Acta Biochim Biophys Sin 2012, 44: 281– 299 | ª The Author 2012. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmr118.
Advance Access Publication 18 January 2012
Review
Structural and functional topography of the human ribosome
Dmitri Graifer and Galina Karpova*
Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, Novosibirsk 630090, Russia
*Correspondence address. Tel: þ7-383-363-5140; Fax: þ7-383-363-5153; E-mail: [email protected]
This review covers data on the structural organization
of functional sites in the human ribosome, namely, the messenger RNA binding center, the binding site of the hepatitis
C virus RNA internal ribosome entry site, and the peptidyl
transferase center. The data summarized here have been
obtained primarily by means of a site-directed crosslinking approach with application of the analogs of the respective ribosomal ligands bearing cross-linkers at the
designed positions. These data are discussed taking into
consideration available structural data on ribosomes from
various kingdoms obtained with the use of cryo-electron
microscopy, X-ray crystallography, and other approaches.
Keywords
human ribosome; photoaffinity crosslinking; mRNA and tRNA binding site; hepatitis C IRES
binding site; ribosomal component
Received: September 22, 2011
Accepted: November 6, 2011
Introduction
Ribosomes are cellular organelles that translate the genetic
information incoming as messenger RNA (mRNA) into the
polypeptide chains of proteins. This function is common to
the ribosomes of all organisms, from eubacteria to humans.
During translation, the ribosome interacts with two types
of specific RNA ligands, the mRNA and transfer RNA
(tRNA). Each species of tRNA carries a specific amino
acid residue for the synthesis of a polypeptide chain.
Insertion of the required amino acid residue into the
growing polypeptide chain at the right site is achieved due
to the recognition of an mRNA codon at the ribosomal
aminoacyl (A) site by the anticodon of the cognate
aminoacyl-tRNA via base pairing. This recognition leads to
the binding of the cognate aminoacyl-tRNA at the A site.
Then the ribosome catalyses formation of the peptide bond
between the amino acid residue of the aminoacyl-tRNA at
the A site and the polypeptide moiety of the
peptidyl-tRNA bound at the neighboring peptidyl (P) site.
Each tRNA is specific to only one amino acid; the correspondence between mRNA codons and amino acid residues
is governed by a genetic code that is practically universal
for cytoplasmic ribosomes from all kingdoms.
The ribosome is a very complicated molecular machine
consisting of two subunits, small and large ones; each contains ribosomal RNA (rRNA) and several dozen ribosomal
proteins. The structures of ribosomes from prokaryotes and
eukaryotes share significant similarity. The secondary
structures of rRNAs possess conserved cores common to
all kingdoms; in eukaryotes rRNAs are longer than in prokaryotes and the conserved cores are interrupted in several
sites by variable regions named expansion segments [1]
(http://www.rna.ccbb.utexas.edu). Eukaryotic ribosomal
subunits contain a complete set of ribosomal proteins homologous to their eubacterial counterparts plus a number of
additional proteins (about one-third of the total number).
The locations of the main functional sites on the subunits
are in general similar for ribosomes from all kingdoms.
The small subunits carry an mRNA binding center, including the decoding site where aminoacyl-tRNA cognate to
the mRNA codon at the A site is selected, and the large
subunits contain the peptidyl transferase center (PTC) responsible for catalysis of peptide bond formation. Three
tRNA binding sites, namely, A, P, and E (E is exit site for
discharged tRNA where it binds before leaving the ribosome) are formed by the interfacial areas of both subunits.
Eukaryotic ribosomes are able to translate not only cellular mRNAs but also viral RNAs, but the mechanisms
of translation initiation of cellular and viral RNAs are
generally different. Cellular mRNAs contain a special
7-methylguanosine cap structure at their 50 -termini that is
recognized by initiation factor eIF4E, which, together with
other initiation factors, promotes the binding of 40S subunits complexed with initiator Met-tRNAMet
and eIFs (i.e.
i
0
the 43S complex) to the 5 -terminus of mRNA. The subsequent movement of a 40S subunit along the mRNA (scanning) leads to positioning of the AUG codon at the P site,
where it interacts with the initiator tRNA [2,3]. An
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 281
Structural and functional topography of the human ribosome
alternative mechanism is utilized by certain uncapped viral
RNAs that contain in their 50 -untranslated region (UTR)
highly structured regions, called IRESs (internal ribosome
entry sites) that can bind to 40S subunits such that the initiation AUG codon is placed immediately at the P site region
[4–6]. In particular, IRES-mediated translation initiation is
inherent to the RNA of hepatitis C virus (HCV), one of the
most dangerous human pathogens. One of the key steps of
the viral life cycle in human host cells is the initiation of
viral RNA translation, which implies that ribosomes are key
players in progression of the viral infection in the cell.
Clearly, knowledge of the ribosome structure and an
understanding of the molecular mechanisms of protein synthesis and its regulation are of basic importance for life
sciences. The structure of the prokaryotic ribosome has
been extensively studied by various biochemical approaches
[7,8]. The accumulated data enabled the later X-ray crystallographic images to be interpreted to reveal the ribosome’s
structure at the atomic level [9–12]. However, eukaryotic
ribosomes, especially mammalian ones, despite their
general similarities to the prokaryotic ribosomes, are much
more complicated and less studied. This can be explained,
in particular, by the fact that several approaches that have
been fruitfully used for studying eubacterial ribosomes are
not applicable yet to mammalian ribosomes, e.g. approaches
based on the reconstitution of active ribosomal subunits
from the total ribosomal protein and rRNA because there is
still no methodology for the reconstitution of eukaryotic
ribosomal subunits in vitro. One of these approaches
includes site-directed mutagenesis to determine the roles of
particular nucleotides of rRNA and amino acid residues of
proteins or site-directed introduction of cross-linking groups
into proteins to identify their inter- or intra-subunit contacts.
Cryo-electron microscopy (cryo-EM) has been applied
widely to eukaryotic ribosomes in the last decade [13 –16]
but it has not yet provided direct visualization of mRNA
codons or the acceptor terminus of aminoacyl-tRNA on the
ribosome, although the locations of eukaryote-specific ribosomal proteins on the subunits have been reported recently
[17]. X-ray crystallography has been applied so far only to
lower eukaryotic ribosomes free from mRNA and tRNAs
[18,19]. The available data on ribosomal structure remain,
to a significant extent, contradictory, especially in the localization of ribosomal proteins that have no eubacterial counterparts. In particular, the structures and locations of
proteins S4e, S17e, S24e, S21e, S26e, and S30e are strongly
or even completely different in X-ray crystallographic [19]
and cryo-EM [17] studies. Moreover, one can find striking
differences between the locations of several ribosomal proteins on the 40S subunit in the structures obtained by X-ray
crystallography in different groups [18,19]. In particular, the
sets of ribosomal proteins that comprise the subunit beak
differ in these studies.
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 282
Direct data on the structural and functional organization
of eukaryotic, in particular, human, ribosomes have been
obtained with the use of a site-directed cross-linking (affinity labeling) approach that had been applied successfully to
eubacterial ribosomes [20–24]. The approach is based on
the use of chemically active analogs of various ribosomal
ligands (mRNAs, tRNAs, and so on) that bear reactive
groups (cross-linkers) to reveal the ribosomal components
contacting these ligands or located near their binding sites.
The cross-linkers are shown not to interfere with the formation of the specific complexes of the analogs with the ribosome. After formation of the cross-links within the
complex and identification of the cross-linked ribosomal
components, conclusions concerning the structure of the
ribosomal ligand binding centers can be drawn. The application of affinity labeling approaches to study ribosomes
was pioneered as early as 40 years ago with chemically
reactive derivatives of tRNA [25] and mRNA [26].
In the present review, we summarize experimental data
on studying the structural and functional topography of
human ribosomes that were obtained with the use of chemically reactive analogs of mRNA, HCV IRES, and tRNA
bearing a cross-linker at designed locations. A comparison
of the cross-linking results with cryo-EM and X-ray data
on the structures of eukaryotic ribosomes made it possible
to draw conclusions concerning the structural organization
of functional sites in human ribosomes and to reveal similarities and differences in the organization of these sites in
mammalian and eubacterial ribosomes.
Reactive Analogs of Ligands Used for
Affinity Labeling of Human Ribosomes
Photoactivatable mRNA analogs
In the majority of studies, derivatives of relatively short (6–
12 nt long) oligoribonucleotides were used as mRNA
analogs. These derivatives bore a p-azidotetrafluorobenzoyl
(ATB) group at the C5 of the selected uridine or the N7 of
the selected guanosine, or the C8 of the selected adenosine,
or at the 50 -terminal phosphate (Fig. 1 ). To introduce the
ATB group in oligoribonucleotides, a general approach was
used based on the initial insertion of a spacer containing an
aliphatic amine group at the designed nucleotide residue
with subsequent selective benzoylation of this amino group
with an N-hydroxysuccinimide ester of p-azidotetrafluorobenzoic acid [27–29]. Application of this type of mRNA
analogs has two major advantages. First, the photoactivatable
group used provides a relatively high yield of cross-links
upon short (1 min) irradiation with mild ultraviolet (UV)
light (l . 290 nm). Perfluoro-aromatic azides excited with
UV light lose a nitrogen molecule and turn into the biradical
nitrene, which is an active intermediate and can give a high
yield of cross-links with both nucleic acids and proteins
Structural and functional topography of the human ribosome
sequence adjacent to the target site. An approach for sitespecific modification of nucleic acids was suggested by
Prof. Grineva and initially was called as ‘complementary
addressed modification’ [37]. Site-specific alkylation
occurs at any RNA nucleotide (except for U) that is
located close to the modified terminus of the
deoxy-oligomer derivative bound in the complementary
complex with the target RNA sequence, leading to the formation of a covalent adduct [38]. After hydrolysis of the
phosphoramide bond in the covalent adduct, an aryl azide
moiety is selectively introduced at the liberated aliphatic
amine group (Fig. 2). The nucleotides alkylated with oligonucleotide derivatives could be determined readily using
the property of reverse transcriptases to make stops or
pauses at the modified nucleotides (see below). Using this
strategy, a set of five HCV IRES derivatives bearing the
cross-linker in the domain II and subdomains IIId and IIIe
were obtained (Fig. 3).
Figure 1 Modified nucleotides that were introduced into short mRNA
analogs, derivatives of oligoribonucleotides
[30–32]. Second, short mRNA analogs can be exactly positioned on the ribosome by the addition of tRNA cognate to
the selected mRNA codon (see below).
In addition to aryl azide derivatives of oligoribonucleotides that provide information on the mRNA environment
on the ribosome, longer mRNA analogs (42–45-mers)
containing single 4-thiouridine (s4U) at designed positions
were used. This type of mRNA analogs can form ‘zerolength’ cross-links reflecting direct contacts of mRNA with
the ribosome. Such analogs had been widely applied
earlier for studying mRNA binding center of eubacterial
ribosomes [8]; s4U-containing mRNA analogs can be
easily prepared by in vitro T7 transcription from synthetic
DNA templates [33,34].
Photoactivatable HCV IRES derivatives
An HCV RNA fragment corresponding to nucleotides
40–372 was used as an IRES element. This fragment was
obtained by in vitro transcription of an appropriate plasmid
using either 32P-labeled or biotinylated nucleoside
triphosphates [35,36]. To introduce ATB groups at the
selected HCV IRES positions, the strategy was based on
the site-specific alkylation of an RNA with [4-(N2-chloroethyl-N-methylamino)benzyl]phosphoramide derivatives of oligodeoxyribonucleotides complementary to a
tRNA analogs
Two types of reactive tRNA analogs were applied to study
the molecular environment of the 30 -terminus of tRNA on
the ribosome: analogs bearing s4U as an additional
30 -terminal nucleotide [39], and an analog with an oxidized
30 -terminal ribose [40]. The s4U-containing derivative was
obtained from yeast tRNAAsp transcript by ligation with
[32P]ps4Up and subsequent dephosphorylation of the
product [41].
Model Complexes of Ribosomes with mRNA
and tRNA Analogs
Cross-linking of mRNA and tRNA analogs with ribosomes
was investigated in model complexes obtained without
translation factors at 208C and Mg2þ concentrations
of 10–13 mM (in several experiments, lower Mg2þ concentrations and the polyamines spermine and spermidine
were used whereever specified). The scheme for complex
formation was based on two main observations. First, it
was generally accepted that tRNAs in all forms have higher
affinities to the ribosomal P site in the absence of translation factors [42 –44]. The second was that the binding of
short (up to 12 nt long) mRNA analogs to 80S ribosomes
depends almost completely on the presence of the tRNA
cognate to one of its codons (i.e. is not detected without
tRNA [27,45–47]). Therefore, in the complex with the 80S
ribosome, an mRNA analog is fixed on the ribosome in a
designed position by interaction with the tRNA anticodon
at the P site. To obtain complexes where, in addition to the
P site, either the A or the E site was occupied with tRNA
molecule too, tRNA cognate to the codon present at the respective site was bound to the ternary complex of 80S
ribosomes with the mRNA analog and tRNA at the P site.
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Structural and functional topography of the human ribosome
Figure 2 Scheme of the site-specific introduction of a photoactivatable group into selected RNA sites It was based on the
complementary-addressed alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides.
Figure 4 Examples of model 80S ribosomal complexes with mRNA
analogs consisting of two to four codons mRNA positions, in which
cross-linkers have been introduced, are marked ( position þ1 corresponds
to the first nucleotide of the P site-bound codon). A, P, and E are the
tRNA binding sites.
Figure 3 Structural organization of the HCV IRES [6] The initiation
AUG codon is underlined. RNA sequences complementary to
deoxy-oligomers used for site-specific modification of the HCV IRES are
marked with thick lines with arrows indicating the 50 -phosphates
derivatized with alkylating groups. Nucleotides of the HCV IRES
modified with the oligonucleotide derivatives are shaded.
Generally, Escherichia coli tRNAs were used instead of
the human species since they are more widely available
and for tRNAPhe it had been shown that the qualitative and
quantitative binding properties of human and E. coli
tRNAs are practically the same [42]. To obtain 80S preActa Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 284
termination complexes, mRNA analogs containing a stop
codon with a cross-linker targeted to the A site were used;
the addition of eRF1 to these complexes led to the formation of the termination complexes [48–51]. Examples of
model complexes of mRNA analogs with 80S ribosomes
and tRNAs are presented in Fig. 4. It is seen that the crosslinker could be placed in a wide spectrum of positions in
the mRNA analog, i.e. in the P site-bound codon, or 50 of
it, and the A site-bound codon, or 30 of it. To obtain comprehensive information about the arrangement of mRNA
binding centers in the human ribosome, we used a large set
of mRNA analogs that varied in their lengths, sequences,
and positions of the cross-linker.
It is not so easy to achieve unambiguous tRNA-directed
positioning on the 80S ribosome with mRNA analogs containing several dozen nucleotides, since polyribonucleotides often have a large intrinsic affinity for ribosomes in
the absence of tRNA. In these cases, the presence of tRNA
does not lead to a change in the location of the mRNA
Structural and functional topography of the human ribosome
the first step of the cap-independent initiation of HCV
RNA translation [5,6]. Introduction of ATB groups at specific positions in domain II and subdomains IIId/e of
HCV IRES did not interfere with its ability to bind to
40S subunits [36].
Model complexes of 80S ribosomes with tRNAAsp
analogs were obtained as described above, and a 12-mer
oligoribonucleotides bearing an Asp codon were used as
mRNA analogs (Fig. 6).
Cross-linking Procedure and Identification
of the Cross-linked Ribosomal Components
Figure 5 Sequences of s4U-containing mRNA analogs used for
cross-linking and an example of the model 80S ribosomal
complex Codons targeted to the P site are marked and underlined.
Positions of s4U with respect to the first nucleotide of the P site-bound
codon are indicated.
Figure 6 Types of complexes of 80S ribosomes with s4U-containing or
30 -terminal oxidized tRNAAsp analogs used in the cross-linking
experiments
analog, shown by unaltered binding levels and crosslinking patterns of these analogs in the ribosomal complexes [52,53]. To overcome this difficulty, special
adenine-rich mRNA analogs were designed whose affinities to 80S ribosomes were relatively low [54,55]. With
these analogs, the addition of tRNA cognate to the selected
codon resulted in an increase in the level of mRNA
binding and the appearance of new cross-links that could
be unambiguously assigned to the complex where mRNA
analog was fixed on the ribosome by codon–anticodon
interaction at the P site. The sequences of longer mRNA
analogs containing s4U residues at various positions and
the types of complexes that they form with 80S ribosomes
are presented in Fig. 5.
The cross-linking of HCV IRES derivatives was
studied in their binary complexes with 40S subunits
obtained at 2.5 mM Mg2þ concentration. Under these conditions, the HCV IRES forms a stable binary complex, in
contrast to all other RNAs; formation of this complex is
All mRNA analogs were 50 -32P-labeled prior to use in
cross-linking experiments; in some experiments,
s4U-containing analogs were labeled at the internal phosphate 30 to the s4U residue. In the latter case, labeled analog
was obtained by ligation of two ‘halves’, the 30 -half being
50 -endlabeled [34]. To obtain cross-links with photoactivatable mRNA and tRNA analogs, the respective ribosomal
complexes were irradiated with mild UV light; the conditions of irradiation were somewhat different between
mRNA analogs bearing aryl azides [47] and s4U [54]. A
tRNAAsp analog with an oxidized 30 -terminal ribose was
cross-linked to ribosomal proteins by incubation of the ribosomal complex with sodium cyanoborohydride. With short
photoactivatable mRNA analogs and with tRNA analogs
(bound to 80S ribosomes in the presence of short mRNA
analogs), the distribution of the cross-linked 32P-labeled
analog between the ribosomal subunits can be examined
easily by centrifugation in a sucrose density gradient
(10%–30%) under conditions, in which 80S ribosomes are
dissociated into the subunits (at 3 mM Mg2þ concentration
and 300–500 mM KCl) [27,28,46,47,49]. Under these conditions, complexes dissociate, so radioactivity in the subunit
fractions indicates covalent attachment of the labeled
mRNA or tRNA analog. Generally, mRNA analogs crosslinked only to the 40S subunits, and tRNA analogs to the
60S subunits. The distribution of the cross-links between
the rRNA and ribosomal proteins in the isolated 40S subunits could be examined easily, after denaturing the crosslinked subunits by incubation in sodium dodecyl sulfate
(SDS) and ethylenediaminetetraacetic acid (EDTA), by
either polyacrylamide gel electrophoresis (PAGE) [47,56] or
centrifugation in a sucrose density gradient in the presence
of SDS and EDTA [45,46,57].
Determination of cross-linking sites in rRNA
The methodology that was applied for identifying the
nucleotides cross-linked in rRNA is based on a two-step
procedure. At first, the rRNA sequences containing sites of
cross-linking of 32P-labeled mRNA or tRNA analogs are
determined using hydrolysis of cross-linked rRNA with
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Structural and functional topography of the human ribosome
RNase H in the presence of deoxy-20-mers complementary
to various rRNA sequences, with subsequent separation of
the resulting RNA fragments by PAGE [46,47,49,57]. The
exact identification of the cross-linked rRNA nucleotides is
performed by primer extension using the cross-linked
rRNA as a template; primers, typically deoxy-20-mers, are
chosen based on the results of experiments with RNase
H. The cross-linking site is generally assumed to be the nucleotide 50 to the primer extension stopping ( pause) site
whose position is determined by PAGE analysis of the
primer extension products in a denaturing gel, with a parallel sequencing reaction on unmodified rRNA. Control
experiments were done with rRNA isolated from unmodified subunits that passed through the same procedures as
the modified ones, but without chemically reactive ribosomal ligands.
Identification of cross-linked ribosomal proteins
With mRNA analogs, total 40S ribosomal protein was isolated from the modified subunits, and labeled proteins were
identified using one- and two-dimensional PAGE in
various electrophoretic systems with subsequent autoradiography of the gels [28,45,47,54,56]. The radioautograms
were superimposed on the respective stained gels and the
positions of the radioactive bands with respect to those of
unmodified proteins were determined. In those cases when
it was possible without loss of the label, prior to electrophoresis, mRNA analogs cross-linked to proteins were
hydrolysed with RNase A to reduce the effect of oligonucleotide on the electrophoretic mobility of the protein. In
some cases, the identity of a cross-linked protein was confirmed by immunoblotting [47], mass spectrometry [54], or
other approaches [47].
To identify ribosomal proteins cross-linked to tRNA
analog oxidized at the 30 -terminal ribose, cross-linked
protein was isolated by one-dimensional PAGE, and the
appropriate piece was excised from the gel, treated with
RNase T1 and proteinase K, and the resulting peptides
were determined by mass spectrometry to identify the
cross-linked protein [40].
Ribosomal proteins cross-linked to HCV IRES
derivatives were identified by one-dimensional and twodimensional PAGE after exhaustive hydrolysis of the crosslinked IRES with RNases A and T1 [35,36]; the identities
of the cross-linked proteins were then confirmed by immunoprecipitation with the use of rabbit antibodies specific to
the candidate proteins [36].
Arrangement of mRNA Binding Site of the
Mammalian Ribosome
The application of a large set of mRNA analogs varying in
their sequence, the nature of cross-linker and the site of its
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 286
attachment allowed us to obtain detailed information about
the molecular environment of mRNA nucleotides in positions from 29 to þ12 with respect to the first nucleotide
of the P site codon on the 80S ribosome (Tables 1 and 2
and Fig. 7). This made it possible to find universal features
of the mRNA binding site in all kingdoms and to identify
peculiarities specific to eukaryotic (or even to mammalian)
ribosomes. Cross-linking data summarized in Table 1
show that cross-linking of mRNA analogs to the 18S
rRNA occurs mainly when modified nucleotides are
located at the A or the P site (in positions from þ6 to
þ1); cross-linking to ribosomal proteins is observed as
well. The yield of the cross-links with ribosomal proteins
is similar to that with the 18S rRNA, while in eubacterial
30S subunits A and P site codons of mRNA contact only
rRNA. mRNA regions 50 of the P site codon or 30 of the A
site codon cross-link presumably to ribosomal proteins.
Moreover, those mRNA nucleotides that are the most
distant from the A and the E sites (both downstream and
upstream ones) neighbor practically only ribosomal proteins. All these indicate that proteins play more significant
roles in the formation of the ribosomal mRNA binding site
in eukaryotes than in eubacteria.
Nucleotides of 18S rRNA at the mRNA binding site
Cross-linking of short ATB-derivatized mRNA analogs to
the 18S rRNA was completely position specific, i.e. it was
not observed in binary mixtures of the analogs with 80S
ribosomes. All cross-linked 18S rRNA nucleotides fall into
the conserved core of the small subunit rRNA secondary
structure (Fig. 8), which makes it possible to find corresponding nucleotide positions in the eubacterial 16S
rRNA. It shows that positions of cross-linked 18S nucleotides almost completely coincide with those of 16S rRNA
nucleotides that interact with or neighbor the respective
mRNA positions known from X-ray data on complexes of
mRNA with eubacterial ribosomes [9–11]. This is clearly
seen when these 16S rRNA nucleotides are marked on the
atomic model of the eubacterial 30S ribosomal subunit
(Fig. 9). Thus, our studies experimentally confirmed the
widely accepted idea on the conserved rRNA ‘core’ of the
ribosome and showed that the small subunit rRNA core is
involved mainly in the binding of mRNA codons interacting with tRNAs at the A and the P sites. It should be noted
that 18S rRNA nucleotides cross-linked to s4U and
ATB-derivatized nucleotides placed at the same mRNA
positions in some cases do not exactly coincide (Table 1).
However, in the 30S subunit spatial structure, 16S rRNA
positions corresponding to these 18S rRNA nucleotides are
very close (within 10 –15 Å) to each other. Thus, crosslinks of perfluorophenyl azide-modified mRNA analogs to
the 18S rRNA actually reflect the immediate neighborhood
of the derivatized mRNA nucleotides in the eukaryotic
Structural and functional topography of the human ribosome
Table 1 Summary of mRNA analogs cross-links with the 18S rRNA in human 80S ribosomal complexes
Position of nucleotide bearing
cross-linker with respect
to the first nucleotide
of the P site codon
Nature of the
modified nucleotide
and the cross-linker
Share of cross-linked 18S
rRNA (in % of the total
amount of cross-linked 40S
components)
Cross-linked 18S rRNA nucleotides
(corresponding nucleotides of
Escherichia coli 16S rRNA
are given in brackets)
References
29
26
24
U, G (ATB)
U, G (ATB)
U, G (ATB)
,2
,3
,3
–
–
–
[47]
23
23
50 p (ATB)
U (ATB)
6–8
7
G1207 (G926)
G961 (G693)
[57]
[46]
23
22
21
G (ATB)
U (ATB)
U (ATB)
n.d.
n.d.
n.d.
G1207
G961
G1207
[58]
23, 22, 21
þ1
þ1
þ1
þ2, þ3
þ4
þ4
þ4
s4U
50 p (ATB)
U (ATB)
G (ATB)
U (ATB)
G (ATB)
U (ATB)
s4U
n.d.
40– 70
50
30
n.d.
70
n.d.
,30
Fragment 1699 – 1704 (1398– 1403)
G1207
G1207
G1702 (G1401)
G1207
A1823 – 1825 (1491 – 1493)
A1823 – 1825
C1696 (C1395)
[55]
[57]
[46]
[47]
[58]
[46]
[47,49]
[54]
þ5
þ5
þ6
þ6
U
G
U
G
30
n.d.
30
n.d.
A1823 – 1825
A1823 – 1825, G626 (G530)
A1823 – A1825
A1823 – A1825, G626
[47]
þ5, þ6
s4U
n.d.
C1696 (1395), C1698 (C1397),
fragment 1820 – 1825
[55]
þ7
G/U (ATB)
20/8
[59]
þ9
G/U (ATB)
10/7
þ12
G/U (ATB)
2
A1823 – A1825, region 605 – 630
(509 – 534), S1698
A1823 – A1825, region 605 – 630,
S1698
A1823 – A1825, region 605 – 630,
S1698
(ATB)
(ATB)
(ATB)
(ATB)
Major cross-links are given in bold; n.d., not determined.
ribosomal complexes. Moreover, molecular environment of
the A site codon practically did not depend on the codon
type (sense or stop codon) and on the presence of termination factor eRF1 [49,63]. Evidently, the application of different types of cross-linkers provided correct and
comprehensive information on the molecular environment
of mRNA on the ribosome. Remarkably, data very similar
to those presented in Table 1 have been obtained in
Pestova’s group, where a panel of mRNA-containing s4U
at positions from 226 to þ11 relatively to the first nucleotide of the AUG codon were used for cross-linking in
rabbit 48S and 80S initiation complexes assembled with
the use of a set of the appropriate initiation factors [64,65].
Thus, the conserved core of mammalian 18S rRNA accommodates mRNA very similarly during all steps of
translation.
Protein environment of mRNA region 30
of the A site codon
Similarities in protein environments of mRNA on eubacterial and mammalian ribosomes concern presumably mRNA
region 30 of the A site codon that neighbors mainly ribosomal protein (rp) S3e (major) and rpS2e (Table 2, Figs. 7
and 9), which is confirmed by the data obtained with
s4U-containing mRNA analogs bound in 48S and 80S initiation complexes [65]. RpS3e and rpS2e are homologous to
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Structural and functional topography of the human ribosome
Table 2 Summary of mRNA analogs cross-links with 40S ribosomal proteins in human 80S ribosomal complexes
Position of nucleotide bearing
cross-linker with respect to the first
nucleotide of the P site codon
Nature of the modified
nucleotide and the
cross-linker
Share of cross-linked ribosomal proteins Cross-linked
(in % of the total amount
ribosomal
of cross-linked 40S components)
proteins
References
[47]
29
29
26
26
24
U (ATB)
G (ATB)
U (ATB)
G (ATB)
U, G (ATB)
.98
.98
.97
.97
.97
S26
S26, S14
S26
S26, S14
S26, S3
23
U (ATB)
93
S3, S26, S2, S5/ [60]
S7, S14
þ1
U (ATB)
50
S3, S26, S2,
S14, S23
[60]
þ4
G (ATB)
30
S15, S2, S30
[28]
þ4
þ5
þ6
U (ATB)
50
70
70
S15, S3
S15, S3
S15, S3
[47]
þ4
s4U
.70
[54]
þ5, þ6
s4U
n.d.
S15, S3, S3a,
S2, S30
S15, S2/S3/
S3a, S30
þ7
G/U (ATB)
80/92
[56]
þ9
G/U (ATB)
90/93
þ12
G/U (ATB)
98
S3, S15, S2,
S30
S3, S2, S15,
S30
S3, S2
Major cross-links are given in bold; n.d., not determined.
Figure 7 Schematic representation of the protein neighborhood of
mRNA on the 80S ribosome Solid lines correspond to the major
neighbors; eubacterial counterparts of human ribosomal proteins are given
in brackets.
prokaryotic rpS3p and rpS5p, respectively, which are
known as components of the ‘ribosomal mRNA entry site’
[66]. Earlier, rpS3p and rpS5p were found to cross-link to
mRNA analogs with s4U residues in positions þ8 and
þ11 [67]. According to X-ray data, these proteins interact
with phosphates of mRNA nucleotides in positions þ11 to
þ15 [68]; in particular, residues Arg131, Arg132, Lys135,
and Arg164 in the rpS3p contact mRNA. Alignment of
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 288
amino acid sequences of Thermus thermophilus rpS3p and
human rpS3e shows that three of these residues (Arg131,
Arg132, and Lys135) are located in the conserved parts of
the proteins [69]. A rough mapping of the mRNA analogs
cross-linking sites on the rpS3e using CNBr-induced cleavage of the cross-linked protein with the subsequent identification of the labeled peptides reveals that these sites are
actually located in the N-terminal half of the protein (amino
acid 2–127) containing all three above-mentioned conserved
amino acid residues [69]. This indicates that arrangements of
mRNA regions 30 of the A site codon on the eubacterial and
mammalian ribosomes share significant extent of similarity.
However, an X-ray crystallographic study of yeast 80S ribosome [18] showed that arrangement of the ribosomal area
corresponding to the region of mRNA entry site was substantially different in 70S and 80S ribosomes. In particular,
distinctions have been found in protein–rRNA and protein–
protein interactions involving rpS3e and rpS2e. Thus, similarities in the organization of binding sites of the mRNA
Structural and functional topography of the human ribosome
Figure 9 Path of mRNA on the eubacterial 30S subunit as revealed
from the X-ray study of a complex of eubacterial 70S ribosome with
mRNA and tRNAs [62] Locations of 16S rRNA nucleotides whose
positions in the secondary structure correspond to those 18S rRNA
nucleotides that cross-linked to mRNA analogs in the human ribosome
(are given in brackets) are marked with yellow circles. The mRNA
positions are presented in red boxes. Locations of several 30S proteins
homologous to cross-linked human 40S proteins are shown.
Figure 8 Positions of cross-linked nucleotides in the 18S rRNA
secondary structure It was taken from http://www.rna.ccbb.utexas.edu.
The results obtained with ATB-derivatized and s4U-containing mRNA
analogs are shown with solid and dotted lines, respectively. Positions of
nucleotides bearing cross-linkers with respect to the first nucleotide of the
P site codon are boxed. Helices of the 18S rRNA containing cross-linked
nucleotides are numbered according to [61].
region 30 of the A site codon on 70S and 80S ribosomes are
limited, and further more detailed mapping of mRNA crosslinking sites on rpS3e and rpS2e could make it possible to
reveal differences in mRNA binding tracks in the 70S and
80S ribosomal entry site regions.
It should be noted that cross-linking of mRNA analogs
to rpS3e and/or rpS2e was detected also in the majority of
other complexes where nucleotide bearing the cross-linker
was in positions from 24 to þ6 (Table 2). Besides, with
all mRNA analogs used, cross-linking to these proteins
was observed in binary complexes of the analogs with 80S
ribosomes that were hardly detectable by nitrocellulose filtration technique due to their lability. Evidently, this crosslinking occurred because of rapid turnover of mRNA
analogs in these complexes leading to relatively high yield
of cross-linking in spite of low stability of the complexes.
Therefore, rpS3e and rpS2e were not assigned to the environment of mRNA nucleotides in positions from 24 to
þ6. Evidently, all mRNA analogs have a pronounced affinity to the 80S ribosomal entry site, where they bind in a
labile manner in the absence of tRNA.
Protein environment of mRNA at the decoding site
The most distinctive feature of the protein environment
of the A site codon on the 80S ribosome is rpS15e.
This protein cross-links to ATB-derivatized nucleotides of
mRNA analogs in positions þ4 to þ7 (the yield of crosslinking decreases significantly when the modified
nucleotide moves from position þ4 to position þ6 [47];
cross-links from position þ7 are minor, and from downstream positions are hardly detectable [56]). mRNA analogs
bearing s4U cross-linked to rpS15e when the photoactivatable nucleotide was in positions þ4 and þ5 in complexes
representing elongation [54] and initiation [64] steps of
translation. All these data imply that rpS15 contacts the first
and second nucleotides of the A site codon and is relatively
close to mRNA nucleotides in positions þ6 and þ7.
RpS15e is homologous to eubacterial rpS19p that is located
mainly on the 30S subunit head away from the mRNA
track, but has a C-terminal tail extending toward the decoding site [68,70]. It was suggested that the tail of mammalian
rpS15e comes closer to the decoding site than that of its eubacterial counterpart rpS19p [47]. RpS15e has been mapped
on the head of 40S subunit in several cryo-EM studies
based on docking of known atomic structure of eubacterial
rpS19p into the 40S density map [15–17], but the unstructured C-terminal tail that has almost no sequence homology
with the tail of eubacterial rpS19p [71], could not be
mapped this way. Moreover, the main part of the
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Structural and functional topography of the human ribosome
tail of eubacterial rpS19p, is able to reach the ribosomal decoding site and interact with the mRNA due to its larger
length and specific location in the 40S subunit. A variant of
location of the C-terminal sequence of human rpS15e with
respect to the mRNA based on similar positioning of the
structured parts of rpS15e and rpS19p in the small ribosomal subunits [15–17] has been proposed (Fig. 10). In the
atomic model of the 40S subunit [19], the last solved
residue, an amino acid in position 130, is 30 Å away from
of the putative site for the A site codon of mRNA (deduced
on the basis of similar positioning of the A site codon with
respect to the conserved rRNA decoding site in prokaryotic
and eukaryotic ribosomes). These are in a good agreement
with our results and with the proposed variant of location of
the C-terminal tail of rpS15e in the 40S subunit (see above).
Figure 10 A proposed variant of the location of the C-terminal
sequence of human rpS15e with respect to the mRNA The scheme is
based on similar locations of the structured parts of rpS15e and rpS19p in
the small ribosomal subunits. Atomic model of the 70S ribosomal
complex was taken from [70] (PDB ID code 1IBM). All components of
the complex except rpS19p and the mRNA were removed from the
model; a minimal distance between these two molecules is shown. The
C-terminal sequence 120– 145 of human rpS15e ( presented in dark red)
was built arbitrarily by sequential attachment of the respective amino acid
residues taken from the model 1IBM, and then attached virtually to the
structured part of rpS19p ( presented in purple) to fit the cross-linking
results. Boxed tetrapeptide 137-HSSR-140 is the most probable candidate
for interaction with the mRNA codon.
unstructured C-terminal tail of rpS15e is lacking in the 40S
subunit atomic structure obtained by X-ray crystallography
[19]. To determine peptides of rpS15e cross-linked to
labeled mRNA analogs in the 80S ribosomal complexes,
cross-linked labeled rpS15 was isolated from the irradiated
ribosomal complexes by SDS–PAGE [71]. Radioactive
bands corresponding to cross-linked rpS15e were excised
from the dried gels and treated with various specific proteolytic agents with subsequent electrophoretic separation of
the resulting modified oligopeptides. Then these oligopeptides were identified based on comparison of the electrophoretic data with the map of rpS15e cleavages. Finally, the
cross-link was mapped to the C-terminal rpS15e decapeptide 131–140 specific to eukaryotes and archaea. The same
results were obtained with an mRNA analog bearing
ATB-derivatized stop codon targeted to the A site in the
presence of termination factor eRF1 [71]. It was suggested
that the C-terminal tail of human rpS15e, in contrast to the
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 290
Protein environment of mRNA region 50
of the P site codon
It is seen that one of the main components of the mRNA environment upstream of the P site codon is rpS26e that is
almost the only neighbor of mRNA nucleotides in positions
24 to 29. These data are mostly confirmed by later results
obtained with the above-mentioned rabbit 48S and 80S initiation complexes [65], which indicates that rpS26e is a keystone component of the ribosomal binding site of mRNA
region 50 of the E site codon, at least at the initiation and
elongation of translation. This protein lacks eubacterial
counterparts, and therefore, molecular environment of
mRNA 50 of the E site codon on the mammalian ribosome
is strikingly different from that on the eubacterial ribosome.
It is worth mentioning here that labeling of this protein has
been reported in early studies of the mammalian ribosomal
mRNA binding site with the use of derivatives of oligo U
bearing an alkylating group at the 50 -terminal phosphate
bound to human and rat ribosomes in the presence of
tRNAPhe [45,72,73]. In these reports, positions of the derivatized mRNA nucleotides on the ribosome remained indefinite; nevertheless, it is evident that they could be either in
the first position of the P site codon or 50 of it. RpS26e has
been mapped on 40S subunits from lower eukaryotes initially by cryo-EM [17] and then more precisely by X-ray crystallography [19] in a region of the platform side facing the
head near the mRNA exit site region. The X-ray study
showed that, in contrast to the flexible 30 end of eubacterial
16S rRNA, the 30 end of the eukaryotic 18S rRNA appears
to be locked in place by tight interactions with rpS26e [19]
and therefore it is not accessible for contacts with mRNA
region 50 of the E site codon. The latter is a good explanation for hardly detectable cross-linking to the 18S rRNA
from the respective mRNA positions. Based on the comparison of our results with the mentioned X-ray data and the
results reported previously [65], one can suggest that rpS26e
directly interacts with mRNA nucleotides in positions –6 to
Structural and functional topography of the human ribosome
Figure 11 Comparison of locations of eIF3 and ribosomal proteins on the 40S subunit (A) Cryo-EM image of the complex of eIF3 with the rabbit
40S subunit (adapted from [74]). (B) X-ray structure of the 40S subunit from T. thermophila where ribosomal proteins are shown [19], an alternative
designation of rpS1e is given in brackets. Locations of rpSA and rpS26e neighboring eIF3 on the cryo-EM image (A) are shown by homology with
positioning of their counterparts on the X-ray structure (B).
–9. Apparently, weaker cross-linking of ATB-derivatized
uridines in positions –4 [47] and þ1 [60] to rpS26e was
detectable because the ATB group used was attached to uridines via a flexible linker enabling this group to reach
targets located close to the mRNA but not making direct
contacts with it. With the same approach as described above
for mapping of cross-link on the rpS15e, it was found that
cross-linking site of mRNA analogs bearing ATB group in
positions 26 and 29 on the rpS26e was located in the fragment 60 –71. Alignment of amino acid sequences of eukaryotic (from lower fungal to mammalian) and archaeal
proteins from the rpS26e family showed that this fragment
contains motif 62-YxxPKxYxK-70 conserved in eukaryotic
but not in archaeal rpS26e.
Possible roles of peptides 131–140 of rpS15e
and 60 –71 of rpS26e in translation
Specific features of the arrangement of mRNA binding site
on the mammalian ribosome found by site-directed crosslinking likely relate to more complex and multistep regulation of protein synthesis in eukaryotes. Analysis of X-ray
structure of the Tetrahymena thermophila 40S subunit [19]
showed that eukaryote-specific motif YxxPKxYxK of
rpS26e is not implicated in the intra-ribosomal interactions,
implying its involvement in the translation process in a
eukaryote-specific manner, e.g. in binding of eukaryotespecific ligands. One of these ligands could be eukaryotic
initiation factor eIF3 that is involved in recruitment of the
mRNA to the 40S subunit and has no counterparts in eubacteria and archaea [2]. This suggestion is based on the
data on overlapping locations of rpS26e [19] and eIF3 [74]
on the 40S subunit (Fig. 11) and the data on contacts of
mRNA nucleotides in position 28 and upstream of it with
eIF3 [65]. The role of the mentioned rpS26e motif could
extend beyond the initiation step by its possible contribution to the maintenance of the mRNA path from the region
of codon–anticodon interactions to the exit site during
translation. Finally, the YxxPKxYxK motif could be a potential target for the action of some kind of regulatory
factors modulating ribosomal function during translation
since it is partly accessible at the surface of the 40S
subunit solvent side [19].
Several hypotheses have been suggested to explain possible roles of the C-terminal eukaryote/archaea-specific
decapeptide of rpS15e found at the decoding site of human
ribosomes [71]. Remarkably, this decapeptide was not
found in the Protein Data Bank in any other proteins than
rpS15e; only short fragments of this sequence (not longer
than penta- or hexapeptide motifs) were traced in several
protein families. The data point to an important role of this
part of the unstructured C-terminal tail of rpS15e in the
translation process in eukaryotes and probably in archaea.
During initiation, as suggested in the previous report [65],
it could be important for the selection of initiation codon
and/or for the stabilization of 48S complexes since rpS15e
contacts mRNA positions þ4 and þ5 in these complexes.
During elongation, it possibly contributes to the fixation of
mRNA codon at the decoding site that, in turn, could
provide higher accuracy of translation and less probability
of a frameshift. During termination, the decapeptide possibly interacts with polypeptide chain release factor eRF1
and thus mediates stop codon decoding. This assumption is
based on the data on shielding of rpS15e from contacts
with mRNA by eRF1 [75]. Finally, the C-terminal
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Structural and functional topography of the human ribosome
decapeptide of rpS15e may be involved in the regulation of
translation in a manner specific for eukaryotes.
HCV IRES Binding Site on the 40S Subunit
The sequence of events leading to the formation of the
HCV IRES-containing 80S initiation complex competent
for the start of the viral RNA translation is well known
[76–78]. The functional roles of individual IRES domains
and subdomains (Fig. 3) have been studied using mutant
IRESes [5,76–80], cross-linking with chemically reactive
derivatives of oligonucleotides complementary to target
RNA sequences [81], and enzymatic and chemical footprinting [79,80,82]. According to the generally accepted
idea, at the first step of the assembly of the 80S initiation
complex, HCV IRES forms a stable binary complex with
the 40S subunit, where the initiation codon is positioned
near the P site without any initiation factors. The binary
complex formation crucially depends on the basal portion
of domain III (mainly hairpins IIId and IIIe) and on the
four-way junction IIIabc (Fig. 3). Thereafter, eIF3 and the
ternary complex (eIF2†Met-tRNAMet
i †GTP) assemble to
form the 48S preinitiation complex; binding of eIF3 is
mediated by domain IIIb and four-way junction IIIabc.
Binding of eIF5 to this complex promotes hydrolysis of
eIF2-bound GTP and the subsequent release of eIF2†GDP,
these are modulated by HCV IRES domain II [78]. Finally,
the release of the remaining factor eIF3 requires eIF5B and
GTP hydrolysis and occurs during joining of the 60S
subunit to form the 80S initiation complex. When eIF2 is
inactivated by phosphorylation under stress conditions,
HCV IRES can direct translation without eIF2 and eIF5
using a bacterial-like pathway requiring as initiation factors
only eIF5B (an analog of bacterial IF2) and eIF3 [83].
Knowledge on the ribosomal components responsible
for binding of HCV IRES to 40S subunits is important to
understand the mechanism of translation initiation of the
viral RNA and could be essential for the development of
novel efficient anti-HCV therapies. However, at the beginning of our studies, the structural organization of the IRES
binding site on the 40S subunit was less studied than functional roles of its specific domains, and the resulting data
were rather contradictory. So, direct UV-induced crosslinking of HCV IRES to the 40S subunit revealed rpS5e as
the only target for cross-linking [84]. Other investigators
using HCV IRES with uridines randomly substituted with
4-thiouridines reported a set of cross-linked proteins
(rpS2e, rpS3e, rpS10e, rpS15e, rpS16e/S18e, and rpS27e),
of which rpS3e, rpS2e, and rpS27e were the major targets
[85]. Finally, with the use of yeast genetics it was shown
that rpS25e is required for translation initiation of HCV
IRES [as well as of cricket paralysis virus (CrPV)] while
the lack of this protein in yeast strains does not lead to
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 292
serious defects in global translation, read-through, ribosome biogenesis, and programmed ribosomal frameshifting
[86]. The above-mentioned studies highlighted a set of proteins that could surround the 50 -terminal part of HCV
RNA, but did not provide information on the specific ribosomal proteins neighboring particular RNA nucleotides.
Cryo-EM visualization of HCV IRES in its complexes
with the 40S subunit [87] and the 80S ribosome [88] provided a general idea of the IRES positioning on the ribosome. So, two ribosomal proteins, namely, rpS5e on the
head of the subunit interface and rpS14e on the platform
were mapped close to the apex of subdomain IIb of the
IRES [88]; recent cryo-EM data on positioning of CrPV
IRES [89] make it possible to propose that N-terminal tail
of rpS25e extends toward rpS5e. However, domain II, in
contrast to subdomains IIId/e and the four-way junction
IIIabc, is not essential for binding of HCV IRES to 40S
subunit (see above). Thus, ribosomal proteins that could
contact keystone structural elements of HCV IRES leading
to its high affinity for the 40S subunit remained unknown.
With the use of HCV IRES derivatives bearing ATB
group at the selected nucleotides, it was shown that ribosomal proteins but not the 18S rRNA take part in the formation of HCV IRES binding site on the 40S subunit. The
summary on our cross-linking results is presented in
Table 3 and Fig. 12. These results are in a good agreement
with location of HCV IRES on the 40S subunit determined
by cryo-EM at a low resolution [87]. Seen from Fig. 12, all
cross-linked proteins are located mainly on the solvent side
of the 40S subunit opposite to the beak around the cleft
between the head and the body, at an area homologous to
the region of mRNA exit site in the 30S subunit and overlapping with the E site. The only pronounced dissimilarity
between our cross-linking results and the cryo-EM data is
that rpS5e appears to neighbor subdomain IIIe [35] rather
than domain II [87]. This in fact is not a real discrepancy
because the absence of cross-link can not indicate lack of
neighborhood or even interaction of the IRES with rpS5e
since the protein could not have a suitable target for crosslinking or ATB group could be oriented unfavorably with
respect to the protein to obtain a cross-link. The same
reasons may explain the lack of cross-linking of HCV
IRES derivatives to rpS25e.
The sets of 40S ribosomal proteins cross-linked to
s4U-containing HCV IRES [85] and to ATB-derivatized
IRESes (Table 3) are significantly different. It is clear now
that a significant part of the set of proteins found previously [85], namely rpS2e, rpS3e, and rpS15e, belongs to the
environment of the mRNA at the decoding site and downstream of it (Table 2, Fig. 7, and the respective discussion
in the text). These proteins most probably did not crosslink to the 50 -UTR of the HCV IRES but did to the part of
the coding sequence that could contain 4-thiouridines in
Structural and functional topography of the human ribosome
Table 3 Ribosomal proteins cross-linked to HCV IRES derivatives bearing ATB groups at selected positions in binary complexes of these
derivatives with human 40S ribosomal subunits [35,36]
Position and nature of nucleotide
bearing cross-linker
HCV domain
(subdomain)
Cross-linked ribosomal
proteins
Eubacterial counterparts of cross-linked
ribosomal proteins
G263
IIId
S3a
S14e
S16e
S3a
S14e
S16e
S3a
S5
S16
p40 (SA)
S14e
S16e
Not detected
No counterpart
S11p
S9p
No counterpart
S11p
S9p
No counterpart
S7p
S9p
S2p
S11p
S9p
A275
A296
IIIe
C83
Apical loop of domain II
G87
Near the apical loop of
domain II
Major cross-links are given in bold.
Figure 12 Location of the HCV IRES site on the 40S
subunit Proteins cross-linked to HCV IRES derivatives and their
eubacterial counterparts ( presented in brackets) are shown on the solvent
side image of the 40S subunit derived from the X-ray crystallographic
study [19]. mRNA entry and exit sites are shown by homology to the
respective sites on the eubacterial subunit. Major part of the mRNA
binding channel including the decoding site is located on the subunit
interface (on the back of this image).
positions þ2, þ10, and þ13 (Fig. 2). Consequently, only
proteins rpS10e, rpS16e/S18e, and rpS27e may be considered as cross-linked to the 50 -UTR of the HCV RNA in the
binary complex. This set overlaps with the set found in
[35,36] only by protein rpS16e. One possible explanation
for this discrepancy is that proteins rpS10e and rpS27e are
cross-linked to 4-thiouridines located in IRES positions far
from nucleotides C83, G263, A275, or A296 that bore perfluorophenyl azides. Finally, identification of one of crosslinked proteins as rpS10e [85] could be mistaken since,
according to the recent X-ray data on the 40S subunit
structure [19], this protein is located at the beak region,
which is incompatible with all the above-mentioned data
on the location of HCV IRES on the subunit.
Our cross-linking results provided significant novel information on the organization of the HCV IRES binding
site on the 40S ribosomal subunit at the first step of translation initiation of the viral RNA, highlighting the key role
played by the ribosomal proteins SA ( p40), S3a, S5e,
S14e, and S16e in the organization of this site. It is worth
mentioning here that location of rpS3a on the 40S subunit
that had been predicted based on the cross-linking results
[36] has been exactly confirmed by later X-ray crystallographic data [19] (Fig. 12). Remarkably, HCV IRES
binding site has only a minor overlap with the mRNA
binding track in the region of the mRNA exit site
(Fig. 12). These data can be used for creation of new types
of antiviral drugs targeted to proteins involved in the formation of only the IRES binding site. It should be noted
here that binding sites of various IRESes on the 40S
subunit could be significantly different. For instance, in
contrast to the HCV IRES, CrPV IRES is located entirely
in the mRNA binding cleft, reaching into the P and the A
sites [90]. Nevertheless, these IRES binding sites share
some extent of similarity, in particular, both CrPV IRES
and HCV IRES neighbor rpS5e and moreover, the CrPV
IRES is located close to rpS16e as one could see upon
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Structural and functional topography of the human ribosome
comparing the models presented in [87,90]. It is reasonable
to suggest also that proteins play a key role in the formation of the 40S ribosomal binding site not only for HCV
IRES but also for other IRESes. However, we cannot
extend this suggestion to the later steps of translation initiation of viral RNAs since in the 80S ribosomal complex
with HCV IRES, close proximity of the IRES with 18S
rRNA helices was detected [88].
Roles of rpSA ( p40) in the 40S Structure
and Translation of Canonical and
IRES-containing mRNAs
Notably that rpSA (mentioned as ribosomal protein p40 in
[35,91] and as precursor of the laminine-binding protein in
other works, e.g. [92] and references therein), one of proteins neighboring HCV IRES (see above), is not a constant
ribosomal component. It is known that binding of rpSA to
40S subunits accompanies formation of polysomes during
active cell growth; in contrst, dissociation of polysomes
leads to a partial loss of rpSA from the 40S subunits [91].
In accordance with these, it was found that 40S subunits
isolated from human placenta varied in the extent of the
rpSA loss, and subunits lacking this protein could directly
bind recombinant human rpSA [93]. Remarkably, affinity
of HCV IRES to 40S subunits strongly depends on their
rpSA content [94]. It was shown that monoclonal antibodies against C-terminal domain (CTD) of rpSA blocked
HCV IRES binding to 40S subunits, but had no effect on
the binding of Phe-tRNA to the poly(U)-programmed ribosomes [94]. To study effect of the antibodies on the translation of various mRNAs in a cell-free system, two mRNAs
were used: one carried 50 -UTR of actin mRNA, another
contained HCV IRES. It was found that binding of the
antibodies to their epitopes in 40S subunits inhibited
mRNA translation independently on the nature of its
50 -UTR, but the effect was larger with the IRES-containing
mRNA [94]. The results obtained with mRNA-containing
50 -UTR of actin mRNA suggest that rpSA has a regulatory
function in the process of translation of cellular mRNAs.
The protein, when bound to the 40S subunit, could
enhance or coordinate its interaction with initiation factor
eIF3, whose binding site is close to site of the conserved
part of rpSA (Fig. 11), and thus activate translation initiation. In contrast, dissociation of rpSA could lead to decrease of the eIF3 affinity to 40S subunits, resulting in the
reduction of the total translation efficacy. This implies that
the conserved part of rpSA on the 40S subunit is involved
in the interaction with the factor that, in turn, is likely
implicated in binding with rpS26e (see above). All these
are in a good accordance with close locations of rpS26e
and the conserved part of rpSA on the 40S subunit [19]
(Fig. 11).
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 294
Multiple sequence alignment of ribosomal proteins
belonging to the rpS2p family that includes rpSA revealed
that the eukaryotic proteins had C-terminal extensions,
whose lengths and sequences are strongly variable; higher
eukaryotic proteins have longer C-terminal tails [95]. In the
model of the lower eukaryotic ribosomal 40S subunit [19],
the CTD of rpS0 (a counterpart of higher eukaryotic rpSA)
is located closer to other proteins of the HCV IRES
binding site than the main conserved part of the protein
(Fig. 12). Likely, much longer CTD of human rpSA comes
just to the HCV IRES binding site, and it is reasonable to
suggest that it is involved in the IRES binding while the
conserved part of rpSA apparently takes part in the regulation of eIF3-dependent initiation of cellular mRNAs translation (see above). With the use of a set of truncated forms
of the recombinant human rpSA, it has been found that
CTD of rpSA is responsible for binding of the protein to
the 40S subunit, and fragment 236–262 is the key player
in this process [95]. Hydroxyl-radical footprinting showed
that helix 40 of the 18S rRNA is involved in the CTD
binding in the 40S subunit [95]. Remarkably, the CTD of
rpSA has another function operating as a receptor in the
binding of extracellular ligands (envelope proteins of
several viruses and prion proteins) by Laminine receptor,
which is a dimer of rpSA. However, this process involves
CTD regions that do not overlap with the region responsible for the binding to 40S subunit [92].
Arrangement of tRNA Acceptor End
on the Mammalian Ribosome
First, the positioning of 30 -terminal of tRNA in the 80S
ribosomal A and P sites was studied using a tRNAAsp
analog that bore s4U attached to the 30 -terminal adenosine
(s4U77–tRNAAsp) [39]. This analog cross-linked exclusively to the 28S rRNA when bound at the A or the P site. All
cross-linked 28S rRNA nucleotides were found in the fragment 4302 –4540 (Table 4) belonging to the highly conserved domain V that in prokaryotic ribosomes is involved
in the PTC formation [1,9,11,96]. However, the direct comparison of cross-linking results obtained with the use of
s4U77–tRNAAsp with available data on the respective prokaryotic ribosomal complexes would be incorrect since this
tRNA analog contained an additional nucleotide at the
30 -terminus. The environment of the s4U77 on the 50S
subunit was predicted [39] by means of a molecular modelling approach using the available X-ray structural data on
50S subunits complexed with a substrate mimicking
30 -terminal part of tRNA [97] and taking into account probable thermal fluctuations of the CCA end. These made it
possible to obtain information concerning nucleotides of
23S rRNA that could appear within 6–7 Å of s4U77 and
could be candidate targets for cross-linking. Few of them
Structural and functional topography of the human ribosome
Table 4 Summary of s4U77– tRNAAsp cross-links with the 28S rRNA in human 80S ribosomal complexes [39]
P site
A site
Human 28S rRNA
Deinococcus radiodurans
numbering
E. coli numbering
Human 28S rRNA
D. radiodurans numbering
E. coli numbering
U4461
U4462
U4502
2534
2535
2575
2555
2556
2596
U4462
U4461
C4507
U4469
2535
2534
2580
2542
2556
2555
2601
2563
Major cross-links are given in bold.
were indeed found to cross-link to the 28S rRNA
(Table 4). It is also essential to note that, according to molecular modelling data, two of the cross-linked 28S rRNA
nucleotides determined [39], U4502 and especially U4469
(2596 and 2563, respectively, in E. coli 23S rRNA), were
located too far from s4U77 to be cross-linked. Therefore,
the cross-linking data suggest that the mentioned 28S
rRNA nucleotides (at least U4469) are located much closer
to s4U77 than the corresponding 23S rRNA nucleotides in
the model prokaryotic complex used for the calculations.
Dissimilarities between the set of the cross-linked 28S
rRNA nucleotides and the set of candidate targets may
result from allosteric effects of ribosomal proteins interacting with domain V on the mutual arrangement of the CCA
end of tRNA and the 28S rRNA even if these proteins are
located far from the PTC region in the mammalian
complex. Such effects of ribosomal proteins on the peptidyl transferase activity of yeast ribosomes were discussed
in detail [98].
The data obtained with s4U77–tRNAAsp somewhat
modified the generally accepted idea that the structure of
the PTC region is the same in all kingdoms based on the
strong conservation of the secondary structure and a substantial part of the primary structure of domain V of
23S-like rRNAs. Thus, the arrangements of the prokaryotic
and the mammalian PTC regions do share significant similarity, but also have pronounced differences that seem to
relate to the more complex structure of the mammalian
ribosome and different dynamic properties of ribosomes
from various kingdoms.
Data complementary to the results obtained with
s4U77–tRNAAsp were reported with tRNAAsp analog
whose 30 -terminal ribose was oxidized (tRNAox) [40]. The
application of this analog targeted to proteins made it possible to find that 30 -terminus of tRNA bound at the 80S
ribosomal P site was located closely to 60S rpL36a-like
(rpL36AL). Thus, for the first time it has been demonstrated that a ribosomal protein is located at a zero distance
to the CCA 30 -terminal adenosine of P site-bound tRNA.
RpL36AL, which is strongly conserved in eukaryotes,
belongs to the L44e family of ribosomal proteins, a representative of which is rpL44e in Haloarcula marismortui.
These results are not in agreement with the structure of the
archaeal 50S ribosomal subunit lacking protein electron
density in a radius of 18Å of the PTC, where L44e is
located at the E site apart from the P site [99]. The mentioned structure is a basis of the current view that the PTC
works as a ribozyme and that ribosomal proteins are not
involved in catalysis of peptide bond formation. The disagreements between cross-linking results with mammalian
80S ribosomes and X-ray data on prokaryotic ribosomal
complexes were discussed previously [40], and one of the
most probable explanations presented there concerned wellknown flexibility of the CCA end of tRNA. This flexibility
could allow 30 -end of the tRNAox positioned at the P site
to migrate from the P- to the E site where it could interact
with nearby proteins.
Conclusions
Application of a site-directed cross-linking approach made
it possible for the first time to reveal features of the structural organization of functional centers of the human ribosome and to evaluate extent of their similarity with the
respective sites of prokaryotic ribosomes. It was found that
the conserved core of small subunit rRNA almost identical
in all kingdoms is involved in the structural organization of
80S ribosomal binding sites of mRNA codons that interact
with tRNA anticodons at the A and the P sites, while ribosomal proteins surround primarily mRNA parts upstream
and downstream of these codons. Moreover, it turns out
that the protein environment of mRNA on the human ribosome has a number of features that have no homology in
the eubacterial ribosome. In particular, eukaryote-specific
and eukaryote/archaea-specific peptides of ribosomal proteins are key players in accommodation of mRNA region
50 of the codons interacting with tRNAs and in the formation of the decoding site, respectively. The application of
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 295
Structural and functional topography of the human ribosome
short mRNA analogs bearing cross-linkers at the designed
positions goes beyond studying the structure of the 80S
ribosomal mRNA binding site. So, recently these analogs
were successfully used to determine peptides of polypeptide chain release factor eRF1 neighboring stop codons in
human 80S termination complexes [50,51] and the data
obtained provide a new insight into the mechanism of stop
signal decoding [51] that remained largely unknown due to
the lack of cryo-EM and X-ray crystallographic data on
80S termination complexes. As for the PTC that is generally assumed to be strongly conserved and composed only of
the rRNA, features distincting it from the respective prokaryotic center were also detected; the most essential one
is the involvement of a ribosomal protein in the accommodation of 30 -terminal nucleotide of tRNA at the P site.
Finally, application of a site-directed cross-linking made it
possible to determine 40S ribosomal components implicated in the formation of the hepatitis C IRES binding site,
a functional center that is specific to the mammalian ribosome. Involvement of ribosomal proteins but not rRNA in
its formation implies participation of eukaryote-specific
proteins or peptides in the HCV IRES binding. Subsequent
studies on the contributions of rpSA into the ribosomal
structure and the IRES binding shed light on the particular
roles of the CTD of rpSA specific to higher eukaryotes in
binding of the protein to the 40S subunit and in binding of
the subunit with the HCV IRES. In general, data obtained
by a site-directed cross-linking approach are in a good
agreement with available X-ray and cryo-EM models.
However, the detailed comparison is difficult because complexes of higher eukaryotic ribosomes with mRNA, tRNA,
and HCV IRES are not studied so far by X-ray crystallography. As for cryo-EM, today this approach does not allow
particular mRNA codons, CCA ends of tRNAs, and
eukaryote-specific peptides of proteins to be directly
visualized. Evidently, cross-linking data provide a biochemical basis for future creating of atomic models of
higher eukaryotic ribosomes and their functional sites with
application of X-ray and cryo-EM approaches, and for
further studying in detail molecular mechanisms of the
work of human translational machinery operating with cellular and viral mRNAs. The next frontier of the research is
to understand functional roles of specific structural elements of higher eukaryotic ribosomal functional sites in
translation and its regulation.
Acknowledgement
We gratefully thank Ian Eperon for critical reading of this
manuscript.
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 296
Funding
This work was supported by the grants from Russian
Foundation for Basic Research (including the current grant
11-04-00597 to G.K.) and the Program ‘Molecular and
Cell Biology’ of the Presidium of the Russian Academy of
Sciences (to G.K.).
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Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 299