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Mechanics of
the ribosome
50S
5’
tRNA
Roger Garrett
Electron-density maps of a cell’s protein-producing machinery — the
ribosome — have been partially solved. This breakthrough shows that
high-resolution structures of the whole ribosome will soon be available.
he ribosome is central to every cell
because it provides the workshop and
tools to synthesize all of the proteins
(see Box 1). The simplest ribosome, from
bacteria, is made up of two subunits known
as 30S and 50S. These are defined by their
apparent sedimentation coefficients, which
are characterized by the Svedberg unit, S, and
reflect the rate at which a molecule sediments in a solvent. Each subunit is made up
of ribosomal RNA (rRNA), along with a
large number of proteins. The 30S subunit,
for example, contains a large rRNA molecule
(16S rRNA) along with 21 proteins. The 50S
subunit, by contrast, consists of large and
small RNAs (23S rRNA and 5S rRNA) and 35
different proteins.
On pages 833 and 841 of this issue,
Clemons et al.1 and Ban et al.2 provide
electron-density maps of the two ribosomal
subunits, obtained by X-ray diffraction at resolutions of 5.5 Å and 5 Å, respectively. The 30S
subunit comes from a hyperthermophilic
bacterium, Thermus thermophilus1, whereas
the 50S subunit is from the extremely
halophilic (‘salt-loving’) archaeon Haloarcula
marismortui 2. For the first time, several proteins of known three-dimensional structure,
and many regions of double-stranded rRNA,
have been located in highly complex electrondensity maps of ribosomal subunits.
This is not a sudden development. Crystals of these ribosomal subunits that diffract
at high resolution have been available for
years, and most of the crystallographic techniques used by Clemons et al. and Ban et al.
have already been successfully tested on
ribosomes3. Moreover, by using extensive
biochemical data, most of the proteins and
rRNA in each subunit have been incorporated into lower-resolution (in the 10–20-Å
range) shapes of the ribosome determined
by cryo-electron microscopy4–6. For the 30S
subunit, at least, this process has also exploited a spatial model of the ribosomal proteins
that was derived from neutron-scattering
data7. Previously, however, the X-ray data
were not of a high enough quality to allow
accurate models to be built.
The enormous effort that has gone into
characterizing and analysing components
of the ribosome5,6 has spawned many new
experimental methods. It has led, for example, to a method for accurately predicting all
T
NATURE | VOL 400 | 26 AUGUST 1999 | www.nature.com
50 Å
AA
3’
mRNA
30S
8
of the double-helical structures in rRNA ,
including those incorporated in the new
models1,2. But progress in understanding
how the ribosome works has been hampered
by two developments — one avoidable and
another that could not be prevented. Avoidable was the tradition of viewing the ribosome as two pieces of rock containing two
(later three) shallow cavities where transfer
RNAs (tRNAs; Fig. 1) carrying amino acids
bind and interact. This produced the erroneous view that the tRNAs, the messenger
RNA (mRNA) template and protein factors
are located at one cavity or another, and that
the ribosome itself is more or less irrelevant
to the process of protein production. This
view negated the concept of ribosomal
movements. But we now know, from studies
of cells containing mutated ribosomes with
altered properties, and from investigations
into how antibiotics block different steps of
protein synthesis, that the ribosome is a
dynamic machine5,6. This evidence is strongly reinforced by physical measurements of
the differences in shape observed for ribosomes in different functional states9,10.
An unavoidable block to progress in
solving the ribosome’s structure was the fact
Figure 1 The ribosome, showing some of the
states through which the tRNA–mRNA complex
moves between the ribosomal subunits. AA,
amino acid.
that, at a very early stage in evolution, the
ribosome became highly complex (fossilized deposits of cyanobacteria and other
bacteria and archaea containing ribosomes
date back up to three billion years11). Efficient proof-reading (to ensure that the
mRNA template was followed correctly)
and a fine balance between the speed and
accuracy of peptide elongation developed.
During this stage, the ribosome also defined
the size of evolving genes, because the capacity to translate larger regions of mRNA more
accurately allowed larger functional proteins to be produced. So, there is no simpler
ribosome structure than the bacterial one
(except for some highly degenerate ribosomes within the cell’s respiration centre,
the mitochondria). One consolation, however, has been that by comparing rRNA
sequences, which are very similar (‘conserved’) between different organisms, reliable evolutionary trees of microorganisms
Box 1: Building proteins
Protein synthesis starts
on the 30S subunit. The
messenger RNA (mRNA)
— the template for a new
protein — binds to this
subunit then unfolds its
highly twisted structure
as it enters. A transfer
RNA (tRNA) molecule
carrying the first amino
acid for the new protein
chain at one end (its socalled 38 terminus) binds
to the 30S subunit and
to the mRNA. It attaches
to the latter by basepairing to a sequence of
three nucleotides in the
mRNA template (a
‘codon’) that specifies
which amino acid needs
to be added.
Next, the 50S subunit
associates with the 30S
subunit, and the 38
terminus of the tRNA is
positioned in a cavity on
the large subunit.
Another tRNA is bound
at an adjacent codon
carrying a second amino
acid, which is also
positioned in the cavity.
A peptide bond is then
formed to join the two
amino acids. During
each of these steps —
which are repeated until
a protein chain
(polypeptide) is
synthesized — the many
protein factors that bind
to the ribosomal
subunits ensure that the
© 1999 Macmillan Magazines Ltd
tRNA and mRNA
molecules are
positioned accurately,
and they facilitate
movement of the
tRNA–mRNA complexes
relative to the ribosome.
As can be seen from
Fig. 1, the 38 end of each
tRNA moves around 100
Å through the ribosome.
When the new
polypeptide is ready,
another protein factor
recognizes a specific
‘stop’ codon, allowing
the protein to be
released along a
channel through the 50S
subunit. The two
ribosomal subunits then
dissociate.
R. G.
811
news and views
812
friendly, producing only GDP and phosphate. The advances in ribosomal modelling
presented by Clemons et al.1 and Ban et al.2
should first be savoured. They should then
signal that this is the time for new thinking,
for the development of new techniques and
for young scientists to get involved. The static models produced by cryo-electron
microscopy and X-ray crystallographic
studies will be a passing phase — the next
decades will be dedicated to studying the
machine’s movements.
Roger Garrett is at the Institute of Molecular
Biology, Copenhagen University, Sølvgade 83H,
1307 Copenhagen, Denmark.
e-mail: [email protected]
1. Clemons, W. M. Jr et al. Nature 400, 833–840 (1999).
2. Ban, N., Nissen, P., Capel. M. S., Moore, P. B. & Steitz, T. A.
Nature 400, 841–847 (1999).
3. Yonath, A. et al. Acta Crystallogr. A 54, 945–955 (1998).
4. Müller, F. & Brimacombe, R. J. Mol. Biol. 271, 545–565 (1997).
5. Hill, W. E. et al. (eds) The Ribosome: Structure, Function and
Evolution (ASM, Washington DC, 1990).
6. Garrett, R. A. et al. (eds) Ribosomes: Structure, Function,
Antibiotics and Cellular Interactions (ASM, Washington DC, in
the press).
7. Capel, M. S. et al. Science 238, 1403–1406 (1987).
8. Fox, G. E. & Woese, C. R. Nature 256, 505–507 (1975).
9. Pettersson-Landen, L., Fredriksson, M. G., Ofverstedt, L. G.,
Skoglund, U. & Isaksson, L. A. Exp. Cell Res. 238, 335–344 (1998).
10. Agrawal, R. K., Heagle, A. B., Penczek, P., Grassucci, R. A. &
Frank, J. Nature Struct. Biol. 6, 643–647 (1999).
11. Pflug, H. D. Syst. Appl. Microbiol. 7, 184–189 (1986).
12. Woese, C. R. & Fox, G. E. Proc. Natl Acad. Sci. USA 74,
5088–5090 (1977).
13. Porse, B. T. & Garrett, R. A. Cell 97, 423–426 (1999).
Astronomy
Life beyond the pulsar death valley
© 1999 Macmillan Magazines Ltd
To
graveyard
De
at
h
ars
Young pulsars
12
tur
ep
uls
R
∗
Ma
apidly spinning, strongly magnetized
neutron stars, which reveal themselves
to astronomers as radio pulsars1, have
been known for more than 30 years. Yet
despite a steady progress in our understanding of the physics of these fascinating objects,
to paraphrase a quote from a 1976 conversation between Sandra Faber and Jonathan
Arons2, “we know how pulsars pulse, but we
do not know how they shine”. This disappointing state of affairs is heightened by the
astonishing discovery of the slowest radio
pulsar detected so far, reported on page 848
of this issue3. The 8.5-second rotation period
of pulsar J2144–3933 is so long that, according to our current thinking about the ways
pulsars shine, this one should not exist as an
observable object.
The key to producing pulses is the rotation of the neutron stars, which have masses
similar to the Sun’s but diameters of only 20
km or less. Pulsars expend their rotational
kinetic energy by emitting electromagnetic
radiation and a wind of relativistic particles,
leading inevitably to a gradual slow-down of
pulsar rotation. Luckily, this slow-down is
measurable and, together with the rotation
period itself, gives a useful estimate of a pulsar’s magnetic field strength and age. Only a
small fraction of the total energy lost is radiated away in the form of beamed radio emission, which appears to originate well inside
the pulsar magnetospheres, in the regions
located just over the magnetic poles. Pulsars
are best described as ‘misaligned’ rotating
magnets in which the magnetic poles do not
coincide with the poles of rotation, so that
the spinning pulsar produces a series of
evenly spaced pulses. The pulses themselves
are beams of radio waves emitted along the
magnetic axis of a predominantly dipolar,
huge (` 1012 Gauss) magnetic field.
The detection of an 8.5-second radio pulsar is likely to send theorists back to the draw-
va
lle
y
Alex Wolszczan
Log pulsar magnetic field (Gauss)
could be constructed for the first time. This
led to the discovery12 of the third domain of
life, the Archaea, in 1977.
What do the latest crystallographic
results actually tell us about the ribosome?
They put the whole model-building exercise
on a surer footing, and promise much more.
At this resolution, a-helices (spirals) in the
protein structures can be readily fitted to the
electron-density maps of the subunits, as can
most double-helical segments (around twothirds) of the rRNA’s structure. Moreover,
known three-dimensional structures of
proteins and protein–rRNA fragments are
placed with some confidence. Clemons et al.1
have even pinpointed the position of a protein called S20 based on the a-helical composition that was predicted from its aminoacid sequence. In all other cases, though, to
localize components of unknown structure
(and this still includes two-thirds of the proteins in the 30S subunit and most of the proteins in the 50S subunit), it is still necessary
to fall back on data from biochemical studies
and cryo-electron microscopy4,6.
The central domain of the 30S subunit
contains about one-third of the 16S rRNA,
and produces a flat projection — the ‘platform’ — that modulates the passage of the
tRNA–mRNA complex through the ribosome. This whole region has now been modelled by Clemons et al.1, revealing new details
of structures that may influence movement
of the tRNA–mRNA complex. In the 50S
subunit, Ban et al.2 have located two main
regions that regulate at least one protein factor (called elongation-factor G), which helps
the tRNA–mRNA complex to move through
the ribosome after the peptide bond has been
formed (see Box 1). One of these regions is a
small stretch of 23S rRNA; the other is a protein–RNA complex. Both of these regions
have previously been isolated from the ribosome and analysed by NMR spectroscopy
and X-ray diffraction6,13. Conformational
transitions in these regions can be inhibited
by antibiotics or toxins, leading to reduced
cell growth or even cell death. For example,
the 23S rRNA segment is modified by several
toxins, including ricin and a-sarcin, which
inactivate the ribosome. The function of the
protein–rRNA complex, on the other hand,
is blocked by the antibiotics thiostrepton and
micrococcin13.
When I first saw these X-ray structures I
thought that this is how it must have felt, for
those involved, to see the final stones being
placed on the first pyramid. But on reflection, although this analogy may be appropriate for the amount of collective effort that
has been expended, it would also repeat the
conceptual mistake that has plagued the
ribosome field (more pieces of rock). The
ribosome, together with its accessories, is
probably the most sophisticated machine
ever made. All of its components are active
and moving, and it is environmentally
10
s
led ar
yc puls
Back from
c
Re
graveyard
8
0.001
0.01
0.1
1
10
Pulsar period (seconds)
Figure 1 Life and death of a radio pulsar. The
distribution of young and old pulsars is shown
according to the strength of their magnetic fields
and how fast they rotate. The two sloping lines
define ‘death valley’, where pulsars are assumed
to turn off their radio emission. Arrows indicate
possible directions of pulsar evolution, with
only superfast ‘millisecond pulsars’ coming back
from the dead as recycled pulsars. The slowly
rotating pulsar with a period of 8.5 seconds
discovered by Young et al.3 (marked by an
asterisk) does not fit in this picture.
ing board for another look at the details of the
way pulsars shine. The most popular idea is
that the observed radio emission arises from
bunches of charged particles streaming out
along the magnetic field lines. These particles
are created over the ‘polar caps’ of the neutron stars and are subsequently accelerated to
ultra-high relativistic speeds by a `1012 V
electric-potential drop along the field lines
close to the magnetic axis. If the magnitude of
the potential drop is greater than a critical
value, the pulsar will keep on pulsing away.
In the lifetime of a pulsar, illustrated in Fig. 1
(see also Fig. 2 on page 848), a young pulsar
will follow a well-worn path as it slows down
(moving to the right in the graph). If the
NATURE | VOL 400 | 26 AUGUST 1999 | www.nature.com