Minireview R41

Electrons and X-rays gang up on the ribosomeChristopher Davies1 and Stephen W White2,3*

In all cells, protein synthesis is coordinated by theribosome, and a number of pivotal structural studies onthis complex have been completed during 1999. Thecombined results of the X-ray crystallography and electronmicroscopy studies have shed new light on themechanism of this molecular machine.

Addresses: 1School of Biological Sciences, University of Sussex,Falmer, Brighton BN1 9QG, United Kingdom, 2Department ofStructural Biology, St. Jude Children’s Research Hospital, 332 NorthLauderdale, Memphis, TN 38105, USA and 3Department ofBiochemistry, University of Tennessee, 858 Madison, Suite G01,Memphis, TN 38163, USA.

*Corresponding author.E-mail: stephen.white@stjude.org

Structure 2000, 8:R41–R45

0969-2126/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

The past half-century has witnessed major advances instructural biology, and it is appropriate that an era whichbegan with the determination of the structure of DNAshould end with the promise of understanding proteinsynthesis at the molecular level. The ribosome has alwaysheld a special fascination for biologists because it lies atthe interface between nucleic acids and proteins, asembodied in the genetic code. It is widely felt that thekey to understanding how modern life evolved lies in itsmolecular mechanism.

By mass, the ribosome contains approximately two thirdsribosomal RNA (rRNA) and one third ribosomal protein.It comprises a pair of subunits, and the two main tasks ofprotein synthesis, or translation, are divided betweenthem [1]. The smaller subunit binds the messenger RNA(mRNA) at the decoding site, and the larger subunit con-tains the peptidyltransferase center (PTC) that attachesthe incoming amino acid to the growing peptide chain.These two ‘active sites’ face each other across the subunitinterface and are functionally linked by the two ends ofthe transfer RNA (tRNA) molecule, which can be viewedas the ribosome’s substrate. The anticodon loop of thetRNA ‘reads’ the mRNA at the decoding site, and thetRNA -CCA 3′ terminus delivers the attached amino acidor peptide to the PTC. Two tRNA molecules move inconcert with the mRNA between the subunit interface(translocation), and shuttle their amino acid and peptidecargoes between the aminoacyl (A) site and the peptidyl(P) site. The exit (E) site accommodates a third emptytRNA molecule at the end of the assembly line. This‘elongation cycle’ (Figure 1) requires two G-protein

factors, elongation factors EF-Tu and EF-G, which bindto the ‘factor binding site’ on the large subunit, andcontrol throughput and accuracy by a mechanism involv-ing conformational switches and GTP turnover. It is nowappreciated that the catalytic machinery of the ribosome isthe RNA component. This has profound evolutionary sig-nificance because of the growing consensus that early lifewas based on RNA.

Structural studies have concentrated on the smaller bacter-ial ribosome. With a molecular mass of approximately2.7 MDa (70S using sedimentation nomenclature) and nointernal symmetry, however, these particles have for manyyears been too large for X-ray crystallography studies andtoo small for electron microscopy (EM). Size is not the onlyproblem. During translation, the ribosome cycles betweenvarious states that mediate the translocation process, andwhich also involve the transient binding of initiation, elon-gation and termination factors. These states appear torequire conformational changes in the ribosome [1], andeach must be individually studied at the molecular level.During the past few years, these problems have started tobe overcome by combining the results of X-ray crystallog-raphy and EM. The two techniques traditionally providestructural information at different resolutions, but advancesin both have allowed the ribosome to be visualized at com-parable resolutions (Figure 2). Recent papers from theFrank and van Heel groups [2,3], the two leaders in theEM field, provide examples of how this process will occur.

EM has played a major role in ribosome research sincethe discovery of the particle in the late 1950s. Earlystudies defined the morphological features of thecomplex and the general locations of many of its compo-nents and active centers [4,5]. A breakthrough occurredduring the 1980s with the development of single-particlereconstruction techniques. The resolution of theseimages reached 25 Å in 1995 [6,7], and has now improvedto the point where regions of double-stranded RNA havestarted to become visible [8]. Using the well-character-ized biochemistry of the ribosome, it has also been possi-ble to trap and visualize individual translational states. Inthis way, the locations of the A, P and E site tRNAs havebeen identified [9–11], as well as the common bindingsite for EF-Tu and EF-G in the large subunit [12–14]. Inaddition, a tunnel in the 50S subunit and a groove in the30S subunit have been identified as suitable conduits forthe passage of nascent peptides and mRNA, respectively.Most significantly, perhaps, has been the demonstrationthat EM can visualize movements within the ribosome,even from a mixed population of particles [15]. Large

st8312.qxd 03/22/2000 11:51 Page R41

conformational changes have been identified in the smallsubunit [14–17] and also within the EF-Tu [12] andEF-G [13,14] molecules.

Low-resolution EM images can provide high-resolutioninformation when the molecular structures of componentscan be recognized and incorporated [18]. The knownstructures of tRNA, EF-Tu, EF-G and initiation factor 3(IF-3) have now been fitted into the images, and thesehave revealed the locations and environments of the ribo-some’s functional centers. Thus, the anticodon loop andthe -CCA 3′ terminus of tRNA have pinpointed thedecoding site and the PTC, respectively [10–12,14], andthe G-protein domains of EF-Tu and EF-G identify thefactor-binding site [12–14]. Recently, images of the smallsubunit initiation complex have revealed the mechanismby which IF-3 prevents subunit association and directs theinitiator tRNA exclusively to the P site [17].

X-ray crystallography of whole ribosomes and ribosomalsubunits began in the early 1980s with the demonstrationby Yonath that suitable crystals can be grown fromsamples isolated from thermophilic bacteria [19]. The suc-cessful analysis of these crystals ultimately depended onthe development of crystal freezing techniques (byYonath as part of this quest) and the advent of brightersynchrotron sources. Crystals of the entire bacterial ribo-some, and the large and small subunits (50S and 30S parti-cles, respectively) have now been reported and hold thepromise of near atomic resolution. Four papers appearedin 1999 that show spectacular advances in their analyses.Interpretable electron-density maps have now beenobtained for the 30S subunit [20,21], the 50S subunit [22]and the entire ribosome [23]. Progress in interpretingthese complex electron-density maps has been most suc-cessful when high-resolution structures of ribosomal com-ponents can be identified and incorporated. This has

R42 Structure 2000, Vol 8 No 2

Figure 1

A simplified diagram of the ribosomalelongation cycle showing the participation ofelongation factors EF-Tu (orange rectangle)and EF-G (purple oval). Amino acids areshown as coloured circles, and theirassociated tRNA molecules as elongatedrectangles of the same colour. The ribosomeis divided into a large (upper) subunit and asmall (lower) subunit, and the tRNA moleculescan occupy three sites, A, P and E, that spanthe subunit interface. The mRNA (thickhorizontal line) is bound to the small subunit.Proposed relative movements of the subunitsduring elongation are indicated. Progress ofthe elongation cycle requires conformationalchanges in both EF-Tu and EF-G (indicatedby the altered shapes of the rectangle andoval, respectively). These conformationalchanges are driven by the turnover of GTP.(The figure was adapted from [28].)

st8312.qxd 03/22/2000 11:51 Page R42

Minireview Structural analyses of the ribosome Davies and White R43

Figure 2

A direct comparison of the latest images ofthe ribosome obtained by X-raycrystallography (left) and electron microscopy(EM; right). In each case, the pairwise viewsare in equivalent orientations showing thesubunit interface surface. (a) The isolatedsmall (30S) subunit. The X-ray image (5.5 Å) isreproduced from [20] with permission. TheEM image was obtained by a separation of thelatest 11.5 Å map of the complete E. coli70S ribosome from the Frank group [29]. Thedecoding site is located at the top of adistinctive vertical extended region of double-stranded RNA that is depicted in a paleryellow on the EM map (see also (c) below).(b) The isolated large (50S) subunit. TheX-ray image (5 Å) is reproduced from [22]with permission, and the EM image (7.5 Å) isreproduced from [3]. Note that, in the X-rayimage, the L1 stalk (on the left hand side) ismissing, but it is clearly visible in the EMimage. (c) The 30S subunit and (d) the50S subunit in the context of the 70Sribosome with the three tRNA moleculesincluded. The X-ray images (7.8 Å) werereproduced from [23] with permission, andthe EM images were obtained by a separationof Frank's 11.5 Å map (see above). In theX-ray images, the A, P and E site tRNAs areshown in green, blue and orange, respectively.In the EM images, the A, P and E sites are inpurple, green and orange, respectively, andare inferred from previous EM studies [11,14].Note that the agreement between thepositions of the tRNA molecules in bothimages is excellent. Also, the A and P sitetRNAs are adjacent, whereas the E site tRNAis further away.

st8312.qxd 03/22/2000 11:51 Page R43

allowed rather detailed models of the 30S decoding siteand the 50S factor-binding site to be constructed.

In the recent paper from the Frank group [2], a novel EMtechnique is described for mapping regions of rRNA onimages of the bacterial ribosome. The idea originates fromthe larger eukaryotic ribosome where additional morpho-logical features represent expansions of the rRNA [24].Frank reasoned that artificial expansions of the bacterialrRNA should be visible in EM images and these, in turn,would pinpoint their locations on the surface. This turnedout to be the case. Frank went further, however; he usedfragments of tRNA to expand defined stem–loop regions,and the EM images clearly showed tRNA-like objects pro-jecting from the ribosome surface. By modeling the tRNAstructure into these projections, it was possible to gaininsights into the local conformation of the rRNA. Thesestudies are confined to the large subunit, but could equallywell be applied to the small subunit. This should prove tobe a valuable technique for identifying surface regions ofthe rRNA that are more distant from the active centers.

The van Heel group [3] reported the 7.5 Å EM structureof the isolated Escherichia coli 50S subunit. This is aremarkable achievement for EM, and it vindicates thepredicted potential of the single particle method for high-resolution analyses of all large biological systems [25].Reassuringly, this image of the subunit closely resemblesthat obtained by X-ray crystallography at 5 Å resolution[22]. However, it nicely complements the X-ray structureby showing regions that are obscured by conformationalflexibility and/or crystal disorder. Most notably, the dis-tinctive projections of the ribosomal proteins L1 andL7/L12 on opposite sides of the 50S subunit are clearlypresent in the EM image, but poorly resolved in the X-raymaps. When compared to images of the entire ribosome, itis also possible to identify conformational changes in the50S subunit that result from its association with the 30Ssubunit. One such change is an apparent opening of adeep cleft on the interface side of the 50S subunit, whichis the site of the PTC. Data can now be exchangedbetween the X-ray and EM groups to speed up the inter-pretations and to resolve differences and ambiguities.

What can we expect from the future? It is now clear thatX-ray crystallography will eventually provide ‘snapshots’of the two ribosomal subunits and the entire complex atnear atomic resolution. Although spectacular, these struc-tures are likely to pose as many questions as they answer.Even high-resolution enzyme structures typically requirefollow up mutagenesis experiments to fully understandtheir mechanisms. Most importantly, the structures areunlikely to provide much information about functionalmovements within the ribosome. EM has demonstratedthat it can fill this gap, however, and it should eventuallybe possible to use the crystal structures to interpret

movements at the molecular level. In the past, EMimages of the ribosome have been useful to X-ray crystal-lographers for obtaining preliminary phase information[26], and their overlapping resolutions will allow thispractical collaboration to continue. For example, crystal-lographers could call upon the latest technique describedby Frank to identify or confirm questionable interpreta-tions of electron density. It is also important to appreciatethat the latest structures would not be possible withoutthe previous efforts of biochemists that have character-ized and mapped the ribosomal components. It will beimportant to maintain these collaborations, and thepresent structures should facilitate these future efforts.For example, the whole particle crystal structures havealready identified ribosomal-protein–rRNA complexesthat can be studied in isolation. In addition, the biochem-ical footprinting and cross-linking techniques that havebeen so vital for mapping the ribosome can now bespecifically targeted to studying conformational changes.

Not surprisingly, the best characterized regions of the ribo-some, biochemically, functionally and now structurally, areits active centers. Although it will eventually be necessaryto determine the entire structure, it is likely that ratherdetailed models of these regions will soon be available.The most important region of the small subunit is centrallylocated on the interface side, and includes the decodingsite and a switch region that appears to undergo a crucialconformational change [27]. This entire region is welldefined in the 30S crystal structure [20,21]. The importantregions of the large subunit are the PTC and the factor-binding site; the latter is already well defined in the 50Scrystal structure [22], and similar definition of the PTCwill no doubt quickly follow. Finally, the 70S crystal struc-ture has started to reveal details of the tRNA-binding sites,the interaction between tRNA and mRNA, and informa-tion on the 50S–30S interface [23]. Thus, the prospectsthat the ribosome will reveal some of its more importantmechanistic secrets in the near future are excellent.

AcknowledgementsThe authors would like to thank Joachim Frank and Marin van Heel for sup-plying the EM views of the ribosome shown in Figure 2. They are also grate-ful to Tina Izard and Hee Won Park for critically reading the manuscript.SWW is supported by National Institutes of Health grant GM 44973,Cancer Center (CORE) support grant CA 21765, and the AmericanLebanese Syrian Associated Charities (ALSAC).

References1. Wilson, K.S. & Noller, H.F. (1998). Molecular movement inside the

translational engine. Cell 92, 337-349.2. Spahn, C.M.T., Grassucci, R.A., Penczek, P. & Frank, J. (1999). Direct

three-dimensional localization and positive identification of RNAhelices within the ribosome by means of genetic tagging and cryo-electron microscopy. Structure 7, 1567-1573.

3. Matadeen, R., et al., & van Heel, M. (1999). The Escherichia coli largeribosomal subunit at 7.5 Å resolution. Structure 7, 1575-1583.

4. Stöffler, G. & Stöffler-Meilicke, M. (1984). Immunoelectron microscopyof ribosomes. Annu. Rev. Biophys. Bioeng. 13, 303-330.

5. Oakes, M.I., Kahan, L. & Lake, J.A. (1990). DNA-hybridization electronmicroscopy tertiary structure of 16S rRNA. J. Mol. Biol. 211, 907-918.

R44 Structure 2000, Vol 8 No 2

st8312.qxd 03/22/2000 11:51 Page R44

6. Frank, J., et al., & Agrawal, R.K. (1995). A model of protein synthesisbased on cryo-electron microscopy of the E. coli ribosome. Nature376, 441-444.

7. Stark, H., et al., & van Heel, M. (1995). The 70S Escherichia coliribosome at 23 Å resolution: fitting the ribosomal RNA. Structure3, 815-821.

8. Mueller, F. & Brimacombe, R. (1997). A new model for the three-dimensional folding of Escherichia coli 16S ribosomal RNA. I Fittingthe RNA to a 3D electron microscopic map at 20 Å. J. Mol. Biol.271, 524-544.

9. Agrawal, R.K., et al., Frank, J. (1996). Direct visualization of A-, P-, andE-site transfer RNAs in the Escherichia coli ribosome. Science271, 1000-1002.

10. Stark, H., et al., & van Heel, M. (1997). Arrangement of tRNAs in pre-and post-translational ribosomes revealed by electron cryomicroscopy.Cell 88, 19-28.

11. Malhotra, A., et al., & Frank, J. (1998). Escherichia coli 70S ribosomeat 15 Å resolution by cryo-electron microscopy: localization of fMET-tRNAfMet and fitting of L1 protein. J. Mol. Biol. 280, 103-116.

12. Stark, H., Rodnina, M.V., Rinke-Appel, J., Brimacombe, R.,Wintermeyer, W. & van Heel, M. (1997). Visualization of elongationfactor Tu on the Escherichia coli ribosome. Nature 389, 403-406.

13. Agrawal, R.K., Penczek, P., Grassucci, R.A. & Frank, J. (1998).Visualization of the elongation factor G on E. coli 70S ribosome: themechanism of translation. Proc. Natl Acad. Sci. 95, 6134-6138.

14. Agrawal, R.K., Heagle, A.B., Penczek, P., Grassucci, R.A. & Frank, J.(1999). EF-G-dependent GTP hydrolysis induces translocationaccompanied by large conformational changes in the 70S ribosome.Nat. Struct. Biol. 6, 643-647.

15. Gabashvili, I.S., Agrawal, R.K., Grassucci, R. & Frank, J. (1999).Structure and structural variations of the Escherichia coli 30Sribosomal subunit as revealed by three-dimensional cryo-electronmicroscopy. J. Mol. Biol. 286, 1285-1291.

16. Lata, K.R., Agrawal, R.K., Penczek, P., Grassucci, R., Zhu, J. & Frank, J.(1996). Three-dimensional reconstruction of the Escherichia coli 30Sribosomal subunit in ice. J. Mol. Biol. 262, 43-52.

17. McCutcheon, J.P., et al., & Frank, J. (1999). Location of translationalinitiation factor IF3 on the small ribosomal subunit. Proc. Natl Acad.Sci. 96, 4301-4306.

18. Holmes, K.C. (1994). Solving the structures of macromolecularcomplexes. Structure 2, 589-593.

19. Yonath, A. (1992). Approaching atomic resolution in crystallography ofribosomes. Annu. Rev. Biophys. Biomol. Struct. 21, 77-93.

20. Clemons, W.M. Jr., May, J.L.C., Wimberly, B.T., McCutcheon, J.P.,Capel, M. & Ramakrishnan, V. (1999). Structure of a bacterial 30Sribosomal subunit at 5.5 Å resolution. Nature 400, 833-840.

21. Tocilj, A., et al., & Yonath, A. (1999). The small ribosomal subunit fromThermus thermophilus at 4.5 Å: pattern fittings and the identificationof a functional site. Proc. Natl Acad. Sci. 96, 14252-14257.

22. Ban, N., Nissen, P., Hansen, J.C., Capel, M., Moore, P.B. & Steitz, T.A.(1999). Placement of protein and RNA structures into a 5 Å resolutionmap of the 50S ribosomal subunit. Nature 400, 841-847.

23. Cate, J.H., Yusupov, M.M., Yusupova, G.Z., Earnest, T.N. & Noller, H.F.(1999). X-ray crystal structures of 70S ribosome functionalcomplexes. Science 285, 2095-2104.

24. Dube, P., et al., & Brimacombe, R. (1998). Correlation of theexpansion segments in mammalian rRNA with the fine structure of the80S ribosome: a cryo-electron microscopic reconstruction of the ofthe rabbit reticulocyte ribosome at 21 Å resolution. J. Mol. Biol.279, 403-421.

25. Henderson, R. (1995). The potential and limitations of neutrons,electrons and X-rays for atomic resolution microscopy of unstainedbiological molecules. Q. Rev. Biophys. 28, 171-193.

26. Harms, J., et al., & Yonath, A. (1999). Elucidating the medium-resolution structure of ribosomal particles, an interplay betweenelectron cryo-microscopy and X-ray crystallography. Structure7, 931-941.

27. Lodmell, J.S. & Dahlberg, A.E. (1997). A conformational switch inEscherichia coli 16S ribosomal RNA during decoding of messengerRNA. Science 277, 1262-1267.

28. Czworkowski, J. & Moore, P.B. (1996). The elongation phase ofprotein synthesis. Prog. Nucleic Acids Res. Mol. Biol. 54, 293-332.

29. Gabashvili, I.S., et al., & Frank, J. Solution structure of the E. coli70S ribosome at 11.5 Å resolution. Cell, in press.

Minireview Structural analyses of the ribosome Davies and White R45

st8312.qxd 03/22/2000 11:51 Page R45