Reviews
K. H. Nierhaus and D. N. Wilson
Ribosome Structure and Translation
The Ribosome through the Looking Glass
Daniel N. Wilson and Knud H. Nierhaus*
Keywords:
amino acids · proteins · ribosomes ·
RNA · translation
Angewandte
Chemie
3464
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200200544
Angew. Chem. Int. Ed. 2003, 42, 3464 – 3486
Angewandte
Chemie
Ribosome Structure and Translation
For almost 20 years crystallographers have sought to solve the
From the Contents
structure of the ribosome, the largest and most complicated RNA–
protein complex in the cell. All ribosomes are composed of a large and
small subunit which for the humble bacterial ribosome comprise more
than 4000 ribonucleotides, 54 different proteins, and have a molecular
mass totaling over 2.5 million Daltons. The past few years have seen
the resolution of structures at the atomic level for both large and small
subunits and of the complete 70S ribosome from Thermus thermophilus at a resolution of 5.5-+. Soaking of small ligands (such as
antibiotics, substrate analogues, and small translational factors) into
the crystals of the subunits has revolutionized our understanding of the
central functions of the ribosome. Coupled with the power of cryoelectron microscopic studies of translation complexes, a collection of
snap-shots is accumulating, which can be assembled to create a likely
motion picture of the bacterial ribosome during translation. Recent
analyses show yeast ribosomes have a remarkable structural similarity
to bacterial ribosomes. This Review aims to follow the bacterial
ribosome through each sequential “frame” of the translation cycle,
emphasizing at each point the features that are found in all organisms.
1. The Wonders of the Translational World
The ribosome is a translator. It uses the information
contained in messenger RNA (mRNA) to produce the
corresponding sequence of amino acids, thus linking the
worlds of nucleic acids (DNA and RNA) and proteins. It does
this by providing the platform on which each codon of the
mRNA is matched with the amino acid it encodes. The
physical link between the worlds of RNA and protein is the
pool of transfer RNAs (tRNAs). One end of each tRNA
species, the anticodon, is complementary to the codon of the
mRNA while the other end, termed the CCA end, is
covalently attached to the amino acid specific for that
codon. The correct aminoacylation of tRNAs with the
appropriate amino acid is equally important for ensuring
the fidelity of translation. This task is performed by synthetases, such that for each of the 20 amino acids there is a
correponding synthetase, which recognizes on the one hand
the amino acids and on the other hand all tRNAs which
decode this amino acid. The job of every ribosome is to ensure
that the mRNA is read in the correct frame and that each
tRNA faithfully follows the code. The ribosome performs this
process with amazing accuracy and at high speed: 10–
20 amino acids are incorporated per second into the growing
nascent chain, with only one error in every 3000 codons
deciphered. To understand how the ribosome achieves this
feat, an understanding of the overall structure of the ribosome
will be necessary.
Angew. Chem. Int. Ed. 2003, 42, 3464 – 3486
1. The Wonders of the
Translational World
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2. The Path of the tRNA through
the Ribosome
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3. Summary and Outlook
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1.1. Common Structural Features of the
Ribosome
All ribosomes are composed of two
subunits of unequal size. Bacterial
ribosomes have a relative sedimentation rate of 70S and can be separated
into a large 50S subunit and a small 30S
subunit. Eukaryotic ribosomes are
larger:
Saccharomyces
cerevisiae
(yeast) ribosomes, for example, sediment at 80S and are separable into 60S
and 40S subunits. Each subunit of a
ribosome is a ribonucleoprotein particle. In the eubacteria Escherichia coli
one third of the mass of a ribosome consists of protein and the
other two thirds of ribosomal RNA (rRNA): The 50S subunit
contains both a 5S (120 nucleotides) and a 23S rRNA (about
2900 nucleotides), while the 30S subunit contains a single
16S rRNA (approximately 1500 nucleotides). The protein
fraction consists of 21 different proteins in the small subunit
and 33 proteins in the large subunit. Eukaryotic ribosomes
have longer rRNAs (because of the insertion of additional
sequences at specific regions termed expansion sequences
(ESs)), an additional rRNA, and 20–30 extra ribosomal
proteins, which together account for the 30 % increase in size
relative to E. coli ribosomes.
The overall three-dimensional shapes of the 70S ribosome
and its component subunits have been characterized by a
variety of electron microscopy techniques since the 1980s. The
small subunit was described anthropomorphically with a
head, connected by a neck to a body with a shoulder and a
platform (Figure 1 a). The large subunit presents a more
compact structure consisting of a rounded base with three
protuberances, termed the L1, central protuberance, and the
L7/L12 stalk (Figure 1 b). A vast improvement in the resolution was achieved by the introduction of single-particle
[*] Prof. Dr. K. H. Nierhaus, Dr. D. N. Wilson
Max-Planck-Institut f%r molekulare Genetik
Ihnestrasse 73, 14195 Berlin (Germany)
Fax: (+ 49) 30-8413-1594
E-mail: [email protected]
DOI: 10.1002/anie.200200544
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3465
Reviews
K. H. Nierhaus and D. N. Wilson
Figure 1. Comparison of the small and large ribosomal subunit from bacteria with those of a lower eukaryote. Cryo-electron microscopic reconstructions of the small (a) and large subunit (b) of the bacteria Escherichia coli are compared with the small (c) and large subunit (d) of the yeast
Saccharomyces cerevisiae and the recent high-resolution crystal structures for the small subunit from the bacteria Thermus thermophilus (e) and
large subunit from the archeon Haloarcula marismortui (f). All subunits are viewed from the interface side with the P-tRNA present in the cryo-EM
reconstructions (green). Additional masses within the yeast 80S ribosome compared with that of a bacterial ribosome are shown in dark yellow
(40S subunit) and purple (60S subunit). Landmarks of the small subunits include: b, body; bk, beak; h, head; lf, left foot; rf, right foot; pt, platform; sh, shoulder; and sp, spur. Landmarks for the large subunit: CP, central protuberance; L1, L1 protuberance; SB, stalk base; St, L7/L12
stalk; H34, helix 34; H38, helix 38; and SRL, sarcin–ricin loop. Cryo-EM images adapted from Spahn et al.[3]
reconstruction of cryo-electron microscopy images.[1] The
general structural features of the ribosome remained at
higher resolution, but more detailed structural features
appeared, such as the beak and toe or spur on the 30S subunit.
More recently, cryo-EM analysis has been used to
examine eukaryotic ribosomes and subunits.[2–5] These reconstructions show that despite the extra size, yeast and
mammalian ribosomes show extensive structural similarity
with their bacterial counterparts (Figure 1 c and d). The
differences, which stem principally from additional rRNA and
proteins in the eukaryote ribosome (shaded darker in
Figure 1 c and d), are located predominantly in the surfaces
of the subunits exposed to the cytoplasm and not to the
intersubunit surface where the subunits interact with one
another. Another feature of all ribosomes is a tunnel that
traverses the large subunit; it starts at the peptidyltransferase
(PTF) center at the interface and exits at the base or
cytoplasm side of the large subunit. The growing polypeptide
chain is believed to travel through the tunnel before exiting
into the cytosol of the cell. The tunnel has a length of
approximately 100 @ and can house between 30 and 50 amino
acid residues of the growing polypeptide chain.
In the case of proteins that are targeted to organelles and
cell compartments, such as the endoplasmic reticulum,
chloroplasts, or mitochondria, the ribosome must dock with
a pore or protein-conducting channel on the surface of the
Knud H. Nierhaus studied medicine and
completed his thesis with Prof. Klaus Betke
in Tbingen. In 1968 he joined the MPI fr
Molekulare Genetik in Berlin, where he currently leads a research group studying different aspects of translation. He is
“außerplanm-ßiger Professor” at the TU,
Berlin and “adjunct Professor” at the
Moscow State University. His main achievements include: the development of a
method to degrade the large subunit from
E. coli ribosomes and the detection of a
third tRNA binding site on the ribosome.
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Daniel N. Wilson studied Biochemistry and
Molecular Biology at Victoria University,
Wellington, New Zealand. He carried out his
PhD in the laboratory of Prof. Warren Tate
in the Biochemistry Department at the University of Otago, Dunedin, New Zealand. In
his thesis he focused on the mechanisms of
translational termination and recoding
events. Following completion of his studies
in 1999, he was awarded an Alexander von Humboldt and currently works in the laboratory of Prof. Nierhaus at the MPI fr Molekulare Genetik in Berlin.
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Ribosome Structure and Translation
organellar membrane, through which the nascent chain is
cotranslationally exported. Such a complex from yeast,
namely the 80S ribosomes of S. cerevisiae bound to the
Sec61 pore protein, a protein involved in protein transport
into the endoplasmic reticulum, has been analyzed by cryoEM. The results of these studies show that the funnel-shaped
pore sits directly over the exit of the tunnel,[4–6] thus
supporting the role of the tunnel as the conduit for the
nascent chain.
The presence of the tunnel in ribosomes of all organisms
implies its importance, yet its function still remains speculative. The tunnel has been proposed to provide an environment
suitable for the early stages of protein folding or to simply
protect the nascent chain from proteases until sufficient
amino acids have been synthesized to enable secondary
structure formation. Recently, a more active role for the
tunnel has been proposed, based on the observation of
sequence-specific recognition by ribosomal components of
nascent oligopeptides within the tunnel, which were shown to
influence both protein elongation and translation termination
(reviewed by Tenson and Ehrenberg).[7]
1.2. The Ribosome up Close
The last few years have seen the arrival of high-resolution
crystal structures of the small subunit from the thermophilic
bacterium Thermus thermophilus (3 @; Figure 1 e),[8, 9] the
large subunit from the archaebacterium Haloarcula marismortui (2.4 @; Figure 1 f),[10] and also more recently the large
subunit from the mesophilic eubacterium Deinococcus radiodurans (3.1 @).[11] A number of excellent reviews followed
shortly after, in which the implications of these structures
were discussed in regard to our extensive knowledge of the
functional aspects of protein synthesis.[12, 13]
With the arrival of high-resolution subunit structures, it
was now possible to correlate the low-resolution features with
particular rRNA helices and/or ribosomal proteins. For
example, the beak of the small subunit consists exclusively
of helix h33, the spur of h6, and the central protuberance is
made up of the 5S rRNA, part of 23S rRNA, as well as
ribosomal proteins L5, L18, L25, and L33. A more detailed
analysis of the subunit structures reveals an immediately
discernible difference in the overall assignment of the
domains of the rRNA secondary structure relative to the
tertiary domains: In the 30S subunit, the rRNA can be simply
divided into tertiary domains, with each domain corresponding with a structural landmark of the 30S subunit; for
example, the 5’-domain of the 16S RNA forms the body
from the toe to the shoulder, the middle domain forms the
platform, and the large 3’-domain the head, and finally the
small 3’-domain runs down the intersubunit surface of the 30S
subunit. In contrast, rRNA in the 50S subunit has a much
more compact interwoven secondary structure, perhaps
suggesting that the 50S subunit is older in evolutionary
terms, and has had more time to evolve such a complex
organization of the domain[14] and/or that the 30S subunit
requires more flexibility to perform its function.
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With the crystal structures of the ribosomal subunits came
20 new and complete structures for both small[8] and large[10]
subunits of ribosomal proteins. A special feature of many of
these ribosomal proteins is the presence of a globular domain,
which is usually bound to the surface of the subunit, as well as
a long filamentous extension, which penetrates deep into the
center of the ribosome, the ribosomal RNA core. Ribosomal
proteins are commonly found bound to junctions between
rRNA helices, thereby often connecting different domains. A
comprehensive analysis of the protein–rRNA interactions in
the small (30S) subunit revealed that the globular proteins
tend to bind early in the assembly process whereas the
proteins with long extensions assemble later.[15]
Perhaps one of the biggest surprises from the crystal
structures was that, despite a tenfold increase in the amount
of RNA structure known, almost all of the secondary
structure motifs had been seen before, which suggests that
the number of motifs is limited. A common feature of the
ribosome is the highly helical nature of the rRNA. Regions
that were predicted to be single-stranded loop regions
actually appear as slightly irregular double-stranded extensions of neighboring helices in the crystal structure. Furthermore, helical regions stack end-to-end to form long quasicontinuous helical structures. The proportion of adenine
residues is significantly underrepresented within these helical
or paired regions (Table 1).[16] The significance, both funcTable 1: Frequency and distribution for each ribonucleotide within
bacterial 16S and 23S rRNAs secondary structure models.[a]
Nucleotide
G
C
A
U
overall frequency
distribution within
helical regions
frequency within
unpaired regions
distribution within
unpaired regions
unpaired/paired ratio
31.4
36.6
22.4
25.7
14.5
20.5
12.5
42.6
22.3
66.2
30.1
0.43/1
0.29/1
1.96/1
41.5
0.71/1
[16]
[a] Data sourced from Gutell et al.
tional and structural, of these residues is evident from the
participation of adenine residues in the so-called “A-minor”
motif, a recurring feature of the ribosome which is important
in the stabilization of the rRNA tertiary structure.[17] In
general, the A-minor motif constitutes an interaction between
an adenine residue and the minor groove of an rRNA helix
and is not limited to ribosome structures, and have been
observed previously in Tetrahymena and hepatitis delta virus
ribozymes.[18] Four variations in this motif have been identified in the ribosome,[17] two of which, type I and II, play a
crucial role in ribosome function by being involved in both
decoding and formation of a peptide bond. Since the adenine
residues involved at both of these sites are universally
conserved, the implication is that the mechanisms of decoding
and formation of a peptide bond are also conserved between
prokaryotes and eukaryotes. The details of these interactions
are discussed more thoroughly in Sections 2.2.2 and 2.3.2,
respectively.
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K. H. Nierhaus and D. N. Wilson
2. The Path of the tRNA through the Ribosome
The “decoding site” is located on the small subunit. This is
the site where the codons of the mRNA are recognized and
deciphered by the complementary anticodon of the tRNA.
There are three sites on the ribosome that tRNAs can occupy
(A, P, and E; Figure 2). The A site is where the aminoacyl
tRNA (aa-tRNA) binds, according to the codon displayed at
this site. It is the aa-tRNA that brings in the new amino acid to
extend the growing polypeptide chain. The P site is where the
peptidyl-tRNA is bound before formation of the peptide
bond. This is the tRNA carrying the nascent polypeptide
chain. The E site is the exit site for the deacylated or
uncharged tRNA. The tRNAs move through each of the sites
sequentially during translation, starting at the A site and
passing through the P site to the E site, before leaving the
ribosome. The exception is the binding of the very first tRNA
(the initiator tRNA), which binds directly to the P site
(Figure 2 a).
Initiator tRNAs decode the start codon, usually AUG, and
carry the amino acid formylmethionine in bacteria or
methionine in eukaryotes (including archaea). The codon
following the start codon is displayed at the A site and
dictates which aa-tRNA will now bind (Figure 2 b). The aatRNAs are delivered to the A site in the form of a ternary
complex consisting of an elongation factor (EF-Tu in bacteria
and EF1a in eukaryotes), GTP, and the aa-tRNA. After GTP
hydrolysis, EF-Tu·GDP is released from the ribosome and the
aa-tRNA docks into the A site (Figure 2 c). The formation of
a peptide bond involves the transfer of the peptidyl moiety of
the P-tRNA to the aminoacyl moiety of the A-tRNA: It is
noteworthy that the whole polypeptide chain is added to the
new amino acid rather than the addition of the new amino
acid to the chain. Formation of peptide bonds occurs on the
large subunit at the PTF center. The formation of the peptide
bond had no significant change in the positions of the two
tRNAs (Figure 2 d), although the P site now contains an
uncharged tRNA and the A site contains a peptidyl-tRNA.
Transfer of the A- and P-tRNAs to the P and E sites is
termed translocation and is mediated by a second elongation
factor (EF-G in bacteria (Figure 2 e) and EF2 in eukaryotes).
In simple terms, the role of the elongation factors is to
accelerate the elongation cycle to the rate of 50 msec per
elongation cycle in vivo. The rate without elongation factors is
about four orders of magnitude slower[20] because of the high
energy barrier (120 kJ mol 1) that separates the pre- and
posttranslocational states (Figure 2 c and f, respectively) in
the E. coli ribosomes.[21] Translocation places the deacylated
tRNA at the E site and peptidyl-tRNA at the P site, thus
freeing the A site for the binding of the next aa-tRNA
(Figure 2 f). Binding of the next A-tRNA releases the EtRNA (Figure 2 g) and so the cycle repeats (that is, back to
Figure 2 c) until a stop codon appears in the A site. At this
point protein termination factors release the completed
polypeptide and dissociate the ribosome into subunits in
preparation for the next round of translation.
2.1. Initiation of Protein Synthesis: Subunit Association and
Intersubunit Bridges
Figure 2. Overview of the translation cycle. Multiple cryo-electron
microscopic studies have determined the binding positions of the
tRNA and elongation factor on the 70S ribosome during different
stages of the elongation cycle (see Ref. [19] and references therein).
The small 30S subunit is in yellow, the 50S large subunit in blue. The
positions of the ribosomal elongations factors have been overlaid onto
a 3D map of the ribosome at a resolution of 11.5 F to generate a schematic overview of the elongation cycle, the details of which are provided in the text. Adapted from Agrawal et al.[19]
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The translation of functionally active protein requires that
mRNA be positioned on the 30S subunit such that the start
codon will be read first and in the correct frame. Initiation at a
codon before or after the start codon would produce either an
extended or truncated protein, either of which may be
inactive. The placement of the start codon must also be
precise: As codons are composed of three nucleotides, this
opens the possibility of initiating translation in an incorrect
frame through selection of the incorrect first nucleotide of the
codon. Thus, the precision and specificity of the initiation
phase is crucial for cell viability.
How does the ribosome select the correct start codon and
ensure the specificity of the interaction with the initiating
tRNA? In fact, there is no single answer to this question, since
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there are so many exceptions to the rule. In general, however,
the positioning of the mRNA utilizes the untranslated region
(UTR) upstream of the start codon, which directs placement
of the mRNA on the small subunit. In bacteria, the
mechanism is relatively simple and involves a stretch of
nucleotides (the Shine–Dalgarno (SD) sequence) that interacts with a complementary sequence within the 3’ end of the
16S rRNA (the anti-SD sequence). This interaction was
directly visualized recently in the Fourier difference maps
between empty 70S ribosomes and those carrying mRNAs.[22]
The situation is much more complex in eukaryotes
because of the increased regulatory mechanisms operating
on translation. In most cases, the 5’-end of eukaryotic mRNAs
are modified with a guanine “cap” structure. This cap
structure is recognized by specific initiation factors, which
direct binding of both the mRNA and the initiator Met-tRNA
to the 40S subunit. The mRNA is then “scanned” downstream
(in the 3’ direction) until the first AUG start codon is found.
The large 60S subunit can now bind and protein synthesis
commences. In certain cases, the UTRs contain complex
secondary structures, which are recognized and bound by
large heteromeric protein complexes. Some of these protein
factors interact directly with the 40S subunit to mediate
mRNA positioning (reviewed recently by Pestova and
et al.).[23]
Despite these differences between translation initiation in
bacteria and eukaryotes, a number of distinct similarities are
also emerging.[24] For example, formation of initiation complexes in both prokaryotes and eukaryotes involves the
binding of the mRNA and initiator tRNA to the small
subunit, such that the initiator tRNA is present at the P site of
the small subunit. This is a unique situation as all subsequent
aa-tRNAs that participate in translation will enter the
ribosome through the A site. As discussed in Section 2.2.2,
it is the codon–anticodon interactions at the A site that are
monitored carefully by the ribosomes to ensure translational
fidelity. Initiation, by allowing direct P-site binding, bypasses
this important “monitoring” step.
How then does the ribosome ensure that the correct
tRNA binds the start codon and that the start codon is placed
exactly at the P site? A number of specialist initiation factors
have evolved to ensure the fidelity of P-site binding during
initiation. In bacteria, this process is mediated by three
initiation factors, IF1, IF2, and IF3 (reviewed by Gualerzi and
et al.).[25] The only ribosome complexes with protein translation factors that have been solved so far are those with
initiation factors IF1 and a domain of IF3.[26, 27]
IF3, which may be the first initiation factor to bind to the
30S subunit, displays dual functions. Firstly, by acting as an
anti-association factor, it prevents formation of the 70S
ribosome by prohibiting association of the 30S subunit with
50S subunits. Secondly, it plays an important role in codon–
anticodon discrimination in the P site. IF3 is composed of two
domains separated by a long lysine-rich linker. The Cterminal domain (CTD) is sufficient for ribosome binding
and fulfills the first function of the factor, while the Nterminal domain (NTD) has been implicated in the second
function. The crystal structure of the CTD on the 30S subunit
has been solved, which allowed the NTD to be docked into
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the structure.[27] The binding site of the CTD of IF3 suggests
that the anti-association function of this domain operates
through conformational changes, not through direct steric
obstruction as previously thought. Furthermore, the NTD is
proposed to monitor correct codon–anticodon interactions at
the P site, not through a direct interaction, but through space
restrictions, such that only correct binding orientations are
possible.[27]
Recently, footprinting experiments using IF3 led to a
contradictory model for IF3 binding on the 30S subunit which
suggested the CTD of IF3 does in fact prevent subunit
association directly.[28] This result raises the question of
whether IF3 has two binding sites on the 30S subunit: perhaps
one associated with initiation, thus checking the codon–anticodon interaction, and the other during ribosome disassembly.
Despite the single unequivocal binding site of IF1 on the
ribosome, it's exact function still remains a mystery. The small
size of IF1 (less than 10 kDa) allowed the factor to be soaked
into crystals of the 30S subunit.[26] IF1 was found to bind in the
A site which suggested that it helps prevent premature
binding of the A site to the initiator tRNA. It was however
shown that factor-free 30S subunits bind tRNA exclusively at
the P site in the presence of a suitable mRNA,[29] so this
additional verification seems unnecessary. The binding of IF1
induces long-range conformational changes within the intersubunit surface, particularly within the helix h44 of the 30S
subunit, which together with IF3 may promote association of
the subunit.[26] Bacterial IF2 binds specifically to initiator
tRNAs and directs them to the 30S subunit. Delivery of the
initiator tRNA by IF2 is enhanced by IF1. Interestingly, the
central region of eukaryotic translation initiation factor
eIF1A forms a so-called “OB fold”, as seen for IF1, and
thus may bind in an analogous fashion to the A site of the
40S subunit.[30] Homologues to IF2 have been found in
eukaryotes and archaea, eIF5B and aIF5B, respectively, and
have been shown to interact directly with eIF1A (see the
review by Pestova et al.[23]). The complex containing the
40S subunit, the initiator tRNA, initiation factors, and the
mRNA with the AUG codon at the P site is called the 43S preinitiation complex. Association of this pre-initiation complex
with the 60S subunit is stimulated by eIF5B. It is likely that
the association of the small and large subunit results in
conformational change that stimulates hydrolysis of GTP by
IF2 or eIF5B and subsequently their release from the
ribosome.
A number of contact points between the small and large
subunit have been identified in the bacterial 70S ribosome,
which are termed intersubunit bridges (Figure 3 a and b).[31, 32]
The functional importance of these intersubunit bridges is
emphasized by identification of corresponding bridges in the
eukaryotic 80S ribosome (yeast; Figure 3 c and d).[3] As well
as being important for association of the ribosomal subunits,
these bridges probably play an important role in movement of
the tRNAs through the ribosome (see Section 2.4) and in
signaling between the decoding center on the small subunit
and the PTF center on the large subunit. Closer inspection of
the bridges within bacterial and yeast ribosomes reveals that
many of the bridges, namely B2a, B3, B5a, and B5b, involve
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overextrapolated. As yet there are no ribosome crystal
complexes with elongation or termination factors. The
reason for this is probably that the large size of the translation
factors (40–80 kDa) prohibits the use of simple soaking
experiments and instead requires more complex cocrystallization experiments. Thus, most of the structural information
for these complexes comes from cryo-EM studies. In contrast,
the soaking of small ligands, such as antibiotics, into the
ribosome crystals has been very successful. The decoding site
is the target for a number of potent translation inhibitors. The
structures of antibiotics, such as tetracycline, paromomycin,
streptomycin, hygromycin, and spectinomycin, have been
solved to atomic resolution in a complex with the 30S subunit.
2.2.1. The Universal Problem of Aminoacyl-tRNA Selection
Figure 3. Comparison of bridging positions between the subunits of
the bacteria and yeast ribosomes. a, b) The 30S (blue) and 50S (gray)
ribosomal subunits of Thermus thermophilus are shown from their
intersubunit sides. The bridges are marked in red and are annotated
according to the nomenclature from Gabashvili et al.[31] Bridges B1, B2,
and B3–5 are labeled in blue, green, and orange, respectively. The
figure was adapted from Cate et al.[32] c, d): The 40S (yellow) and 60S
(blue) ribosomal subunits of Saccharomyces cerevisiae are shown from
the interface side. The bridges are labeled in red. Those common to
T. thermophilus are labeled in blue (B1), green (B2), and orange (B3–
B5), while additional intersubunit connections in the yeast ribosome
are labeled eB8–eB11 in red. Note that bridges B6 and B7 are not
included for simplicity. Adapted from Spahn et al.[3]
contacts with h44, a helix of the 30S subunit intimately
involved in decoding.
2.2. The A Site: Decoding, Mimicry, and Antibiotic Interaction
With the exception of initiation, the ribosomal A site is
the entry point for charged aa-tRNAs. Here the ribosome
must determine whether the incoming tRNA is correct with
respect to the codon of the A site or not, that is, whether it
should be accepted into the A site or rejected. The molecular
details of this “decision” have been recently revealed and
confirm a 20-year-old hypothesis.[33a] The A site also contributes to the binding site of the elongation factors. During each
stage of protein synthesis—initiation, elongation, and termination—translation factors interact with the ribosomal A site.
Structures for a number of these translation factors have been
determined. Regions of these structures display an overall
similarity with one another, but more specifically they exhibit
a remarkable similarity with the dimensions of a tRNA, the
“true” substrate of the A site. This phenomenon, in which a
protein factor mimics the A-tRNA substrate, is an example of
molecular mimicry—a reoccurring theme in the translation
(see the review by Nissen et al.[34]) that may well have been
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Following dissociation of the initiation factors, the ribosome is now primed with an aa-tRNA bound at the P site. The
codon displayed in the vacant A site is specific for a single
species of tRNA that has a perfectly complementary anticodon, the cognate tRNA. However, there are many other
tRNA competitors that can interfere with this selection
process: 41 tRNAs with different anticodons exist in the
bacterium Escherichia coli and even more in the eukaryotic
cell. To complicate matters further, three to five or six of these
tRNAs (near-cognate tRNAs) will have an anticodon similar
to the cognate tRNA. The remaining 90 % have a dissimilar
anticodon and are termed noncognate tRNAs. The problem is
compounded further when one considers that the aa-tRNAs
are delivered in the form of a ternary complex, that is, in a
complex with the elongation factor EF-Tu and GTP. The
ribosome must therefore discriminate between relatively
large ternary complexes (72 kDa), which present multiple
potential interaction sites to the ribosome, on the basis of a
small discrimination area, the anticodon (1 kDa).
The ratio of the large surface area and the small
discrimination surface defines the corresponding energy
ratio: binding is dominated by a relatively large free energy,
with only a tiny fraction corresponding to the discrimination
energy. The unusual molecular selection problem of the
ribosome consists of the fact that a large part of the binding
energy of the 41 different ternary complexes (E. coli) is
identical, and thus the discrimination is based on the tiny
fraction corresponding to the discrimination energy. The
discrimination potential of the discrimination energy can only
be reached under equilibrium conditions. In this case where
the binding energy is relatively large, the equilibrium can only
be reached after long time periods—in other words, this
process must be slow to be accurate.
Since we know that protein synthesis is a relatively fast
and accurate process, the ribosome must overcome this
hurdle. But how? A model has been proposed which overcomes this problem by simply dividing the occupation at
site A into two distinct events: a decoding step followed by an
accommodation step (see the review in Ref. [35]). During the
initial decoding step, the A site is in a low affinity state, which
reduces interaction of the ternary complex to codon–anticodon interactions, thus excluding general contacts of the
tRNA and elongation factor. By restricting the binding
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surface of the ternary complex to the discriminating feature,
that is, the anticodon, the binding energy is both small and
approximately equivalent to the discrimination energy. Since
the binding energy is small, equilibrium can be rapidly
attained, thus ensuring that the efficiency of the reaction is
retained. The second step, accommodation in the A site,
requires release of the aa-tRNA from the ternary complex.
This step utilizes the nondiscriminatory binding energy to
dock the tRNA precisely into the A site and the attached
aminoacyl residue into the PTF center on the 50S subunit in
preparation for formation of a peptide bond. As we will see
later, accommodation of the aa-tRNA in the A site is
accompanied by release of the E-tRNA. Evidently, this
second step of A-site binding involves gross conformational
changes within the ribosome[21] and thus can be thought of as a
relatively slow process relative to the decoding step. A-site
binding occurs through a coupled reaction system consisting
of a fast initial or decoding step and a slow accommodation
step. This has the important consequence that the initial
reaction operates at equilibrium even when under steadystate conditions. The complete process—the fast decoding
step with a subsequent slower accommodation step—results
in the discriminatory potential of codon–anticodon interactions being efficient and the rate of proton synthesis high.
Recently, the first step of A-site binding (low affinity
A site) was visualized by cryo-EM analysis of ternary complexes stalled the A site with the antibiotic kirromycin.[36, 37]
Although kirromycin allows GTP hydrolysis of EF-Tu, it
inhibits the associated conformational changes in EF-Tu that
are necessary for dissociation from the ribosome. The cryoEM reconstructions suggest that the anticodon stem loop
(ASL) of the tRNA is kinked to allow codon–anticodon
interaction, and thus overcomes the unfavorable incoming
angle of the tRNA to the A site as dictated by the ternary
complex (see Figure 2 b).[36]
As already indicated, the accommodation of an aa-tRNA
into the A site involves the dissociation of EF-Tu·GDP from
the ribosome, a process which is coupled with the hydrolysis
of GTP. It is interesting to note that in E. coli up to two GTPs
are hydrolyzed during the incorporation of cognate-tRNAs
and up to six GTPs during the incorporation of near-cognatetRNAs, whereas noncognate-tRNAs do not trigger EF-Tudependent GTP hydrolysis at all.[38] This observation adds
further weight to the argument that the tRNA discrimination
is governed predominantly by anticodon–codon interactions
during the initial binding step. The next question is: How are
the cognate and near-cognate tRNAs discriminated? This is a
question that can now be answered at the molecular level, as
is discussed in the next section.
2.2.2. Decoding of Aminoacyl-tRNAs
A model for the discrimination between cognate and nearcognate aa-tRNAs was proposed by Potapov about 20 years
ago.[33a] According to this model, the decoding center of the
ribosome recognizes the anticodon–codon duplex, in particular, sensing the stereochemical correctness of the partial
Watson–Crick base pairing and the positioning of the
phosphate–sugar backbone within this structure. A test of
Angew. Chem. Int. Ed. 2003, 42, 3464 – 3486
this hypothesis was performed with an mRNA that carried a
DNA codon at one of the three ribosomal sites.[33b] If the
stability of the base pairs, that is, the hydrogen bonds between
codon–anticodon bases of the Watson–Crick base pairs, is the
sole requirement for the recognition step, then a 2’-deoxy
base in the codon should not affect the decoding process. If,
however, the stereochemical correctness of the base pairing is
tested, that is, including the positioning of the sugar group,
then a 2’-deoxy base should impair the decoding process. It
was found that a deoxycodon at the A site was disastrous for
tRNA binding at this site, whereas a deoxycodon at the P site
had no effect on tRNA binding to the P site. This result was in
agreement with earlier data showing that DNA could not
perform the same function as an mRNA (see Potapov
et al.[33b] and references therein).
Recently, the components of the ribosome directly
involved in decoding were identified by crystallography at a
resolution of 3.1 @.[39] The crystal packing of the 30S subunit
of Thermus thermophilus showed that the spur (h6) of one
subunit was placed fortuitously into the P site of another, thus
mimicking the anticodon stem loop of a P-tRNA. Another
surprise was that the base-pairing partner to the P-tRNA
mimic was the 3’-end of the 16S rRNA, which extended into
the decoding center by folding back upon itself. This situation,
with the P site filled, enabled Ramakrishnan and co-workers
to then soak an ASL fragment (ASL-tRNA) and a complementary mRNA fragment into these crystals to study aatRNA decoding.[39]
The binding of mRNA and cognate aa-tRNA induces two
major rearrangements within the ribosomal decoding center:
the universally conserved residues A1492 and A1493 flip out
of the internal loop of h44, while the universally conserved
base G530 switches from a syn to an anti conformation.
Through this process A1493 recognizes the minor groove of
the first base pair of the codon–anticodon helix in the A site.
The first base pair between ASL-tRNA and the mRNA
consists of position A36 and U1 in Figure 4 a, respectively, and
recognition takes place through a type I A-minor motif. Three
hydrogen bonds are formed between A1493 and the first
position of the codon–anticodon duplex (two with the 2’-OH
groups of A36 and U1 and another with the O2 of U1). It is
noteworthy that the third hydrogen bond is not sequencespecific as might be expected, since the O2 position of the
pyrimidines and the N3 position of purines occupy equivalent
positions in the minor groove of a double helix and both are
hydrogen-bond acceptors.
The second base pair (A35-U2) is also monitored by 2’OH interactions, but this time by two bases, namely A1492
and G530 (this type II A-minor interaction is seen in
Figure 4 b). A1492 and G530 are locked in position by
secondary interactions with protein S12 (serine 50) and
another universally conserved residue C518. Thus, it seems
that the monitoring of the middle base pair of a codon–
anticodon duplex is more rigid than the first base pair. This
finding fits well with the observation that the middle base pair
plays the most important role in coding an amino acid,
followed by the first base pair. In contrast, the third position is
less rigorously monitored and plays no role in the decoding of
mRNA information. This less rigorous checking of the third
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Figure 4. The principles of decoding in the A site of the ribosome.
a) The first base pair of a codon–anticodon interaction (position 1)
exemplifies a Type I A-minor motif: A1493 binds to the minor groove
of the A36-U1 base pair through H bonds (dotted lines). b) Position 2
illustrates a type II A-minor motif: A1492 and G530 act in tandem to
recognize the stereochemical correctness of the A35-U2 base pair
using H bonds. c) The third (or wobble) base pair (G34-U3) is less
rigorously monitored. C1054 stacks against G34 while U3 interacts
directly with G530 and indirectly with C518 and proline 48 of S12
through a magnesium ion (magenta). All nucleotides involved in
monitoring positions 1 and 2 are universally conserved. Adapted
from Ogle et al.[39]
position allows latitude for wobble interactions (Figure 4 c).
This is evident in the third base pair (G34-U3) where the
minor groove remains exposed, despite direct interactions
with C1054 and G530 and indirect metal-mediated interactions with C518 and proline 48 of the ribosomal protein S12.
Taken together, these results clearly confirm the Potapov
hypothesis and explain how decoding operates through the
recognition of the correct stereochemistry of the A-form
codon–anticodon duplex. Furthermore, since the ribosomal
components involved are universally conserved, this suggests
that the mechanism of decoding is likely to be similar for all
ribosomes.
Prior to the Potapov hypothesis, it had been proposed that
the ribosome utilized a “proofreading mechanism” to
improve the accuracy of translation.[40, 41] This mechanism
was suggested to operate by re-selection of the correct
substrate during a so-called “discarding step”, after the initial
binding of the A-tRNA. Since re-selection is dependent upon
release of the tRNA from EF-Tu and is accompanied by GTP
cleavage, the GTP consumption for the incorporation of a
cognate and near-cognate amino acid provides a measure of
the power of proofreading. Insofar as the crystal structure of
EF-Tu and the ribosome are concerned, the proofreading
mechanism does not have its own active center; instead it can
be described in terms of a kinetic effect that occurs after the
release of the binary complex EF-Tu·GDP.[42] Thus, a simple
model for kinetic proofreading is the following: The binding
energy during the decoding step (first step of A-site binding,
see Section 2.2.1) is lower for the near-cognate aa-tRNA than
for the cognate one, therefore, the probability of triggering
the gross-conformational change required for A-site accom-
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modation of the aa-tRNA (second step of A-site binding) is
lower than for the near-cognate. This in turn prolongs the
resting time of the near-cognate aa-tRNA at the low-affinity
A site and provides an additional chance for the near-cognate
aa-tRNA to fall off the low-affinity A site.[43] Re-binding of
this near-cognate aa-tRNA is unlikely in the presence of
competing ternary complexes that have an affinity two to
three orders of magnitude higher for the A site than the
naked aa-tRNA.[44]
The importance of the proofreading step can be quantitatively determined by taking advantage of the fact that the
proofreading mechanism requires EF-Tu-dependent GTP
hydrolysis. The accuracy of aa-tRNA selection in the presence
of EF-Tu and a noncleavable GTP analogue was determined
to be 1:1000,[45] an accuracy only a factor of 3 lower than that
seen in vivo (1:3000). The same threefold difference was also
determined for the GTP consumption per incorporation of
cognate versus near-cognate amino acids.[38] Thus, it is clear
that the significant contribution to the accuracy of translation
(1000-fold) lies within the stereochemical monitoring of the
codon–anticodon duplex by the ribosome—as predicted by
Potapov—and that the “proofreading mechanism” plays only
a minor role in improving the accuracy. This view was
qualitatively confirmed by direct measurement of the discrimination power of the initial binding without proofreading,
where the binding of cognate and near-cognate ASL-tRNA
fragments to the A site of 70S ribosomes were compared. The
accuracy was found to be between 1:350 to 1:500, further
emphasizing that the “lion's share” of the ribosomal accuracy
is carried by the initial binding.[46]
2.2.3. Mimicry at the Ribosomal A Site
The A site is not restricted to binding tRNAs exclusively.
During the various stages of the elongation cycle a number of
translational factors interact at the A site. The first structures
determined for these translational factors were those of the
translational factors EF-G[47, 48] and EF-Tu.[49, 50] Interestingly,
the structure of the latter, in the form of a ternary complex
EF-Tu·GTP·tRNA,[51] had a striking similarity to that of EFG·GDP, such that domains 3–5 of EF-G closely mimic the
tRNA in the ternary complex (Figure 5 a and b; see the review
by Nissen et al.[34]). This observation suggested that the
binding pocket of the A site constrains the translational
factors binding there to conform to a tRNA-like shape. In the
past few years, structures for various termination factors, also
thought to interact with the A site, have generally supported
this concept. The recent structures of the ribosome recycling
factor (RRF) perhaps presented the most convincing tRNA
mimic, exhibiting a similar “L” shape and dimensions as a
tRNA (Figure 5 c).[53] Although the structures of the bacterial
RF2 factor (Figure 5 d)[56] and eukaryotic human eRF1[57]
factor deviate significantly from the simple tRNA structure,
they do reveal overall domain arrangements that were
proposed to span the ribosome in an analogous fashion to
tRNA: One domain extends into the decoding site of the
small subunit and another reaches towards the PTC on the
large subunit. However, a recent study has contradicted these
results: Cryo-EM microscopy analyses of the termination
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is mediated by EF-G, which has been proposed to translocate
RRF from the A to the P site, thus simulating its tRNA
translocative role during elongation.[61]
Mimicry of RNA by a protein may be a more common
feature in ribosomes than first realized. Organellar ribosomes
generally have shorter rRNA components than E. coli.
Recent analyses of the chloroplast and mitochondrial ribosome components suggests that these rRNA losses are
compensated for by both increases in size of the ribosomal
protein homologues and the presence of additional organellespecific ribosomal proteins.[62] Mitochondria represent an
extreme example in that the protein component of the
ribosomes represents two-thirds of the mass (instead of onethird as in E. coli ribosomes). The rRNA that remains consists
predominantly of universally conserved residues that are
located at the active centers of the ribosome, that is, the
decoding center on the 30S subunit and the PTF center on the
large subunit,[63] thus reinforcing the importance of these
regions.
2.2.4. Antibiotic Antagonists of A Site Decoding
Figure 5. Molecular mimicry of tRNAs by translation factors. Comparison of the crystal structures for a) EF-G·GDP with domains 3–5 in gold
(pdb1fmn),[52] b) EF-Tu·GTP·tRNA (pdb1ttt),[51] c) (pdb1eh1),[54] and
d) RF2 (pdb1gqe).[56] e) Cryo-EM reconstruction of RF2 (red) bound to
the 70S ribosome of E. coli (30S in yellow and 50S in blue). DC: decoding center, GAC: GTPase-associated center, P: P-site tRNA, PTC: peptidyltransferase center. f) Modeling of the RF2 crystal structure into the
electron density of RF2 seen in (e). In (d) and (f) the Roman numerals
indicate the RF2 domains which are colored accordingly, and the GGQ
and SPF motifs are indicated in gray and pink, respectively. The
dashed white line delineates the RF2 electron density from the ribosome electron density. The pictures of the crystal structures were
generated with Swisspdb viewer[55] and rendered with POVRAY.
Cryo-EM data adapted from Rawat et al.[59]
To date, the structures of seven antibiotics, namely
tetracycline, paromomycin, spectinomycin, streptomycin,
pactamycin, hygromycin B, and edeine, have been solved in
complexes with the 30S subunit.[27, 39, 64, 65] Although the primary binding sites of these antibiotic are distinct from one
another, they all target functionally important regions of the
30S subunit, mainly rRNA-rich regions associated with tRNA
interaction or movement through the ribosome. Here we will
focus our attention on tetracycline and paromomycin, both
being antibiotics that bind within the decoding site.
Independent studies identified two common tetracycline
binding sites on the 30S subunit (Figure 6 a).[27, 65] In both
complex of RF2 bound to the ribosome revealed that RF2
undergoes dramatic rearrangement upon ribosome binding
(Figure 5 e).[58, 59] This conformational change was not totally
unexpected since two regions within the protein factor, the so
called “tripeptide anticodon” and the GGQ motif were only
23 @ apart in the solution structure of RF2 (Figure 5 d). Since
these regions had been associated with decoding of the stop
codon and hydrolysis at the PTF center, respectively, they
would need to be separated by about 70 @ to make the
appropriate interactions (Figure 5 f).[58, 59]
The structural mimicry of a tRNA by RRF was also
brought into doubt when hydroxyl radical probing experiments suggested that the RRF is oriented upside down on the
ribosome when the structural similarities with a tRNA were
considered.[60] Confirmation of this orientation, however,
awaits cryo-EM analysis of the ribosomal RRF complexes.
Whether RRF undergoes similar structural rearrangements as
seen for RF2 seems unlikely, but it is clear that functional,
rather than structural mimicry, is a more appropriate term in
this case: After release of the polypeptide chain by the
termination release factors, the RRF binds the ribosome and
is involved in dissociating the ribosome into subunits, thus
recycling them for the next round of translation. This process
Figure 6. Binding of tetracycline (Tet) to the 30S subunit of T thermophilus. a) Overview of the primary and secondary Tet binding sites
(red). b) Close-up view of the primary binding site, with the position of
the A-site tRNA (red) and mRNA (yellow). Helix 18 (brown), h31
(green), h34 (blue), and h44 (cyan) are represented in ribbon format.
c) The secondary binding site includes h11 (purple) and h27 (yellow).
The switch described in the text involves interconversion between the
base-pair configuration from red to green. Figure (a) was generated
from pdb file 1HNW using Swisspdb viewer[55] and rendered with
POVRAY. Figures (b) and (c) are adapted from Brodersen et al.[65]
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cases, the site with the highest occupancy, which is located in a
crevice between the head and shoulder of the 30S subunit,
was taken to be the primary binding site (Figure 6 b). In this
position, tetracyclin, which has a system of four fused rings,
interact predominately with the 16S rRNA through the
oxygen atoms located along one side of the molecule. The
oxygen atoms form hydrogen bonds with the exposed sugar
phosphate backbone of helix h34. Thus, the hydrophilic side
of the tetracycline interacts with the 16S RNA while the
hydrophobic side is sited in the lumen of the A site. This is
surprising since interaction between two molecules is usually
through their hydrophobic regions. The primary binding site
of tetracyclin can be expected to overlap with the position of
the A site tRNA, and thus the mechanism of action of
tetracycline most probably results from a direct inhibition of
aa-tRNA during the accommodation step of A-site binding.
For two reasons it seems unlikely that the initial binding of the
aa-tRNA would be affected: 1) the tetracyline binding site is
located on the opposite side of the tRNA from the site of
initial anticodon–codon interaction, 2) during the delivery of
aa-tRNA to the A site, the anticodon- stem loop of the aatRNA of EF-Tu was kinked and presented at an angle,[36, 37]
which would be predicted to initially avoid contact with the
bound tetracycline.[27, 65] This is in line with the observation
that tetracycline does not inhibit the EF-Tu-dependent
GTPase hydrolysis,[66] a step that occurs after the initial
binding of the ternary complex. The rRNA bases of the
primary binding site of tetracycline are poorly conserved
between prokaryotes and eukaryotes, and its universal mode
of action can be explained since the interaction of tetracyclin
is almost exclusively made with the sugar–phosphate backbone. In contrast, the binding sites of pactamycin, edeine, and
hygromycin B are highly conserved, and in these cases the
main interactions are with the conserved bases.
The secondary binding site of tetracyclin is sandwiched
between h11 and h27 in the body of the 30S subunit
(Figure 6 c). Although h27 has been proposed as a conformation switch modulating the translational fidelity of base pairs
in E. coli,[67] it seems unlikely that this secondary site plays a
role in tetracyclin inhibition. The protein Tet(O) removes
tetracycline from the ribosome and thus mediates resistance
against this drug. Probing experiments with dimethyl sulfate
(DMS) in the presence of Tet(O) demonstrated that tetracycline was removed from the primary rather than from the
secondary binding site.[68]
The aminoglycoside paromomycin is well known for
increasing the rate of translational misreading. The binding
site for paromomycin, determined at a resolution of 3 @, was
observed to involve contacts exclusively with h44 of the
16S rRNA.[39, 64] Paromomycin binding induces the universally
conserved residues A1492 and A1493 to flip out of h44 in a
fashion reminiscent of that observed during the binding of aatRNA to the A site. This conformational change is brought
about by the insertion of one of the four rings (ring I) of
paromomycin into h44. In this position, ring I mimics a
nucleotide base: it stacks with G1491 and hydrogen bonds
with A1408. The stability of this conformation is further
reinforced by hydrogen-bonding interactions between ring I
and the backbone of the flipped out A1493. Significantly,
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rings I and II of paromomycin are found in a number of other
aminogylcosides, such as the antibiotics neomycin, gentamycin, and kanamycin families, which suggests that misreading
by these antibiotics operates through a similar mechanism.
As we have seen in Section 2.2.2, the formation of
appropriate codon–anticodon interactions is monitored by
the formation of A-minor interactions between A1492 and
A1493 with the codon–anticodon helix. Presumably the
energy required to flip out A1492 and A1493 during decoding
is compensated for by interactions established with the
codon–anticodon helix, thus stabilizing this conformation. In
the presence of near-cognate tRNA, the prediction is that
these compensatory interactions are not sufficient to stabilize
the flipping out of A1492 and A1493, and thus accommodation of the A site does not occur. However, in the presence of
paromomycin, the normally uncompensated losses of energy
are absorbed by the paromomycin that has already induced
A1492 and A1493 to flip out and stabilized them in this open
conformation. The outcome is that a near-cognate tRNA
becomes fully accommodated into the A site in the presence
of paromomycin and thus results in mis-incorporation of an
amino acid.
2.3. The P Site and the Peptidyltransferase Center
Historically, much controversy has surrounded the question regarding the catalytic “heart” of the ribosome, the
peptidyltransferase (PTF) center. Specifically, the questions
posed related to whether this active site was predominantly
protein or rRNA. The answers to these questions arrived with
the X-ray structure of the 50S subunit, in particular the
50S subunit complexed with a transition intermediate of the
PTF reaction.[69] The location of the transition intermediate
immediately suggested that, in fact, the catalytic center of the
ribosome is exclusively composed of RNA. Furthermore,
analyses of the residues within proximity to the CCA end
analogues of the tRNA led to the proposal of an acid–base
catalysis mechanism for the PTF reaction involving the
universally conserved A2451—a proposal that came under
immediate attack from a number of research groups who
presented biochemical and genetic data to the contrary.[70–72]
The ongoing debate as to the exact catalytic contribution of
the ribosome and the residues involved is the topic of a recent
review.[73] Thus, within a short space of time the debate had
moved from whether or not RNA or a protein constituted the
catalytic domain, onto more detailed mechanistic questions.
In Section 2.3.1 we analyze the interactions of the
peptidyl-tRNA at the P site—certainly the tRNA binding
site on which most information from different species has
been gathered—and illustrate the universal features. The
mechanism of PTF is examined in Section 2.3.2, with particular focus on how the ribosome assists or catalyzes the
transfer of the growing polypeptide chain from the P-tRNA to
A-tRNA. The extreme conservation of residues within the
PTF center suggests that the mechanism of PTF is universally
conserved. Finally, the PTF center and its immediate vicinity
are the target for a number of antibiotics, the structures of
some clinically important examples of which, such as chlor-
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amphenicol, clindamycin, and a number of macrolides, have
been solved in complexes with the 50S subunit.[11, 74] These
structures provide not only an insight into the mechanism of
antibiotic inhibition and ribosome function, but may pave the
way for the design of specific antibiotics to combat the
increase in antibiotic-resistant bacteria.[75]
2.3.1. Peptidyl-tRNA Contacts at the P Site of the Ribosome
In the past, the tRNA positions on the ribosome were
studied using a large number of biochemical approaches that
have identified a number of rRNA nucleotides that are
associated with each tRNA site (see Table 3 in Ref. [13]).
After a crystal structure of the Thermus thermophilus 70S
Figure 7. P-tRNA contacts with the 70S ribosome. The region of the Pribosome in complex with three tRNAs was solved with a
tRNA that makes contact with the 30S subunit (15 %) and the 50S sub[76]
resolution of 5.5 @,
it became clear that the identified
unit (85 %) are shown in blue and red, respectively.[77] The ribosomal
residues either directly contact the tRNA or their altered
components within a 10-F radius of the P-tRNA are indicated for the
modification pattern could be explained by conformational
30S subunit (rRNA: green, proteins: cyan), and for the 50S subunit
(rRNA: yellow, proteins: orange). H: helices of the tRNA, S and L: prochanges within binding regions. An excellent example illusteins of the small and large subunits, respectively. The figure was gentrating this correlation is the analysis of cleavage patterns
erated from pdb files 1GIX/1GIY[76] using the program RASMOL[78] and
from ribosome-bound phosphorothioated tRNAs.[77] The
rendered with POVRAY.
contact patterns suggested that only 15 % of the P-tRNA
(nucleotides 29–43, which comprise the
anticodon loop and two adjacent stem
Table 2: Comparison of ribosomal intersubunit bridge components between the bacteria Thermus
base pairs) interact with the 30S subunit
thermophilus[32] and the yeast Saccharomyces cerevisiae.[3]
while the remaining 85 % is in contact with
Subunit tRNA posi- Ribosomal com- rRNA contact
Ribosomal component
rRNA conthe 50S subunit (Figure 7). The fact that the
tion[a]
ponent
(T. thermophilus) (S. cerevisiae)
tact
protection patterns cover the entire tRNA
(T. thermophilus)
(S. cerevisiae)
again dispels the earlier assumptions that
small
28
16S h30
1229
tRNAs bind the ribosome using only their
29/30
16S h30
1229
18S h30
1229/1230
extremities, that is, through anticodon–
32
RpS16
Tyr 141/
codon interactions in the small subunit and
Arg 142
34
16S h31
966
18S h31
966
the CCA end in the large subunit. Further35
S9
Arg 128
more, the ribosome contacts all three
36
S13
116–120
tRNAs at universally conserved parts of
38 (39)
16S h24
790
(18S h24)
(790)
[76, 77]
their structure.
Phosphorothioate pro(40) 41
(16S h42)
(1339)
18S h43
1338
tection studies have also suggested a con(41) 42
(16S h42)
(1338)
18S h43
1339
formational change of the P-tRNA upon
binding to the ribosome.[79] Such a change is
large
2
RpL10
Lys 101
evident for the P-tRNA in the 70S/tRNA3
3
23S H80
2255–2256
25S H80
2285
crystal structure, where the P-tRNA is
4
25S H80
2286
slightly kinked around the junction of the
11–13 (12– (23S H69)
(1908–1909)
25S H69
1908–1910
D loop and anticodon stem.[76] A similar
13)
conformational change was necessary to
14
25S H69
1924
dock the crystal structure for the yeast
25–26
23S H69
1922–1923
51/52/63
RpL10
Arg 24
tRNAPhe into the P-site electron density
56 (56–57) (RpL5)
(55–66)
RpL11
Tyr 51
during the cryo-EM reconstruction of a
71–72
25S H93
2594
complex of P-tRNA bound to the 80S
73 (75)
23S H93
(2602)
25S H93
2602
[3]
subunit of a yeast.
74/76
23S P loop/L90– 2252/2585
We have chosen to analyze P-tRNA
93
binding interactions with the yeast ribo[a]
tRNA
positions
from
yeast cryo-EM analysis are approximate because of limitations in the resolution.
some[3] in detail because of the availability
of data, with a view to highlight the
positioned in the major groove of the noncanonical helical
conservation in the interaction. A comparison of the contacts
structure at the base of h44 and is fixed with a number of
between rRNA and the P-tRNA in both bacteria and yeast
“ribosomal fingers” mainly to the sugar–phosphate back(Table 2) illustrates the similarity in the contacts.[3, 32] The Pbone.[32] A similar position for the P-tRNA and arrangement
tRNA is fixed very tightly on the bacterial 30S subunit
through approximately six interactions with the rRNA (a–f in
of contacts is also observed for the yeast ribosome (FigFigure 8 a). The codon–anticodon duplex in the P site is
ure 8 b). Two of the rRNA interactions with the P-tRNA in
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the CCA end to base 2602 in the yeast ribosome complex,
which in the bacterial ribosome complex contacts position 75,
the penultimate base of the CCA end of the P-tRNA.
It is clear from the multiple tRNA contact points with the
ribosome that positioning of the tRNA involves a complex
network of interactions. The distinct similarity in the arrangement and make-up of the ribosomal components that interact
with a P-tRNA, despite the billions of years separating yeasts
and eubacteria, suggests an important role for these components during translation. Accurate positioning of tRNAs is
essential for ribosomal function. As seen in Section 2.2, the
process of decoding is governed by the stereochemical
arrangement of the tRNAs relative to the mRNA and the
ribosome. Many of the contact points are components of
intersubunit bridges, which suggests that these interactions
not only “lock” the tRNA in position but may be involved in
transporting it through the ribosome (see Section 2.4).
Furthermore, tight fixation of both A- and P-tRNAs may be
the prerequisite for efficient formation of the peptide bond, as
will be described in the following section.
2.3.2. The Ribosome is a Ribozyme
Figure 8. Details of P-tRNA interactions with the small and large subunit of Thermus thermophilus and Saccharomyces cerevisiae ribosomes.
The P-tRNA (red) contacts made with the 30S (a) and 50S (c) subunits
from T. thermophilus are similar to those made by the P-tRNA (green)
with the 40S (b) and 60S (d) subunits from S. cerevisiae. The small
subunit rRNA is colored cyan (a) or yellow (b), while the ribosomal
proteins are colored either purple (a) or red (b). The large subunit
rRNA is colored gray (c) or blue (d), while the ribosomal proteins are
colored magenta (c) or red (d). The spheres in (a) and (c) represent
rRNA bases that are protected from chemical probes upon tRNA binding. In (a) the six ribosomal fingers that hold the anti-codon stem–
loop (ASL) in place are indicated by a–f. The figure is adapted from
Yusupov et al.[76] and Spahn et al.[3]
Thermus thermophilus are strengthened by interaction with
C-terminal ends of ribosomal proteins S9 and S13. The Cterminal end of S9 is highly conserved and contains a
universally conserved arginine residue that appears to contact
the phosphate group of position 35 in the anticodon loop of
the P-tRNA. Again, a similar interaction is seen in yeast with
rpS16, the homologue of bacterial S9. In contrast, rpS18, the
homologue of S13, does not have a corresponding C-terminal
sequence and, on the basis of its position in the 80S ribosome,
is probably not involved in P-tRNA fixation. Interaction
between the large subunit and the P-tRNA also involves
contacts between H69 and the D loop, while the T loop
contacts rpL11 in eukaryotes and the homologue L5 in
bacteria (Table 2 and Figure 8 c and d). Interestingly, both
these ribosomal components are involved in the formation of
bridges between subunits, which suggests they may play a
dynamic role in translation, for example, translocation of the
tRNAs (see Section 2.4.2). Although the single-stranded
CCA end of the P-tRNA is not resolved in the 80S complex,
its position in the crystal structure suggests there is interaction
within the PTF center. This is evident from the proximity of
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The PTF reaction is the central enzymatic activity of the
large subunit. It occurs when a pretranslocational (PRE) state
is reached, that is, when a peptidyl-tRNA is located in the
P site and an aa-tRNA is in the A site. The two L-shaped
tRNAs at the P and A sites form an angle of about 408,[19, 76, 80]
while the acceptor stems of both tRNAs are parallel to each
other, such that they can move in a translational movement
relative to each other. In contrast, the CCA ends of both
tRNAs at the PTF center have rotation symmetry, being
arranged at an angle of approximately 1808 to each other. The
twist needed to accomplish this rotation occurs almost
entirely between nucleotides 72 and 74 of the tRNA.[81]
Recently, this rotational symmetry of the A- and P-tRNA
CCA ends was shown to be complemented by two sets of PTF
nucleotides surrounding each of the CCA ends that are also
related by a rotational symmetry.[82] This ribosomal structure
might play an essential role in guiding the CCA ends during
translocation from the A and P sites to the P and E sites,
respectively.
During PTF the a-amino group of the A-tRNA attacks
the carbonyl group of the peptidyl residue of the P-tRNA,
which is linked through an ester bond to the tRNA moiety.
This results in the formation of a tetrahedral intermediate,
which leads to formation of a peptidyl bond. As a result, the
aa-tRNA becomes a peptidyl-tRNA extended by one aminoacyl residue, and the former peptidyl-tRNA is stripped of the
peptidyl residue to become a deacylated tRNA (Figure 9).
A long-standing debate within the field of translation
concerned whether, or not, the PTF reaction was catalyzed by
proteins or rRNA. This question was answered with the
identification of the PTF center. A putative transition state
analogue of the PTF reaction was soaked into crystals of the
50S subunit from Haloarcula marismortui.[69] This analogue
(the so-called Yarus inhibitor) is a mimic of the CCA end of a
P-tRNA attached to puromycin in the A site (Figure 9), and is
a strong competitive inhibitor of the A site substrate.[84] The
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Figure 9. The peptidyltransferase reaction. The picture bottom right shows the Yarus inhibitor CCdAp-puromycin (CCdApPmn) that was used to
identify the PTF center of the ribosome. The interactions of the Yarus inhibitor with the rRNA were deduced from the 50S crystal structure of
H. marismortui ribosomes after soaking the inhibitor into the crystals. It was concluded that the protonated N3 atom of A2451 makes a H bridge
to the O2 atom that was thought to mark the position of the oxyanion of the tetrahedral intermediate formed during peptide-bond formation[69]
(see the schematic representation in step b). The figure shows the four possible steps of peptide-bond formation according to recent crystallographic and biochemical data.[69, 73, 81, 85] The essential features are: a) C74 and C75 of the P-site tRNA (green) form a Watson–Crick base pair with
G2252 and G2251, respectively, of the P loop (blue). Likewise, C75 from the A-site substrate (red) forms a Watson–Crick base pair with G2553
(A loop). The a-amino function of the A-site aa-tRNA is an ammonium ion at pH 7.[83] b) Deprotonation of the ammonium ion triggers the nucleophilic attack of the a-amino function on the carbonyl group of the P site substrate, which results in the tetrahedral intermediate T . The secondary
NH2 group forms a hydrogen bond with the N3 atom of A2451 and a second with either the 2’OH group of the A76 ribose at the P site (shown
here) or alternatively with the 2’OH group of A2451. The oxyanion of the tetrahedral intermediate points away from the N3-A2451[81] and thus
cannot, in contrast to the previous proposal, form a H bridge.[69] c) Further deprotonation of the secondary a-NH2 group leads to the tetrahedral
intermediate T and the PTF reaction is completed by an elimination step (d). The peptidyl residue is linked to the aminoacyl-tRNA at the A site
through a peptide bond.
region bound by the inhibitor is densely packed with highly
conserved bases of the 23S rRNA, mainly derived from the
so-called PTF ring of domain V. Although there are 15 proteins that interact with domain V of the 23S rRNA, only the
extensions of proteins L2, L3, L4, and L10e come within 20 @
of the active site (Figure 10). The fact that the active center of
the ribosome is made exclusively from RNA means that the
ribosome is a true ribozyme.
The debate has now turned to whether the PTF reaction
simply utilizes a physical principle or whether an additional
chemical principle also applies, that is, whether besides the
accurate stereochemical arrangement of the substrate (physical principle), a chemical principal, such as a general acid–
base catalysis, is also involved.
The universally conserved residue A2451 of domain V is
the nearest base to the transition analogue (Figure 9). It was
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thought to be a good candidate for a general acid–base
catalyst, since its N3 atom is about 3 @ from the oxygen atom
and 4 @ from the nitrogen atom of the phosphoamide in the
Yarus inhibitor (Figure 11 a). This proposal was strengthened
when the pKa value of the A2451 residue was found to be
abnormally high at neutral pH (pKa = 7), which is six pH units
higher than expected.[71, 85] This property is essential for
acid–base catalysis, as it allows for easy donation and
withdrawal of a proton from the a-amino group of the aatRNA at the A site. According to the same model,[69]
protonation of A2451 would also allow formation of a
hydrogen bond with the carbonyl oxyanion of the tetrahedral
transition state analogue (Figure 9). However, modification
of position 2451 with dimethyl sulfate showed that pH dependence was only displayed by inactive ribosomes, not by
the active ribosomes.[86] Several research groups reported
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Figure 10. The A- and P-site products (red and green, respectively),
bound at the peptidyltransferase center of the 50S subunit. The proteins that reach within about 20 F of the PTF center include proteins
L2 (purple), L3 (blue), L4 (red), and L10e (cyan). This figure was generated from pdb file 1KQS[87] using Swisspdb viewer[55] and rendered
with POVRAY.
shortly after that A2451 was not essential for formation of the
peptide bond, since ribosomes bearing mutations at position 2451 exhibited only modest (2- to 14-fold) decreases in
the rate of peptidyl transfer[72] and were instead shown to be
defective in substrate binding.[70]
The next step in the elucidation of the PTF reaction was
identifying the position of the products and was obtained by
soaking A- and P-site substrates into enzymatically active
H. marismortui 50S crystals.[87] The structure determined to a
resolution of 2.4–3.0 @ was that after formation of the peptide
bond but before translocation, and showed that the deacylated CCA bound to the P site had its 3’OH group in
proximity to the N3 atom of A2451 (Figure 11 b).[87]
Evidence followed, however, that A2451 was not involved
in stabilizing the transition-state analogue: If the oxyanion of
the tetrahedral intermediate is hydrogen bonded to the
N3 atom of A2451, then this N3 atom must be protonated at
around pH 7 and therefore should loose its proton at pH >
7.3. In this case, one would expect the affinity of the Yarus
inhibitor to be strongly pH-dependent, since the hydrogen
bond would contribute significantly (up to three orders of
magnitude) to the affinity. To test this hypothesis, Strobel and
co-workers determined the affinity of the Yarus inhibitor for
the 50S subunit between pH 5 and 8.5, and found that it
remained unchanged.[85] This result is inconsistent with the
idea that the oxyanion is stabilized by a hydrogen bond to the
N3 atom of A2451. The same conclusion was drawn by
subsequent crystallographic studies showing that the oxyanion of the tetrahedral intermediate points away from the
N3 atom of A2451, thus excluding the possibility of the
formation of a hydrogen bond between these two atoms.[81]
Furthermore, the Yarus inhibitor is not an honest mimic of
the transition state. The distance between the O2 atom of the
Yarus inhibitor and the C2’ atom of the deoxy-A76 (dA76)
ribose at the P site is only 2.8 @ (arrowed in Figure 11 a). A
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Figure 11. Tight fixation of the CCA ends of the P- (green) and AtRNAs (red) observed in the 50S subunit from H. marismortui in a
complex with a) the Yarus inhibitor and b) the products following formation of the peptide bond. The N3 atom of A2451 (dark blue) is
3.4 F from the O2 atom of the Yarus inhibitor (see also Figure 9). The
same O2 atom is only 2.8 F from the 2’-deoxy position of A76
(arrowed). Selected rRNA residues of domain V of the 23S rRNA are
colored light blue, including the A- and P-loop bases that participate in
fixation of the CCA ends of the A and P sites (E. coli numbering). In
(b) C74 and C75 of the P site have been omitted for clarity. Dashes
indicate hydrogen bonding and rRNA nucleotides use the following
color scheme: O red, P yellow, N blue, C dark blue. Figures (a) and (b)
were generated from pdb files 1FFZ[69] and 1KQS,[87] respectively, using
Swisspdb viewer[55] and rendered with POVRAY.
physiologically P-site substrate contains a 2’OH group at this
C2’ atom which is essential for formation of a peptide bond[88]
and would sterically clash with the O2 atom of the Yarus
inhibitor positioned as observed in the crystal. The essential
nature of this 2’-OH group might be explained by the
observation that the a-NH2 group of the A site possibly
forms a hydrogen bond with this 2’-OH group (illustrated in
Figure 9 b).
The general base catalysis debate flared up again when
Rodnina and co-workers presented evidence that formation
of a peptide bond depends on two ionizable groups, one with a
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pKa value of 6.9 and the other with a pKa value of 7.5.[89] The
former value was shown to be associated with the a-NH2
group of puromycin used in the kinetic experiments, while the
latter value seemed to be associated with the ribosome. The
ionizable group seemed to belong to A2451, since the
formation of a peptide bond was about 130 times slower
than normal when catalyzed by a ribosome bearing an
A2451U mutation, and had lost the pH dependence. However, an alternative explanation suggested by the authors was
that the protonated group is part of the A2450:C2063 base
pair lying directly behind A2451 (shown in Figure 11 b). In
this case, the ionizable group would be the N1 atom of A2450.
Although a distance of 7 @ from the N1 atom to the a-NH2
group is too long for hydrogen transfer, a postulated
conformational change of the PTF center might bring
A2450 within range.[89] The assumption of conformational
changes again increases the number of possible candidates
that might play a role in the kind of chemical catalysis that is
advocated here. We note that evidence was presented that
His 229 of protein L2 might also be involved in this catalysis,[90] although current atom maps place this residue more
than 20 @ away from the tetrahedral intermediate of the
transition state.
At the moment it can be said that a direct role of A2451 in
a general acid–base catalysis can hardly be reconciled with the
observation that A2451 in active ribosomes, in contrast to
inactive ribosomes, does not contain a titratable group at this
pKa value.[86]
In fact, the ribosome need not use any direct chemical
involvement in the catalysis of the PTF reaction, such as the
formation of a transient covalent interaction between the
substrate (tRNAs) and the enzyme (the ribosome, or more
specifically in this case the rRNA). The template model
predicts that tight stereochemical arrangement of substrates
relative to one another would be sufficient to provide the
dramatic acceleration of the reaction rate needed for the
formation of a peptide bond (see the review in Ref. [91]). In
this case the role of A2451 would be to remove a proton from
the free nucleophilic a-NH2 group of the A-site substrate or
form a hydrogen bond with the a-NH2 group, thus promoting
formation of a peptide bond through proper positioning of the
NH2 group. The reaction scheme would be something like that
presented in Figure 9 and described in more detail in the
corresponding legend.
Tight fixation of the CCA ends of the P- and A-tRNAs is
exactly what is observed both in the analogue-soaked 50S
crystal structure (Figure 11 a) and the products containing a
peptide bond following soaking of the A- and P-site substrates
(Figure 11 b). The CCA end is locked into position in the
P site by formation of two Watson–Crick base pairs (C74 and
C75 with G2252 and G2251, respectively). A76 stacks on the
ribose of A2451 (shown clearly in Figure 11 b). The CCA end
of the aa-tRNA in the A site is fixed by: 1) Watson–Crick
base pairing between C75 and G2553, 2) a type-I A-minor
motif between A76 and the G2583-U2506 base pair, and 3) an
additional hydrogen-bonding interaction between the 2’-OH
group of A76 and U2585. Tight fixation of the CCA ends of
both A- and P-tRNAs at the PTF center underlines the
importance of the (purely physical) template model in
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formation of a peptide bond, namely, that precise stereochemical fixation is predominantly responsible for the
enormous acceleration of the reaction. The rate of formation
of a peptide bond on the ribosome at about 50 s 1 was
estimated to be approximately a factor of 105 faster than the
uncatalyzed reaction (the rate in the absence of ribosomes).[91]
The ribosomal PTF reaction without chemical catalysis (that
is, when the ionizable group of the ribosome is protonated at
pH < 7) occurs with a rate of about 0.5 s 1,[89] which is still
more than 1000 times faster than the uncatalyzed reaction. If
this estimation is correct then the purely physical mode of
peptide-bond formation (the exact stereochemical arrangement of the reactants) represents approximately 90 % of the
reaction rate, with the catalytic component making up the
remaining 10 %.
2.3.3. Antibiotic Action on the 50S Subunit
The functional importance of rRNA at the ribosome
active centers is reiterated by the sites of interaction of a
variety of clinically relevant antibiotics, such as chloramphenicol (Cam) and clindamycin, which inhibit PTF activity.[11]
Cam is well-known as an specific bacterial inhibitor of the
CCA-aa end of an A-site tRNA[92] but does not inhibit the
CCA fixation of a P-site tRNA.[93] The binding site of Cam
was determined to a resolution of 3.5 @ by soaking the
antibiotic into crystals of the 50S subunit of D. radiodurans
and shown to involve interaction with seven nucleotides
within the PTF center.[11] A number of these interactions are
indirect as they are mediated through putative Mg2+ ions.
Since moieties important for the antibiotic action of Cam
constitute these interactions, this result suggests that the
presence of the ions are of extreme importance for antibiotic
binding and PTF inhibition. The position of the “tail” of Cam
within the PTF center is such that it reaches towards, and may
even displace, the CCA end of the A-site tRNA. This
observation is in agreement with the result that although
tRNAs can bind in the presence of Cam, they cannot undergo
formation of a peptide bond, probably because the CCA-aa
end of the A-site tRNA is displaced from the correct position.
Cam inhibition in vitro is dependent on the nature of the
peptidyl residue and the A-site substrate. In particular, Cam is
a less effective inhibitor against aromatic amino acids such as
phenylalanine. This leads to the conclusion that these amino
acids can actually displace Cam during formation of the
peptide bond by competing with the phenyl group of the drug
for binding. The major overlap between Cam and an amino
acid in the A-site tRNA lends credence to the idea that Cam
operates predominantly by displacing the aminoacyl residue
of the A-site tRNA, which probably indirectly displaces the
CCA end.
In contrast with chloramphenicol, the binding site determined for the lincosamide clindamycin spans between both
the A and P sites at the PTF center.[11] The majority of the
interactions involve hydrogen bonds between hydroxy groups
on the sugar moiety and bases within the PTF center; these
contacts are in agreement with most of the available mutation
resistance data. The proline group of clindamycin overlaps
with the position of the phenyl group of Cam, which is in line
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with the A-site nature of clindamycin inhibition. The
C8’ atom on the proline moiety of clindamycin comes within
2.5 @ of the N3 atom of C2452 and is thus in proximity to a Psite-bound tRNA. Thus, the binding position of clindamycin
traverses both the A and P sites, and would be expected to
disturb the positioning of amino acid moieties at both sites.
The positions of no less than seven distinct members of
the macrolide family of antibiotics have been solved in
complexes with the 50S subunit.[11, 74] This large family of
antibiotics can be divided into three classes on the basis of the
size of the lactone ring. Erythromycin and two erythromycin
derivatives (clarithromycin and roxithromycin) are representatives of the 14-membered-ring class, all of which have been
solved in complexes with the 50S subunit of D. radiodurans.[11]
From the two larger 15- and 16-membered classes, the binding
sites of azithromycin, spiramycin, tylosin, and carbomycin A
have been solved in complexes with the 50S subunit from
H. marismortui.[74]
The binding positions determined for these macrolides are
generally in good agreement with one another, located within
the polypeptide tunnel in proximity to the PTF center. In this
position, the macrolides would be expected to block the
tunnel, thus preventing passage of the nascent polypeptide
chain (Figure 12). This proposal can be reconciled with the
observation that macrolides cannot inhibit actively translating
ribosomes, since the presence of the polypeptide chain in the
tunnel precludes the macrolide from binding. Ribosomes
prebound with macrolides are able to synthesize oligopeptides up to five amino acids in length, depending on the
macrolide present. Since the lactone ring of the bound
macrolides are positioned in the tunnel such that the sugar
extensions from the C5 position extend towards the PTF
center, the length of the side chain dictates the number of
peptide bonds that can be formed; for example, tri- or
tetrapeptides can be formed in the presence of erythromycin,
which has a monosaccharide at the C5 position, whereas only
dipeptides are formed in the presence of tylosin and
spiramycin, which bear C5-disaccharides. Carbomycin A,
which has an additional isobutyrate group on the disaccharide, even prevents formation of the first peptide bond. The
isobutyrate group of carbomycin A reaches so far into the
PTF center that it occupies the position that the amino acid
moiety of the A-site substrate would normally occupy.[74] The
direct interaction of the macrolides with position 2058 makes
it easy to understand how modifications and mutations at this
position in E. coli provide resistance to this class of antibiotics. In contrast, erythromycin (or its derivatives) and
azithromycin make no direct contact with ribosomal proteins L4 or L22, which suggests that the mutations within
these proteins that confer resistance must do so indirectly
through conformational changes of the 23S rRNA.
A further interesting discovery was the formation of a
reversible covalent bond between the acetaldehyde substituent at position C6 of the 16-membered ring macrolides and
the N6 atom of A2062 (E. coli).[74] The formation of this
carbinolamine is specific for the 16-membered group, since
the smaller macrolides do not contain a corresponding
aldehyde functional group. Lastly, it is important to note
that although the overall binding sites for each macrolide are
in general agreement between the two studies discussed here,
there are some significant differences in the orientation and
conformation of the lactone ring and cladinose sugar moiety
of the macrolides bound on the ribosome. In one study
interaction with the tunnel wall is made through the hydrophobic face of the lactone ring, whereas in the other
interaction is made through a series of hydrogen bonds.
Whether these discrepancies arise because of differences in
interpretation, differential binding to ribosomes from particular species, or actual differences in the binding of the
macrolides themselves is unclear. The last possibility would
be surprising since azithromycin and erythromycin differ only
by the absence of a ketone oxygen atom and the addition of
methyl nitrogen atom. Re-analysis of these structures and the
duplication of identical antibiotics for each ribosome species
should resolve this problem.
2.4. P- and A-tRNA Translocation within the Ribosome
Figure 12. The macrolide carbomycin A bound in the tunnel of the
H. marismortui 50S subunit. The rRNA and proteins are represented as
violet ribbons and carbomycin A as a red space-filling model. The 50S
subunit is viewed from the cytoplasmic side (backside) looking up the
tunnel to the PTF center. This figure was created from pdb file 1K8A[74]
using Swiss-pdb viewer[55] and rendered with POVRAY.
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The position of the tRNAs remains unchanged after
formation of the peptide bond. This has been demonstrated
by cryo-EM analyses of E. coli ribosome complexes[19] and
most strikingly by soaking A- and P-site substrates into active
50S H. marismortui crystals and solving the structure of the
reaction products.[87] After formation of the peptide bond the
ribosome must transfer the products—the peptidyl-tRNA in
the A site and deacylated tRNA in the P site to the P and
E sites, respectively–-thus, shifting the ribosome from the preto the posttranslational state (Figure 2 c and 2 f respectively).
This process is termed translocation. It must be extremely
accurate at both ends of the tRNA molecule: The anticodon–
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codon complex must be moved exactly 10 @ (the length of
one codon), longer or shorter movements would result in the
ribosome losing the reading frame. At the other end of the Asite peptidyl-tRNA, the CCA end must also be moved
precisely into the P site so as to set up the next PTF reaction
with the incoming aa-tRNA. Incorrect placement of the
peptidyl-tRNA at the P site could be disastrous for the
formation of a peptide bond and result in the abortion of
translation.
Ribosomes have an innate translocase activity, but it is
more than one order of magnitude slower than that of the EFG-catalyzed reaction.[94] This observation implies that the
structures necessary to move the tRNAs reside in the
ribosome and that the role of EF-G/EF2 is to reduce the
activation energy barrier that separates the two sets of tRNA
positions (A plus P and P plus E). Important questions
remaining unanswered are: How does EF-G/EF2 mediate
translocation and which ribosomal components are involved
in the transfer of the tRNAs?
2.4.1. Conservation in the Binding Site of Elongation Factor-G
The crystal structure of EF-G has been solved in the
absence of the nucleotide[48] and in the complex with GDP
(Figure 5 a).[47] The GTP form is the active one, which binds to
the ribosome and triggers translocation. Hydrolysis of GTP
inactivates EF-G and dissociates it from the ribosome (see the
review by Kaziro).[95] EF-G belongs to the same subfamily of
G proteins as IF2, RF3, and EF-Tu, the latter of which has
been crystallized in both GTP (active) and GDP (inactive)
forms which exhibit large domain shifts relative to one
another.[50]
Cryo-EM reconstructions of EF-G bound to bacterial
70S ribosomes at a resolution of 17.5–20 @[96–98] and EF2 to
eukaryotic 80S ribosomes at 17.5 A resolution[99] show similar
binding sites for both factors (Figure 13 a and b). Antibiotics
Figure 13. Comparison of cryo-electron microscopic analyses of the
EF-G·70S complex from E. coli (a) and the EF2·80S complex from
S. cerevisiae (b); the small subunit is on the left side and the large subunit on the right. The same orientation is seen on the left of an empty
80S ribosome (yellow: 40S, blue 60S). The relative arrangements of
EF-G and P-tRNA (c) and EF2 (red) and P-tRNA (d) are illustrated. The
abbreviations of the features are as in Figure 1, while roman numerals
on EF-G and EF2 refer to the domains of these factors. The figure was
modified from Gomez-Lorenzo et al.[99]
Angew. Chem. Int. Ed. 2003, 42, 3464 – 3486
were used to trap the elongation factor on the ribosome in
these complexes. EF-G was trapped on the 70S ribosome
using the antibiotic fusidic acid. This fixation allows translocation and GTP hydrolysis, but blocks the switch into the
GDP conformer of the factor, thus preventing dissociation
from the ribosome. The eukaryotic eEF2 was locked onto the
yeast ribosome using the antifungal sordarin, which is thought
to function analogously to fusidic acid.[99] The complex was
formed with a typical pretranslational state, that is, A- and PtRNAs were present. As expected, the tRNAs were translocated to the P and E sites, but of special interest is that the
tip, domain IV, of EF-G was shown to occupy the position of
the A site. Similarly, EF2 in the EF2–ribosome complex also
occupied the A site and came very close to the position of the
P-tRNA (Figure 13 c and d). EF-G-mediated translocation is
also possible in the presence of nonhydrolyzable GTP
analogues, such as GDPNP, which suggests that binding of
EF-G alone is sufficient for translocation and that hydrolysis
is necessary for the conformational change and release of EFG·GDP.[95]
For classical G proteins, a GTPase-activating protein
(GAP) stimulates the G-protein-mediated hydrolysis of
GTP. In the case of EF-G/EF2, the GAP is provided by
components of the ribosome. There are certainly gross
changes visible upon the binding of each elongation factor
to the ribosome. One of the most striking changes is seen
within the “stalk” region; there is no electron density for this
region in the empty 70S and 80S ribosomes but becomes
ordered upon EF-G/EF2 binding,[96, 97, 99] which supports its
universal role in factor binding. Perhaps the best candidates
for the GAP role are a region of the 23S rRNA termed the
sarcin–ricin loop (SRL) and the pentameric stalk complex of
the ribosomal proteins L10·(L7/L12)4. The SRL is so named
because cleavage after G2661 of the bacterial 23S rRNA
within this region by a-sarcin inhibits all activities dependent
on the elongation factor.[100] Similar effects are seen after
removing the neighboring base A2660 (E. coli nomenclature)
in the 26S rRNA of yeast by the N-glycosidase activity of the
ricin A-chain.[101] Furthermore, this region contains the
longest (12 nucleotides) universally conserved stretch of
rRNA, which underlines its functional importance.
Recently, hybrid ribosomes were constructed in which the
proteins at the GTPase center from E. coli (L7/L12 and L10)
were replaced with their eukaryotic counterparts from rat P1/
P2 and P0, respectively.[102] Both the in vitro translation and
GTPase activity of the resultant hybrid ribosomes was strictly
dependent on the presence of the eukaryotic elongation
factors, EF2 and EF1a. This result reflects not only the
specificity of the interaction between the stalk proteins and
the elongation factors from each species, but also the
importance of the stalk proteins in mediating elongation
factor GTPase activity.
The ribosomal protein L11 (and associated L11 binding
site on the 23S rRNA) is often considered as a candidate for
taking over the function of the GAP. This is because
mutations in both L11 and its binding site on the 23S rRNA
can confer resistance against the antibiotic thiostrepton, a
potent inhibitor of EF-G- and EF-Tu-dependent GTPase
activities.[103] However, the direct involvement of L11 in the
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2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3481
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K. H. Nierhaus and D. N. Wilson
factor-dependent GTPase is not very likely, since mutants
lacking L11 are viable, although extremely compromised,[104]
and the IF-2-dependent GTPase is stimulated rather than
blocked by thiostrepton.[105] Furthermore, replacement of
either the L10·(L7/L12)4 complex or L11 with the equivalent
rat protein showed that the P0·(P1·P2)2 complex, but not the
eukaryotic counterpart to L11 (RL12), was responsible for
factor specificity and associated GTPase dependence,
although addition of L11 or RL12 did stimulate protein
synthesis significantly.[102] Thus, the role of L11 is unclear. L11
is in any case in proximity to the elongation factors: Cryo-EM
analyses of EF-G bound to 70S ribosomes revealed that the
N-terminal domain of L11 is shifted upon binding of EF-G so
as to form an arclike connection with the G domain of EFG.[106] This arclike connection is also observed in the EF2–80S
complex although it is broader and fused to a greater
extent.[99] Thus it seems likely that EF-G binding stimulates
conformational changes in the ribosome, probably through
the L10·(L7/L12)4 complex, which triggers translocation of
the A- and P-tRNAs. The question remaining is how are the
tRNAs actually moved?
2.4.2. Dynamics within the Ribosome
In the a-e model for translocation a moveable domain
within the ribosome is hypothesized to carry the A- and PtRNAs during translocation (see the review in Ref. [107]).
Evidence for this model comes from testing the accessibility
of phosphate groups on the tRNAs in the pre- and posttranslational states. The essential observation was that the
protection patterns of A- and P-tRNAs differ from one
another, but the corresponding tRNAs exhibit the same
protection patterns in the pre- and the posttranslational
states. This observation suggests that distinct ribosomal
components are involved in carrying the tRNAs from the
pre- to the posttranslational state. There are a number of
candidates that may play a role in the translocation of the
tRNAs or even constitute portions of the moveable domains.
Distinct regions within the crystal structures are disordered, which most likely reflects the flexibility of these
components. A classic example is the stalk region, which, as
already mentioned, only becomes ordered upon binding of
the elongation factor. Another is the L1 region, the flexibility
of which may regulate release of E-tRNA (see Section 2.5).
There are also certain structures that become either ordered
or rearranged upon association of the subunit. Most of these
elements are constituents of the bridges between the subunits.
One striking example is the universally conserved helix H69
in domain IV of the larger RNA subunit. H69 is the major
element of bridge B2a, the largest inter-subunit bridge (see
Figure 3), and is disordered in the structure of the 50S subunit
of H. marismortui, but ordered in the 50S subunit of D. radiodurans. Comparison of the latter structure with the 70S
structure of T. thermophilus shows that H69 swings out
towards h44, another very flexible element, upon association.
In this extended conformation H69 would be predicted to
make contact with both A and P tRNAs.[108] Another element
that is not fully resolved in either of the 50S structures is H38
of domain II, a constituent of bridge B1a and often called the
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2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
“A-site finger” because it contacts the A site of tRNA. As
previously mentioned, both B1a and B2a bridge elements
have corresponding counterparts within the 80S yeast ribosome, thus strengthening their candidacy for a role in
translocation.
2.5. The E Site and Translational Fidelity
2.5.1. Discovery of a Third Universal tRNA Binding Site on
Ribosomes
Despite identification of the E site in bacterial ribosomes
in the early 1980s,[109, 110] it is only in recent years that it has
appeared in some textbooks. The E site has also been
discovered in archea[111] and in eukaryotes, both yeast[112]
and mammals,[113] which suggests that it is a universal feature
of ribosomes. The E site is specific for deacylated tRNA and
will not bind peptidyl- or aminoacyl-tRNAs. This result is in
accordance with the idea that the E site only accepts the
deacylated tRNA from the P site following transfer of the
nascent chain to the A-tRNA. At least part of the controversy
over the existence of the E site stems from its instability under
certain buffer conditions (see the review in Ref. [107]). Stable
binding in the E site has been shown to be dependent on
physiological buffer conditions, such as in the presence of a
low magnesium concentration (3–6 mm) and polyamines.
Nonphysiological buffer conditions dissociate the E-tRNA,
as nicely illustrated by the visualization of tRNAs in
posttranslational complexes under different buffer conditions
using cryo-EM.[114, 115] Under nonphysiological conditions, the
E-tRNA electron density was lost from the E site itself and
extra electron density appeared contacting the L1 stalk (the
E2 site). It was thought that this position might reflect the
path of a tRNA that has left the E site as it dissociates from
the ribosome and that the very flexible L1 stalk might play a
role in dissociating the E-tRNA from the ribosome. This idea
has gained recent support from a comparison of the crystal
structure of the D. radiodurans 50S subunit with the T. thermophilus 70S structure, where the position of the L1 arm
differs by 308 (Figure 14). In the latter structure, the L1 arm is
closed and contacts the elbow of the E-tRNA, which prevents
its release from the ribosome. In contrast, the L1 arm in the
D. radiodurans structure is open, which may serve to release
the E-tRNA during translation.[108] In fact, this mechanism
may be universal. In yeast, there is an enormous 70 @
difference in the position of the L1 stalk in the P-tRNAbound 80S ribosome (pseudo-pretranslational state) and a
stalled translating ribosome (artificially induced posttranslational state).[3, 4] In light of the conformational flexibility of the
L1 stalk (see below), the E2 electron density may not even
represent a tRNA, which may have totally dissociated from
the ribosome; instead it may merely reflect a buffer-induced
conformational change within the L1 region.
The existence of the E site was also confirmed by the
presence of three tRNAs in the crystal structure of the
70S subunit of T. thermophilus.[76] The E-tRNA results predominantly from the presence of endogenous E-tRNA that
co-purified with the ribosomes, thus illustrating the stability of
the binding despite the lengthy purification procedure. The
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Chemie
Ribosome Structure and Translation
Figure 14. Movement of the L1 arm. A region of the crystal structure of
the D. radiodurans 50S subunit (D50S) showing the 23S rRNA (gray)
with the L1 arm highlighted (yellow). The position of the L1 arm in the
crystal structure of the T. thermophilus 70S subunit (T50S) is superimposed (green) with the putative pivot point for the L1 arm and is
marked with a red dot. The positions of the A- (cyan), P- (blue), and EtRNAs (pink) are included, with the anticodon arms pointing out of
the plane of the paper. Adapted from Harms et al.[108]
existence of the E site is finally being accepted, but its
importance has yet to be fully recognized.
2.5.2. Importance of the E-Site: Fidelity and Reading Frame
Maintenance
The fact that the E site has been discovered in the
ribosomes of both prokaryotes and eukaryotes suggests that it
should play an important role during translation. The
presence of a deacylated tRNA at the E site has been
shown to modulate aa-tRNA selection at the A site (see the
review in Ref. [116]). In particular, an occupied E site induces
a low affinity A site, such that interaction of the ternary
complex is dictated by anticodon interactions. This allows
only cognate and near-cognate tRNAs to bind, thus eliminating 90 % of the competing noncognate tRNAs (the importance of this was discussed in Section 2.2.1). In contrast, when
the E-tRNA is absent, the A site has high affinity, which
permits interaction of all tRNAs, including the erroneous
incorporation of noncognate tRNAs. These two situations
have been demonstrated in vitro.[117] Simply, in the presence
of P-tRNA alone, both noncognate Asp-tRNA and cognate
Phe-tRNA could bind to an A site UUU codon (Figure 15 a),
but in the presence of an E-tRNA (and P-tRNA), only the
cognate Phe-tRNA could bind the A site (Figure 15 b and c).
Binding of the A-tRNA releases the E-tRNA from the E site,
so that at any one time during translation, except during the
first decoding step, there are never more than two tRNAs
present on the ribosome. Furthermore, the E-tRNA must be
cognate to the E site codon, as a near-cognate E-tRNA could
not prevent erroneous incorporation of the Asp-tRNA.[117] It
is significant that a cognate tRNA must be present at the
E site; this observation indicates that codon–anticodon interAngew. Chem. Int. Ed. 2003, 42, 3464 – 3486
Figure 15. The role of the E site in the accuracy of decoding. Ribosome
complexes were prepared using poly(U)-mRNA where the P site was
occupied with AcPhe-tRNA. A-site binding of cognate Phe-tRNA
(codon UUU) and noncognate Asp-tRNA (codon GAC/U) was performed in the absence (a) or presence (b) of an E-tRNA. Binding of
the Phe-tRNA or Asp-tRNAs to the A site was monitored by measuring
the formation of the dipeptides AcPhe-Phe or AcPhe-Asp, respectively
(c). Both AcPhe-Phe and AcPhe-Asp were formed in the absence of EtRNA, which indicates that erroneous incorporation of Asp-tRNA at the
A site occurred, while normal amounts of AcPhe-Phe with background
levels of AcPhe-Asp were formed in the presence of E-tRNA, thus indicating that the cognate Phe-tRNA predominantly bound the A site.
Data taken from Geigenm%ller et al.[117]
actions at the E site is the signal that tells the ribosome to
adopt the posttranslational state and display a low affinity
A site. Further evidence for anticodon–codon interactions at
the E site comes from the gag-pol recoding site of HIV-1.
Ribosomes recode this site using a posttranslocational
slippage mechanism, that is, slippage into the 1 reading
frame occurs after translocation, where both P- and E-tRNAs
slip simultaneously in the 1 direction.[118] Another recoding
site illustrating the importance of the E site is the + 1
frameshifting site of the termination factor RF2. At codon
position 26 within the RF2 mRNA a + 1 frameshift is
required to avoid an internal UGA stop. This internal stop
codon is specifically recognized by the RF2 protein and thus
provides an autoregulatory mechanism, such that when RF2
protein levels are high in the cell, RF2 termination at the
frameshift is favored and a truncated and inactive RF2 protein is produced which is rapidly degraded. When RF2 levels
are low in the cell, termination loses out to frameshifting and
the full-length active RF2 protein is produced. One important
feature that stimulates frameshifting in this case is an
upstream Shine–Dalgarno (SD) type sequence that interacts
with a complementary anti-SD sequence within the
16S rRNA of the 30S subunit. Interestingly, the SD-anti-SD
interaction includes the first position of the E-site codon.
Recently it could be demonstrated that formation of the SDanti-SD duplex displaces the E-tRNA from the ribosome and
by doing so promotes frameshifting.[119] Thus the implication
is that the presence of the E-tRNA at the E site is necessary
for maintaining the correct reading frame during translation.
3. Summary and Outlook
Although we have taken a huge leap forward during the
past years in our understanding of ribosome structure and
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2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3483
Reviews
K. H. Nierhaus and D. N. Wilson
function, there is still much that eludes researchers. While
some of these questions remain fundamental to the process of
translation, for example, those pertaining to the mechanism of
peptidyltransferase, translocation, or the role of the tunnel,
there are many questions that are perhaps more peripheral,
being associated with translational regulation. This is particularly true in the case of eukaryotic translation, with its
multitude of factors, additional ribosomal proteins, and rRNA
expansion elements. Some of the answers to these questions
will arrive with higher resolution structures of ribosomal
complexes and translational intermediates. As high-resolution structures are dependent on the production of diffracting
crystals it is hard to predict with any certainty the time scale
for the preparation of eukaryotic ribosome crystals or
prokaryotic ribosome complexes, such as pre- and posttranslocational states, or with large translational factors bound.
Unfortunately, these are static structures but the ribosome is a
highly dynamic machine, therefore, dissection of its mechanism will require a concerted approach from many different
angles, both biochemical and biophysical. Given that protein
synthesis is fundamental to all life forms and provides such
insight into general molecular interactions, surely the pursuit
for further knowledge must be one worth striving for!
Abbreviations
A site
A-tRNA
CTD
E site, exit site, specific for
deacylated tRNA
E-tRNA
GAP
NTD
P site
P-tRNA
POST
PRE
PTF
SD
SRL
UTR
aminoacyl(aa)-tRNA site
tRNA in the A site
carboxy-terminal domain
tRNA at the E site
GTPase activating protein
amino-terminal domain
peptidyl-tRNA site before
peptide-bond formation
tRNA at the P site
posttranslocational state
pretranslocational state
peptidyltransferase
Shine–Dalgarno
sarcin–ricin loop
untranslated region of an
mRNA
We would like to thank Christian Spahn for supplying highresolution images of Figure 1 a–d, 3 b, 8 b and d, 8zlem Tastan
for kindly providing Figure 7, and Meredith Ross, Oliver
Vesper, and Sean Connell for critical reading of the Manuscript. D.N.W. would like to thank the Alexander von Humboldt foundation for support. K.H.N. acknowledges the
support of the Deutsche Forschungsgemeinschaft (Ni174/8-2
and Ni174/-9-2).
Received: July 23, 2002 [A544]
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