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Ribosome Structure and Shape

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Ribosome Structure
and Shape
Joachim Frank, Wadsworth Center and State University of New York, Albany, NY, USA
Rajendra K Agrawal, Wadsworth Center, Albany, NY, USA
Adriana Verschoor, Wadsworth Center, Albany, NY, USA
The ribosome is a macromolecular machine, composed of RNAs and proteins, that
synthesizes proteins by stringing together amino acids according to genetic instructions.
Introduction
The ribosome is a large complex of ribonucleic acids
(RNAs) and proteins, ubiquitous in all cells, that translates
genetic information stored on the messenger RNA
(mRNA) into polypeptides. The functional ribosome is
formed by association of its two subunits (the large and
small subunits), and it essentially provides a scaffold for the
binding of mRNA, transfer RNA (tRNA), and a number
of protein factors involved in promoting the different steps
of protein synthesis. Although the principal functions and
complex composition of the ribosome were known by the
1960s, until recently very little information has been
available on its structure. Because of the complexity of its
architecture, comprising three or more ribosomal RNA
(rRNA) molecules and more than 50 protein molecules,
progress toward obtaining a high-resolution X-ray crystal
structure has been slow. However, X-ray and nuclear
magnetic resonance (NMR) structures of some of the
components of the bacterial ribosome have been solved at
the atomic scale. Electron microscopy (EM), particularly
cryoelectron microscopy, has become the method of choice
for studying ribosome structure. Cryoelectron microscopy
of single ribosomes, combined with techniques of three-
dimensional reconstruction, has provided the first detailed
maps of this organelle. Recently, the publication of an X-
ray crystal structure of the large subunit of an archae-
bacterial ribosome, at 0.9-nm resolution, has marked
progress toward atomic resolution.
Size, Composition and General
Structure
Size and composition
The ribosome is approximately globular, its average
diameter ranging from 2.5 nm (Escherichia coli) to 2.8 nm
(mammalian). The small subunit comprises one RNA and
between 21 (E. coli) and 42 (rat liver) proteins, while the
large subunit comprises two or three RNAs, with the
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Introductory article
. Introduction
Article Contents
. Size, Composition and General Structure
. Conservation of Structure
. Abundance of Ribosomes in Different Organisms and
Cell Types
. Techniques for Studying Ribosome Structure
number of proteins ranging from 34 (E. coli) to 46 (rat
liver). The sizes and molecular weights of ribosomes from
some species characteristic of the three kingdoms are listed
in Table 1.
General structure
All known ribosomes are constructed of a somewhat flat,
elongate small subunit and a roughly hemispherical large
subunit (see Figures 1 and 2), which associate at the
initiation step and dissociate upon termination of protein
synthesis. The ribosome serves as a physical scaffold, to
help bring together in correct orientations the necessary
components (mRNA, representing a transcript of the
DNA segment to be translated; tRNA, bearing a specific
amino acid as well as a recognition site (anticodon) for the
corresponding mRNA codon; guanosine triphosphate
(GTP), a molecule supplying energy and a host of protein
factors that operate during initiation, elongation and
termination). Most importantly, it has catalytic activities,
located in the peptidyltransferase centre, that are required
to transfer the tRNA-bound amino acid to the nascent
polypeptide in each step of elongation.
The small subunit (30S in Figure 2) shows a one third/
two-thirds distribution of mass into a head and body
portion. The upper part of the body bears a broad shelf-like
protrusion termed the platform, which extends along the
interface with the large subunit. The roughly triangular
head tapers off into a scythe-shaped extension, curved
away from the large subunit, that is particularly pro-
nounced in eukaryotes and is termed the beak, or bill.
The large subunit (50S in Figure 2) is roughly hemi-
spherical, and bears three prominent extensions that serve
as landmarks. In E. coli, the central extension, termed the
central protuberance (CP), is mainly formed by 5S RNA,
while the flanking ones have been identified by immunoe-
lectron microscopy as protein L1 and a complex formed by
proteins L7 and L12 (‘L7/L12 stalk’, St in Figure 2),
respectively. The L7/L12 stalk in E. coli, and its analogue in
ribosomes from archaea and eukarya, is highly mobile, and
is thought to play an active role in the binding and
1
 Ribosome Structure and Shape

Table 1 Sizes and molecular masses of ribosomes from some

In their mutual association, the subunits form a partially
representative species
open tent-like space, termed the intersubunit space
Approximate Number of Percentage (Figure 1), which provides the sites of interaction between
Subunit total mass (kDa) proteins of protein the ribosome and tRNA molecules. The sites of codon–
Eubacteria anticodon interaction are in the region of the cleft between
Escherichia coli (anaerobic Gram-negative) platform and head of the small subunit (Figure 2), while
those associated with the catalysis of the peptide bond are
30S 900 21 34–40
located in the interface canyon of the large subunit, in close
50S 1500 33–34 30–36
proximity to the tunnel entry. During protein biosynthesis,
70S 2400 54 38–39 the tRNA traverses the intersubunit by going through
Archaebacteria three primary binding positions, termed the A site (i.e. the
Methanobacterium formicicum (Euryarchaeota) binding site of the aminoacylated tRNA), the P site (the site
of peptidyl tRNA, or the tRNA bound to the polypeptide)
30S 800 22–23 N.A.
and the E site (exit site, where the deacylated P-site tRNA
50S 1600 32 N.A.
moves after peptide bond formation). At any time, only
70S 2400 54–55 N.A. two of these sites are occupied: A and P (in the
Sulfolobus solfataricus (Crenarchaeota) pretranslocational state, shown in Figure 3) or P and E (in
30S 1100 27–28 N.A. the posttranslocational state).
50S 1800 34–35 N.A.
70S 2900 61–64 N.A.
Eukaryotes Conservation of Structure
Saccharomyces cerevisiae (Fungi)
Consistent with the remarkable functional conservation of
40S 1500 ~31 ~55
the ribosome and its principal ligands (mRNA, tRNA and
60S 2500 44 ~47 protein factors) among all kingdoms, its structure is also
80S 4000 ~75 ~50 conserved. Despite the great increases in translational
Pisum sativum (angiosperms) control and fidelity in higher eukaryotes, involving many
40S 1500 32–40 53–54 additional components and steps, the basic functions of
60S 2500 ~47 46–49 initiation, elongation and termination closely resemble
80S 4000 83 ~49 those in E. coli, and the topography of the intersubunit
space appears almost identical in eubacterial, archaeal and
Rattus rattus (cytoplasmic) (Mammalia)
eukaryotic ribosomes. Interkingdom differences among
40S 1500 32 ~55
ribosomes tend, therefore, to be most notable in peripheral
60S 3000 43 ~45 regions, away from the intersubunit space. The head of the
80S 4500 75 ~49 small subunit of eukaryotic ribosomes has a pronounced
N.A., not available. beak, its body has additional lobes (‘feet’) at its bottom. In
higher eukaryotes, the large subunit has evolved from a
hemispherical to a more hemiellipsoidal shape. In general,
these differences involve insertions of RNA segments and
additions of proteins compared to the ribosomes of
eubacteria.
movement of tRNAs during the steps of elongation. The
subunit’s interface surface is relatively flat, but it is
interrupted by a deep groove termed the interface canyon Archaebacterial ribosomes
(IC). The interface canyon deepens into a tunnel (not Ribosomes from archaebacteria appear intermediate in
visible in Figure 2; hidden by the front canyon wall) that size and structure between those of eubacteria and
traverses the subunit to exit on its back. Evidently, the eukaryotes. In size they may resemble eubacterial ribo-
nascent polypeptide chain is threaded through this tunnel somes more closely, but sequencing studies of their small-
to exit from the ribosome (Figure 3). The tunnel, from the subunit rRNAs indicate their closer affinity with eukar-
site where the polypeptide is synthesized, to the point where yotes. Of the different branches of archaebacteria, the
it exits, appears to provide a tightly sealed compartment. In methanogens (for the most part) and halophiles show
eukaryal cytoplasmic ribosomes engaged in the synthesis closer affinities to the eubacteria in ribosome structure,
of exported proteins, the sealed environment extends while the methanococcales and sulfolobales more closely
through the protein export channel to which the ribosome resemble eukaryotes.
attaches.

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 Ribosome Structure and Shape

Figure 1 Cryoelectron microscopy maps of prokaryotic (Escherichia coli, left) and eukaryotic (yeast, right) ribosome, with small subunit coloured in
yellow, large subunit in blue. The maps are positioned in such a way that the large subunits are in corresponding orientations. The leader lines point to the
intersubunit space. E. coli ribosome map from Malhotra A, Penczek P, Agrawal RK et al. (1998) Escherichia coli 70S ribosome at 15 Å resolution by cryo-
electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. Journal of Molecular Biology 280: 103–116; yeast ribosome map from A
Verschoor, D Bubeck, R Grassucci, JPG Ballesta and J Frank, unpublished.

Figure 2 Topography of the intersubunit space of the Escherichia coli ribosome. The ribosome map was computationally separated into the two
subunits by severing several connecting bridges. Landmarks: small subunit: h, head; pl, platform; cl, cleft. Large subunit: L1, L1 protein; St, L7/L12 stalk;
IC, interface canyon; CP, central protuberance.

Eukaryotic ribosomes smaller than cytoplasmic, in general, and bear some

Most eukaryotes contain two distinct types of ribosomes:
cytoplasmic and organellar. Cytoplasmic ribosomes show
marked variation with the complexity of the organism: the
yeast ribosome is relatively small and globular, resembling
a eubacterial ribosome except for its greater size, while
higher eukaryotes have larger and more ellipsoidal
ribosomes. Most of the variation is due to the size increases
of components of the large (60S) subunit.
Organellar ribosomes from mitochondria (found both in
animals and plants) and plastids (found in plants only) are
resemblance to the eubacterial ribosomes, consistent with
the hypothesis that these organelles have a eubacterial
origin (‘endosymbiosis’). While chloroplast ribosomes
have been measured at 67–68S, mitochondrial ribosomes
show a wide variation, from 55S (mammalian) to 77S
(yeast). Structural work on organellar ribosomes, how-
ever, lags far behind that for E. coli or even eukaryal
cytoplasmic ribosomes.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3

 Ribosome Structure and Shape

Figure 3 Cryoelectron microscopy map of the Escherichia coli ribosome (yellow, small subunit; blue, large subunit) depicted in the pretranslocational
state. The path of the mRNA, the positions of tRNA in A (pink) and P (green) sites, and the exit path of the polypeptide chain are indicated as inferred by
other cryo-EM experiments. This positioning of A- and P-site tRNAs, with their anticodons bound to adjacent codons of the mRNA, ensures the placement
of their amino acids in the correct order as they are strung together to form the polypeptide chain. From Frank J (1998) How the ribosome works. American
Scientist 86: 428–439.

Abundance of Ribosomes in Different
Organisms and Cell Types
In a prokaryote such as E. coli, or a lower eukaryote such as
yeast, a cell may contain several thousand ribosomes. In a
higher eukaryote (e.g. rat liver), this number may reach five
million. The highest abundances of ribosomes are in cells
or tissues in which rapid growth is taking place, e.g.
embryonic tissue in animals and young leaves in plants.
Ribosomes in eukaryotes exist in two populations, free and
membrane-bound; evidently, the pools exchange freely.
Generally, the proportions vary widely, depending on such
factors as cell or tissue type, stage of differentiation and
biological conditions. Rapidly growing cells or tissues have
higher proportions of free ribosomes, while highly
differentiated cells as well as secretory cells show higher
proportions of membrane-bound ribosomes.
Approximately 75% of the ribosomes in liver tissue are
membrane bound, while the proportion is much smaller
(22%) in reticulocytes, which synthesize principally a
single, nonexported protein, globin. In brain tissue, almost
none of the ribosomes are membrane-bound.
When several ribosomes are associated with a polycis-
tronic mRNA, they are referred to as polyribosomes or
polysomes. Formation of polysomes helps in speeding up
the rate of protein synthesis. Polysomes can be either
cytoplasmic or bound to the membrane of the endoplasmic
reticulum. In the latter case, they generally produce
secretory proteins.
Techniques for Studying Ribosome
Structure
The main techniques used in structural analysis of whole
ribosomes and their subunits are neutron scattering,
electron microscopy and X-ray crystallography. In addi-
tion, individual proteins and RNA fragments are being
studied by X-ray crystallography and NMR spectroscopy.
Through model-building guided by experimental data and
chemical bonding constraints, three-dimensional models
of ribosomal RNA have been obtained.
Neutron scattering
The distribution of proteins within the ribosomal subunits
has been determined by neutron scattering. In this
approach, the subunit is reconstituted from its RNA and
protein components, two of which have undergone

4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

 Ribosome Structure and Shape

hydrogen–deuterium substitution. Neutron scattering, in
solution, of subunits with pairwise deuterated proteins
yields a table of pairwise distances, from which the
constellation of proteins (i.e. the relative positions of their
centres of gravity) has been inferred.
Electron microscopy
Transmission electron microscopy is used to produce
projection images of biological specimens. Projection
images showing the ribosome from different directions
may be combined to form a three-dimensional density
map.
Negative staining method
An aqueous suspension of purified ribosomes is mixed with
1–2% heavy metal salt such as uranyl acetate and applied
to the electron microscope grid. After excess liquid has
been blotted away, the suspension is allowed to dry. The
stain forms a cast around the molecule, bringing out its
boundaries with high contrast. Much of the earlier work,
e.g. the study of the morphology of subunits and
localization of ribosomal proteins by immunoelectron
microscopy was done in this way. However, as the
specimen is dehydrated during grid preparation, the native
structure of the ribosome is not well preserved.
Cryoelectron microscopy
In cryoelectron microscopy, the sample applied to the EM
grid is rapidly cooled to liquid-nitrogen temperature, so
that the macromolecule is preserved in a thin layer of
vitrified ice under conditions that are close to those in its
native state. Visualized in the transmission electron
microscope, typically at a magnification of 50 000  using
a very low radiation dose, the image field shows many
‘copies’ of the molecule, lying in different orientations.
Depending upon the nature of the interactions between the
molecule and the water surface, certain orientations may
occur preferentially, or they may be close to random. Use
of mathematical techniques to align and classify images,
and to find their relative orientations makes it possible to
reconstruct a three-dimensional image, or map, of the
molecule from these projections. The extremely low signal-
to-noise ratio of the molecule images requires collection
and processing of several thousand ribosome images. To
date, the most detailed maps of the ribosome from several
species (prokaryotes: E. coli, Thermus thermophilus;
archaebacteria: Haloarcula marismortui, eukaryotes: Sac-
charomyces cerevisiae (yeast), rabbit reticulocytes and rat
liver) have been obtained in this way, and the binding
positions of tRNA and elongation factors have been
mapped on the E. coli ribosome.
Many ligands that interact with the ribosome in the
course of the translation process, initiation factors, tRNA,
elongation factors and release factors, are large enough to
be visualized by cryoelectron microscopy of ribosome–
ligand complexes. Comparison of such maps with control
maps reveals the three-dimensional position of the ligand.
If the X-ray structure of the ligand is known, its position in
the cryo-map can then be established with high accuracy by
fitting.
For a ligand localization study to be successful, the
occupancy of the ligand in the complex must be high (in the
range of 80% or above). This is because the three-

Figure 4 Three-dimensional binding positions of tRNA (a), elongation factor G (b) and Sec61 protein channel (c) as visualized using cryoelectron
microscopy. (a) 70S ribosome of Escherichia coli with initiation-tRNA bound at the P site (green). Adapted from Malhotra A, Penczek P, Agrawal RK
et al. (1998) Escherichia coli 70S ribosome at 15 Å resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein.
Journal of Molecular Biology 280: 103–116. (b) 70S ribosome of E. coli bound with EF-G GDP (purple). Adapted from Agrawal RK, Penczek P, Grassucci RA
et al. (1998) Visualization of elongation factor G on Escherichia coli 70S ribosome: the mechanism of translocation. Proceedings of the National Academy of
Sciences of the USA 95: 6134–6138. (c) 80S ribosome of yeast with Sec61 channel complex (red) attached near the polypeptide tunnel exit. Adapted from
Beckmann R, Bubeck D, Grassucci R et al. (1997) Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278:
2123–2126. Note: The ribosomes are shown in different orientations to best reveal the binding positions of ligands.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5

 Ribosome Structure and Shape
dimensional map is obtained from many projections of
different ribosomes, but it is difficult to ascertain, by the use
of image processing, whether a particular ribosome is
occupied. Thus far, the positions of tRNA on the ribosome
have been mapped in the pre- and posttranslocational
state, and the binding positions of the ternary complex,
EF-G, and the Sec 61 channel (for the yeast ribosome) have
been obtained (Figure 4).
X-ray crystallography
Only recently has significant progress been made in X-ray
crystallographic analysis of ribosomes, which have tradi-
tionally been considered to be too large and to pack in
arrays too disordered for such analysis to be feasible. The
greatest successes have been in solving the structures of
purified individual ribosomal proteins and initiation and
elongation factor complexes. The large size, sensitivity to
buffer conditions, and conformational variability of the
ribosome and its subunits all pose obstacles to crystal-
lization. The first low-resolution (0.9-nm) X-ray map of the
large ribosomal subunit has been recently obtained for a
halophilic archaebacterium, Haloarcula marismortui,
where the phasing of the X-ray data was facilitated by
the availability of a three-dimensional cryo-EM map.
Model building
Experimentally guided model building is a promising
approach to ribosomal RNA structure. Within the
ribosome, the RNAs form a distinct structural framework
in which helical double-stranded segments alternate with
single-stranded regions and more complicated structural
6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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motifs. For many species, the primary sequences of rRNA
are known, from which the secondary structure can be
inferred according to rules of base pairing and other atomic
interactions, combined with results of biochemical studies.
Using a variety of data from biophysical experiments
including neutron scattering, chemical crosslinking, fluor-
escent resonance energy transfer and chemical footprint-
ing, the three-dimensional folding of rRNA present in the
small subunit of the E. coli ribosome has been modelled by
several groups. Most recent approaches to modelling have
also incorporated constraints provided by the envelopes
and internal density distributions of cryo-EM maps.
Further Reading
Bielka H (ed.) (1982) The Eukaryotic Ribosome. Berlin: Springer Verlag.
Brimacombe R (1995) The structure of ribosomal RNA: a three-
dimensional jigsaw puzzle. European Journal of Biochemistry 230:
365–383.
Dube P, Wieske M, Stark H et al. (1998) The 80S rat liver ribosome at
25 Å resolution by electron cryomicroscopy and angular reconstitu-
tion. Structure 6: 389–399.
Frank J (1998) How the ribosome works. American Scientist 86: 428–
439.
Green R and Noller HF (1997) Ribosomes and translation. Annual
Review of Biochemistry 66: 679–716.
Hill WE, Moore P, Dahlberg A, Schlessinger R, Garrett R and Warner J
(1990) The Ribosome: Structure, Function, and Evolution. Washington
DC: American Society for Microbiology.
Matheson AT, Davies JE, Dennis PP and Hill WE (eds) (1995) Frontiers
in Translation. Canada: National Research Council.
Verschoor A, Warner J, Srivastava S, Grassucci RA and Frank J (1998)
Three-dimensional structure of the yeast ribosome. Nucleic Acids
Research 26: 655–661.