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news and views 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