GTP. h, GTP hydrolysis leads to a transition state for translocation where the ribosome disengages from the tRNA-mRNA complexes to allow
movement of the anticodons of the P- and A-site tRNAs.
i, This movement results in full tRNA occupation of the
E and P-sites, and an empty A site, ready to translate the
next codon.
Aminoglycosides
Thiostrepton
tRNA–mRNA complex binds within a wider and
shallower site on the 30S subunit than the P site14.
G. g
H.h
I.
i
translocation
The interactions with the ribosome are much less
EF-G
GTP
extensive than in the P site (Fig. 5), consistent with
OH
OH
GDP
OH
GTP
biochemical data, and the lower affinity of A-site
tRNA–mRNA complexes for the ribosome40.
Pi
GDP
Ribosomal components of the A-site include portions of the 530 loop, Helix 34 in the head, and
P/E A/P
P/E A/P
E P/P
A1492/93 at the base of the long penultimate stem.
All of these regions of 16S rRNA had been previousjuts out of the 30S particle (corresponding to Helix 6, nts 61–106 ly implicated in A site function by biochemical experiments39.
in E. coli), into the P site of the neighboring subunit. There is also Again, it is striking how well biochemical experiments identified
electron density for mRNA in the P site. This density may derive the critical components of the A site. Only protein S12, which
from the very 3′ end of 16S rRNA which is not observed in the has been extensively implicated in decoding by classic genetic
structure. Based on this P-site position, the authors modeled the experiments42,43, may directly participate in contacting the A-site
positions of adjacent A-site and exit site (E-site) tRNAs. Yonath tRNA–mRNA complex. In contrast, the E site is composed mainand coworkers use the prior crystal structure of functional com- ly of proteins, including S7 and S11.
The 30S subunit participates actively in tRNA selection. A
plexes9 to arrive at similar tRNA positions. In contrast to the large
subunit active site, these tRNA binding sites on the 30S subunit are large body of evidence suggests that there is an active site for
composed of elements from more than one structural domain. decoding, in which the 30S subunit discriminates correct from
Movement of the domains, mediated by tRNA interactions, pro- incorrect tRNAs in the A site. Once a tRNA is selected and peptein factors, and GTP hydrolysis, is likely an essential feature of 30S tide bond formation has occurred, the A-site tRNA must translofunction during decoding and translocation. Yonath and cowork- cate into the P site. The structures of the 30S subunit provide
ers propose that the interdomain closure of the head and body of positions for the A- P- and E-site tRNAs but do not immediately
the 30S subunit latches the mRNA onto the ribosome.
reveal how either tRNA selection or the translocation process
Using the spur interaction as a model, Ramakrishnan and occurs. The tRNA anticodons bind within a cleft that forms
coworkers13,14 propose details of P-site tRNA interaction with the between the individual domains, and relative movements of
30S subunit (Fig. 5). The P-site tRNA interacts with the 30S sub- these domains are likely involved in both decoding and translounit mainly along the minor groove of its anticodon helix. The cation. Antibiotics that target the small subunit interfere with
interactions occur with nucleotides 1338–1341 and 1229–1230 these processes, and provide a means to access functional infor(E. coli numbering) in the 3′ major domain, as well as with por- mation on the 30S subunit.
tions of proteins S13 and S9. A loop residue of the spur, equivaThe ribosome is the target of many clinically important antibilent to position 34 of a P-site anticodon, stacks upon C1400, the otics44 (Fig. 7). The small subunit is the binding site for aminosite of a high efficiency UV crosslink between a P-site tRNA anti- glycosides such as paromomycin, gentamicin, streptomycin and
codon and 16S rRNA37. The regions observed in contact with the spectinomycin, as well as the tetracyclines45. These antibiotics
P site mimic agree with prior biochemical studies of the P site38,39. interfere with critical functions of the ribosome, and thus their
The extensive 30S subunit–P-site tRNA interaction is consistent mode of action sheds light on ribosome function. Spectinomycin
with the high affinity of the P site40 and with hydroxyl radical pro- binds near Helix 34 of 16S rRNA and interferes with EF-G functection experiments41. This makes sense intuitively since the P site tion during translocation. The aminoglycosides paromomycin
must hold on tightly to the peptidyl-tRNA while decoding and and streptomycin bind to the 30S subunit near the decoding site
peptide bond formation are taking place. Dissociation of the P- in distinct areas; in fact, their binding is cooperative46. In their
presence, there is a decrease in fidelity of translation47,48. The
site tRNA would have drastic consequences.
Fidelity of translation in the A site requires specificity of tRNA affinity of tRNA for the A site is increased by aminoglycoside
selection over simple binding affinity. The modeled A-site binding49 and the rate constants for kinetic steps of tRNA selecViomycin
860
nature structural biology • volume 7 number 10 • october 2000
© 2000 Nature America Inc. • http://structbio.nature.com
© 2000 Nature America Inc. • http://structbio.nature.com
review
tion are affected50. NMR studies on the RNA binding site for
paromomycin in the presence and absence of the antibiotic
revealed how aminoglycosides bind to rRNA51. The structure
suggested that the drugs stabilize the local conformation of
A1492 and A1493, which are involved in decoding. However, the
global implications of drug binding to the 30S subunit could not
be determined by NMR.
Ramakrishnan and coworkers have solved the structure of the
30S subunit with spectinomycin, paromomycin and streptomycin simultaneously bound14. The binding sites found for all
three antibiotics agree with prior biochemical and genetic studies. Spectinomycin binds to Helix 34, and is near protein S5 in
which resistance mutations are located52. Binding of the drug
may stabilize the RNA conformation of the head, and prevent
movement of the head domain that may occur during translocation. Streptomycin binds near the decoding site, and links four
regions of rRNA involved in A-site tRNA binding: Helix 44,
Helix 1, Helix 27, and the 530 loop. In addition, protein S12
directly interacts with the antibiotic, consistent with the high
level streptomycin resistance that can occur upon mutation of
S12. Streptomycin stabilizes the structure of RNA around the
decoding site. In particular, the base pairing of H27 is in a configuration that leads to miscoding (ram mutations, for ribosomal
ambiguity mutations)53. In this conformation, H27 docks tightly
into the long penultimate stem (H44), and this docking likely
affects the conformation of the A site. Paromomycin in the
three-drug complex binds in the major groove of H44, as
observed by NMR51. Upon binding, A1492 and A1493 are extensively flipped out into the minor groove. Apparently, antibiotics
that act on the 30S subunit affect ribosome function by stabilizing particular conformations of ribosomal RNA.
The ribosome must distinguish correct versus incorrect
codon–anticodon pairs by structure-specific, sequence independent interactions. Based on NMR and biochemical studies,
Yoshizawa et al.54 proposed that the mRNA backbone was contacted by A1492 and A1493. Ramakrishnan and coworkers13,14 propose
a similar molecular basis for ribosomal decoding. The complex of
the 30S subunit with the antibiotic represents a high affinity form
for tRNA–mRNA interaction in the A site. The flipped-out bases of
A1492 and A1493 would be positioned within the minor groove of
the A-site codon-anticodon helix. A reasonable hypothesis is that
A1492 and A1493 sense the width of the minor groove, which
would be distorted in non-cognate codon–anticodon pairs (Fig. 6).
As suggested by NMR, paromomycin induces miscoding by stabilizing the flipped-out conformation of A1492 and A1493. The 30S
subunit structure suggests that these nucleotides may represent
some of the moving parts of this molecular machine.
Conclusions
The structures of the 30S and 50S subunits are solved. As anticipated, the ribosome is composed of active site clefts, catalytic
centers and channels. Most importantly, the ribosome is a
ribozyme. This is an amazing and satisfying result.
Decades of biochemistry and genetics can now be interpreted
in atomic detail. Remarkably, these approaches identified the
vast majority of the players in the active sites. As a general rule in
the ribosome, and perhaps in other RNPs, nucleotides that are
accessible to base-specific chemical probes have a high probability of being involved in critical functions — indeed, the likelihood improves if highly accessible and highly conserved
nucleotides are considered.
The euphoria of these results yields to a more sober reality.
There are still many unanswered questions about the mechanature structural biology • volume 7 number 10 • october 2000
nism of translation. How do the large and small subunits communicate between their ‘active sites’ and how do they interact
with factors to promote the directional movements of translocation? The visceral power of structure can be overwhelming.
However, the ribosome is a dynamic engine (Fig. 7), and we
must now reconcile the static structures solved by crystallography with the multiple states that the ribosome must sample
during the translational cycle. The answers to these questions
will come with further mechanistic and structural studies. But
the field of translation is now permanently changed.
Hypotheses about ribosome function must now be framed on
the molecular level.
Acknowledgments
The authors thank E. Viani Puglisi and C. Merryman for useful discussions,
M. Levitt for assistance generating full backbone coordinates for the 50S subunit
proteins, and V. Ramakrishnan for providing 30S subunit coordinates prior to
publication.
Received 8 September, 2000; accepted 12 September, 2000.
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