A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. fitting the...

J. Mol. Biol. (1997) 271, 524±544

A New Model for the Three-dimensional Foldingof Escherichia coli 16 S Ribosomal RNA. I. Fittingthe RNA to a 3D Electron Microscopic Map at 20 AÊ

Florian Mueller and Richard Brimacombe*

Max-Planck-Institut fuÈ rMolekulare GenetikAG-Ribosomen, Ihnestrasse 7314195 Berlin, Germany

Abbreviations used: EM, electronimmune EM; 3D, three-dimensiona

0022±2836/97/340524±21 $25.00/0/mb

Recently published models of the Escherichia coli 70 S ribosome at 20 AÊ

resolution, obtained by cryo-electron microscopy (cryo-EM) combinedwith computerized image processing techniques, exhibit two features thatare directly relevant to the in situ three-dimensional folding of the rRNAmolecules. First, at this level of resolution many ®ne structural details arevisible, a number of them having dimensions comparable to those ofnucleic acid helices. Second, in reconstructions of ribosomes in the pre-and post-translocational states, density can be seen that correspondsdirectly to the A and P site tRNAs, and to the P and E site tRNAs, re-spectively, thus enabling the decoding region on the 30 S subunit to belocated rather precisely. Accordingly, we have re®ned our previousmodel for the 16 S rRNA, based on biochemical evidence, by ®tting it tothe cryo-EM contour of ribosomes carrying A and P site tRNAs. For thispurpose, the most immediately relevant evidence consists of new site-di-rected cross-linking data in the decoding region, which de®ne sets of con-tacts between the 16 S rRNA and mRNA, or between 16 S rRNA andtRNA at the A, P and E sites; these contact sites can be correlated directlywith the tRNA positions in the EM structure. The model is extended toother parts of the 16 S molecule by ®tting individual elements of thewell-established secondary structure of the 16 S rRNA into the appropri-ate ®ne structural elements of the EM contour, at the same time takinginto account other data used in the previous model, such as intra-RNAcross-links within the 16 S rRNA itself. The large body of available RNA-protein cross-linking and foot-printing data is also considered in themodel, in order to correlate the rRNA folding with the known distri-bution of the 30 S ribosomal proteins as determined by neutron scatteringand immuno-electron microscopy. The great majority of the biochemicaldata points involve single-stranded regions of the rRNA, and therefore,in contrast to most previous models, the single-stranded regions areincluded in our structure, with the help of a specially developed model-ling programme, ERNA-3D. This allows the various biochemical datasets to be displayed directly, in this and in the accompanying papers, ondiagrams of appropriate parts of the rRNA structure within the cryo-EMcontour.

# 1997 Academic Press Limited

Keywords: electron microscopic reconstruction; computer modelling;rRNA secondary structure; intra-RNA cross-linking; structure ®tting*Corresponding author

Introduction

In order to be able to understand the intricateprocesses involved in protein biosynthesis on ribo-somes at the molecular level, it is essential to ob-

microscopy; IEM,l.

971210

tain a correspondingly detailed knowledge of thestructure of the ribosome itself. A great deal of ef-fort has been expended on this topic, over the lastthree decades. On the physical side, although con-siderable progress has been made in the crystalliza-tion of ribosomal particles (Von BoÈhlen et al., 1991),a determination of the structure of a ribosome atatomic resolution by X-ray crystallographic anal-

# 1997 Academic Press Limited

A New 3D Model for E. coli 16 S Ribosomal RNA 525

ysis still remains a long-term goal. This problem isof course not speci®c for the ribosome, but is ofgeneral concern for most structural studies onlarge biological macromolecular complexes. Re-cently, however, dramatic advances in the analysisof such complexes by cryo-electron microscopy(cryo-EM) combined with image processing tech-niques (e.g. see Serysheva et al., 1995; Frank et al.,1995) have made it possible to generate computer-ized reconstructions of the particles at increasinglyhigh resolution. The method exploits the randomspatial orientations of the individual macromolecu-lar complexes within the embedding matrix (amor-phous ice), and obviates the need for tilting thespecimen at different angles to obtain the EM data.Several thousand images are considered, usingpattern-recognition approaches to de®ne classaverages of characteristic views. After the a poster-iori determination of the different projection angles(angular reconstitution) a ®rst 3D reconstructioncan be obtained, which is subjected to iterative re-®nement procedures. So far no direct theoreticalresolution limits for this single particle approachare in sight (Henderson, 1995), and in the case ofthe bacterial ribosome the current resolution of thestructures obtained has reached a level of ca 20 AÊ

(Stark et al., 1995, 1997).On the biochemical side, a unique feature of

research into ribosomes, particularly the ribosomeof the eubacterium Escherichia coli, is the vast bodyof low-resolution structural information that hasaccumulated over the years. The available data setsinclude determinations of the spatial distributionof the ribosomal proteins by neutron scattering orimmuno-electron microscopy (IEM), analyses ofcontacts or neighbourhoods between ribosomalproteins and rRNA by cross-linking and foot-print-ing techniques, analyses of neighbourhoods withinor between the rRNA molecules by intra or inter-rRNA cross-linking, and studies on the location ofthe rRNA relative to functional ligands such asmRNA and tRNA, again with the help of cross-linking and foot-printing methods (for reviews,see Brimacombe, 1995; Noller et al., 1995). Anumber of research groups (Expert-BezancËon &Wollenzien, 1985; Brimacombe et al., 1988; Naganoet al., 1988; Stern et al., 1988; Mitchell et al., 1990;Hubbard & Hearst, 1991; Malhotra & Harvey,1994; Fink et al., 1996) have made use of thesetypes of data to fold the well-established secondarystructures of the 16 S and 23 S rRNA moleculesinto three dimensions. The resulting 3D models areessentially ``cartoon'' structures of the ribosomalsubunits, in which the various helical elements ofthe rRNA have been manoeuvred relative to theribosomal proteins so as to satisfy as many of thetopographical constraints as possible. The modelsdiffer from one another according to the weight orsigni®cance that the individual authors have as-signed to the various data sets, and also of courseaccording to the availability of the data sets con-cerned at the time when a particular model wasconstructed. Among the published models, only

one (Malhotra & Harvey, 1994) has so far includedthe speci®c use of an earlier lower-resolution EMstructure of the 30 S subunit (Verschoor et al., 1984)as an additional constraint on the folding of the16 S rRNA molecule, (although it should be notedthat the latter structure has been criticized; VanHeel & StoÈ f¯er-Meilicke, 1985).

The advent of the EM reconstructions at ca 20 AÊ

resolution discussed above (Stark et al., 1995, 1997)has opened a new chapter in the rRNA modellingstudies. These reconstructions show a great deal ofhitherto unobserved ®ne structure, including anumber of features with dimensions suggestive ofrRNA helices, which can be correlated with speci®celements of the 16 S rRNA (Stark et al., 1995). Inanother study, Agrawal et al. (1996) have publishedreconstructions of ribosomes carrying three un-charged tRNA molecules, in which density corre-sponding to these tRNA molecules could be seen.Furthermore, in the most recent reconstructions(Stark et al., 1997), made from 70 S ribosomes inthe pre and post-translocational states, it waspossible to directly visualize the individual tRNAsin the ribosomal A and P sites, or P and E sites, re-spectively. Thus, nucleotides in the 16 S rRNA,which have been implicated by cross-linking orfoot-printing as lying close to tRNA or mRNAwithin the decoding area of the 30 S subunit, cannow be placed with some con®dence at the appro-priate sites in the EM structure. This developmentis ideally suited to our model-building strategy forthe 16 S rRNA, which has for some time beenbased on the principle of building up the modelaround the ``functional complex'' of mRNA andtRNA in the decoding area (for discussion, seeBrimacombe, 1995). The structure surrounding thefunctional complex is then extrapolated to otherand more remote regions of the 16 S molecule bymaking use of the constraints imposed by thebiochemical data described above (intra-RNAcross-links, RNA-protein interactions, etc.), andthose imposed by the secondary structure itself,using our previous cartoon model (cf. Brimacombeet al., 1988; Brimacombe, 1995) as a guide. Further-more, and most importantly, the ®ne structure ofthe EM contour now provides an additional set ofrigorous constraints on the folding of the 16 SrRNA molecule, and many of the individual 16 SrRNA helices can be ®tted into appropriate ®nestructural features, thus giving the cartoon model adirect ``physical'' basis.

The new three-dimensional model for the 16 SrRNA that we have derived is described here andin two accompanying papers. In this ®rst paper,we simply present the completed 16 S model, andshow how the secondary structure of the 16 S mol-ecule ®ts to the cryo-EM reconstruction of ribo-somes containing A and P site-bound tRNAs(Stark et al., 1997), at the same time satisfying theintra-RNA cross-linking data. Only those cross-links are considered that were formed in situ inintact subunits, and where the cross-link sitescould be localized to within a few bases. We also

526 A New 3D Model for E. coli 16 S Ribosomal RNA

consider the distribution of modi®ed nucleotides inthe model, and the positions of bases that havebeen located by IEM on the surface of the 30 S sub-unit. On the other hand, we have not speci®callytaken into account the results of ``higher orderstructure'' analyses of the 16 S rRNA (e.g. seeMoazed et al., 1986); the accessibility (or lack there-of) of a particular nucleotide is obviously highlysensitive to local ®ne structure, interactions withproteins, etc., with the result that, in our opinion,this type of data will become really useful in de-tailed model-building studies only when a higherlevel of resolution has been reached. In the secondpaper (Mueller & Brimacombe, 1997), we add theneutron scattering map of the 30 S ribosomal pro-teins (Capel et al., 1988) to the model, and describein detail the ®t to the large body of RNA-proteininteraction data for the individual proteins. In thethird paper (Mueller et al., 1997), we come back tothe question of the functional complex, and de-monstrate how the model ®ts to the correspondingdata, including cross-links between 16 S rRNA andmRNA, cross-links and foot-prints to tRNA, andthe positions of sites known to interact with riboso-mal factors or antibiotics. In a structure of thiscomplexity, and with the vast amount of infor-mation available, there are inevitably some discre-pancies, both within the individual data sets andbetween the different types of experimental data.Such discrepancies are all too often played downor even ignored in the ribosomal literature. Here,on the other hand, we emphasize them as andwhere they arise, because it is precisely the discre-pancies that may help to reveal important newconcepts. In particular, experiments made underdifferent conditions may re¯ect differing con®gur-ations of the ribosome, and in the context of thefunctional data there is one region of the 30 S sub-unit where the various observations can be ex-plained only in terms of major conformationalchanges within the functioning ribosome. In allthree papers, the data are presented with the helpof the computer programme ERNA-3D, which wasspecially developed for the modelling of the largeribosomal RNA molecules (Mueller et al., 1995).

Results

Secondary structure and intra-RNAcross-linking data

Figure 1 shows the secondary structure of theE. coli 16 S rRNA (cf. Brimacombe, 1995), andindicates the numbering system used to identifythe various helical elements in the model. Many ofthe helices are given subsidiary numbers (cf.Malhotra & Harvey, 1994; Fink et al., 1996), whichdivide the helix concerned into regions separatedby intra-helical loops and bulges. For example,helix 7 (Figure 1, lower left) is divided into threeseparate regions marked 1, 2 and 3, and these arereferred to as 7.1, 7.2 and 7.3, respectively. Alterna-tively, in the all-atom stereo computer diagrams

describing the model (see below) the helices aredenoted h7d1, h7d2 and h7d3. In such cases theintra-helical loops were used to introduce slightbends into the overall helix structure, so as tofacilitate ®tting the rRNA to the EM contour.

Data from in situ intra-RNA cross-linking exper-iments (Atmadja et al., 1986; Stiege et al., 1986,1988) are also included in Figure 1, marked byRoman numerals. These data, although publishedsome time ago, are directly relevant to the three-di-mensional folding of the 16 S rRNA, and moreovera number of the cross-links have since beenindirectly corroborated by other ®ndings. Forexample, nucleotides 693 and 794 (cf. cross-link V)are both foot-print sites for P-site bound tRNA(Moazed & Noller, 1990), and nucleotides 1125 to1130 and 1278 to 1281 (cf. cross-link VII) are simi-larly foot-print sites for protein S9 (Stern et al.,1988). A further cross-link, connecting residues 118and 288-289 (Stiege et al., 1986), is identical withthe more recently added base-pair 118-288 (cf.Haselman et al., 1989), which constitutes helix 6b(Figure 1). It is important to remember that theintra-RNA cross-link sites could not, in general, belocalized to a single nucleotide, but rather only toa short oligonucleotide sequence, and these se-quences are listed in Table 1. Table 1 (and Figure 1)also include the sites of inter-RNA cross-linksidenti®ed between 16 S and 23S rRNA (Mitchellet al., 1992).

In addition, Figure 1 shows some potential base-pairs from alternative versions of the 16 S rRNAsecondary structure (cf. Brimacombe, 1995; Nolleret al., 1995), denoted by broken lines, which havenot been included in our model. Of these, the pairs61-63/104-106 (extending helix 6 at its base) and521-522/527-528 (extending helix 18 at the loopend) would be possible to accommodate in themodel without dif®culty; the same applies to po-tential base-pairs (Noller et al., 1995) betweennucleotides 921-922 and 1395-1396 (extending helix28; not shown in Figure 1). On the other hand, thepairs 437-440/494-497 (which extend helix 17 atthe base and are absent from the latest secondarystructure reported by Noller et al., 1995) are notcompatible with our model in its present form, be-cause the respective termini of helices 16 and 18are too far apart (see below). Similarly, the pairs885-887/910-912 (Lodmell et al., 1995) can beformed only at the expense of opening the pseudo-knot structure (helix 2); it has been suggested(Lodmell et al., 1995) that these latter base-pairs,which are well-supported phylogenetically, rep-resent an alternative conformation of this area ofthe 16 S rRNA. Finally, in the context of the 16 Ssecondary structure, it should be noted that thelocations of post-transcriptionally modi®ed basesare also given in Figure 1.

Constructing the 16 S model with ERNA-3D

One of the central features of our modellingprogramme ERNA-3D (see Materials and Methods)

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Table 1. Summary of intra- and inter-RNA cross-linking data

Cross-link Location in 16 S rRNA Reference

I h3 (31) :h4 (48) Stiege et al. (1986)II h3 (31) :h12 (306) Atmadja et al. (1986)III h15 (366-369) :h4 (397-399) Stiege et al. (1986)IV h11 (243-244) :h27 (891-894) Stiege et al. (1988)V h23 (693-696) :h24 (794) Atmadja et al. (1986)VI h37 (1090-1094) :h40 (1161-1164) Stiege et al. (1986)VII h39 (1125-1127) :h41 (1280-1281) Stiege et al. (1986)23 S h44 (1408-1411) :h69 (23S RNA; 1912-1920) Mitchell et al. (1992)

h45 (1518-1520) :h69 (23S RNA; 1912-1920)

The Roman numerals assigned to the cross-links are the same as in Figure 1. The 16 S loca-tions show the helix number (Figure 1) in bold face, with the precise nucleotide(s) concernedin parentheses. The cross-links from Stiege et al. (1986, 1988) were induced in vivo by directUV-irradiation, whereas those from Aal. (1986) and Mitchell et al. (1992) wereinduced by treatment of 30 S subunits or 70 S ribosomes with bis-(2-chloroethyl)-methyl-amine, nitrogen mustard.


is the dynamic docking of single strands. After thehelical elements (cf. Figure 1) have been de®ned inthe form of standard A-double helices, this dockingtakes place by appropriate iterative rotations alongthe sugar-phosphate backbone of the single-stranded sequences. Once docked, the singlestrands remain attached to their adjacent heliceswhile the latter are being moved within the model,provided of course that the maximum possible dis-tance for a particular single strand is not exceeded.Individual helices, or selected groups of helices,can be translated or rotated about any point. As aresult, the 16 S rRNA helices can be manipulatedvery rapidly within the EM structure, which is it-self displayed as a semi-transparent contour. The16 S model presented here has in fact already un-dergone several cycles of re®nement, as improvedEM reconstructions or new biochemical data havebecome available (cf. Stark et al., 1995).

Once the helices have been positioned in themodel, the con®gurations of the single-stranded re-gions can be adjusted, so as to satisfy a particularbiochemical constraint such as a cross-link. It mustbe stressed, however, that at the current level ofresolution the con®gurations of the single strandsare still very arbitrary, and the model is not in-tended to do more than convey a general indi-cation of where a particular single-strandedresidue is located. For this reason, we have notconsidered it worthwhile at this stage to attemptany energy minimization of the structure, forexample to resolve van der Waals con¯icts withinthe single-stranded regions. Energy minimizationwould not alter the overall arrangement of therRNA model.

With the help of ERNA-3D, the rRNA model canbe displayed in a number of different ways. Theoptions include display of the helical elements ascylinders (cf. Brimacombe et al., 1988; Malhotra &Harvey, 1994), or of the sugar-phosphate backboneas a continuous tube. Alternatively, the whole 16 SrRNA, or selected regions thereof, can be presentedas an all-atom ``wire-frame'' structure, with indi-vidual nucleotides being highlighted as ball-and-

stick or CPK models. This latter method of rep-resentation is particularly useful for displaying thebiochemical data, such as cross-links or foot-prints.The great majority of these biochemical data pointslie within single-stranded regions of the rRNA,and in most previously published rRNA modelsthe single-stranded regions have not been shown,although they comprise ca 40 % of the molecule.As a result, it is very dif®cult for the reader tojudge how good or bad the ®t to the biochemicaldata really is in a particular model. Here, for dis-play purposes, we show the single-stranded re-gions in the all-atom mode, so that the reader canindeed see the quality of the ®t to the experimentaldata directly. In our opinion this detailed displaygives a better ``feeling'' for the data than the use ofpseudo-atoms to represent structural elements (e.g.see Malhotra & Harvey, 1994), although once againwe emphasize that the detailed con®gurations ofthese regions are still undetermined at the currentlevel of resolution.

Overall features of the 16 S rRNA model

The EM reconstruction that we have used to de-rive the 16 S rRNA model is that of the 70 S ribo-some in the pre-translocational state (Stark et al.,1997), carrying tRNAs at the A and P sites. A gen-eral stereo view of the complete 16 S molecule,®tted to the 30 S subunit moiety of this 70 S recon-struction, is shown in Figure 2(a). The A and P sitetRNAs are included in the Figure as ``backbonetube'' models. In this and in the following Figures,the EM structure is displayed as a semi-transparentenvelope, and due to the transparency the hue ofthe ribosomal particle depends on how many``layers'' of the envelope are visible at any position;with increasing thickness, the envelope takes on anincreasingly golden appearance. The 16 S rRNA inFigure 2(a) is shown as an all-atom structure, withthe helical elements (cf. Figure 1) in white, inter-he-lical single strands in red, intra-helical loops ingreen and hairpin loop ends in blue; more detailedviews are shown in Figures 5 to 9, below. As withthe EM envelope itself, the colours of the RNA

Figure 2. Overall features of the 16 S rRNA model. (a) Stereo view of the 16 S rRNA in the 70 S ribosome, viewedfrom the L7/L12 side (cf. Stark et al., 1997). The 16 S molecule is colour coded as described in the text. The A and Psite tRNAs are represented by the light blue and green tube models, respectively. (b) Stereo view of the 16 S rRNA inthe 30 S subunit, viewed from the solvent side of the latter. The colour coding is as in (a). (c) The double helicalelements of the 16 S rRNA in the 30 S subunit; a stereo view from the interface side of the subunit. The rRNAdomains are coloured 50 dark blue, central red, 30 light blue, 30-minor yellow.



structure change according to how deeply they liewithin the semi-transparent contour. It is import-ant to note that objects lying within the contourare displayed with reduced contrast, and areslightly ``fogged''; this is how the computer gener-ates the illusion of semi-transparency. It shouldalso be noted at this stage that all the stereo dia-grams in this series of papers are intended to beviewed with a standard 6 cm magnifying stereo-scope.

In Figure 2(b), the 30 S subunit has been excisedfrom the 70 S EM reconstruction of Figure 2(a), andis shown, together with the complete 16 S rRNA,from the solvent side of the subunit. The pattern ofbridges and cavities in the central region of thestructure is very stable in the EM reconstruction,and was also seen in both ``empty'' ribosomes andthose in the post-translocational state (Stark et al.,1995, 1997); the ®ne structure in this region is ofparticular importance in ®tting the 16 S rRNA. Thepronounced ``toe'' at the bottom right-hand cornerof the 30 S subunit in Figure 2(b) is also a stablefeature (cf. Agrawal et al., 1996), whereas in con-trast the regions that are shown devoid of rRNA inthe head of the subunit (upper right in Figure 2(b))and in the side lobe (centre left) were more vari-able with regard to both size and shape in thevarious reconstructions (Stark et al., 1995, 1997).

The distribution of the different domains of the16 S rRNA is illustrated in Figure 2(c). Here thesingle-stranded regions are omitted, and the colourcode is dark blue for the 50-domain of the rRNA(helices 1 to 18 in Figure 1), red for the central do-main (helices 19 to 27), light blue for the 30-domain(helices 28 to 43), and yellow for the 30-minordomain (helices 44 and 45). It can be seen that theoverall distribution is the same as that in previous16 S models (Brimacombe et al., 1988; Stern et al.,1988; Malhotra & Harvey, 1994), with the 50-do-main corresponding to the body of the subunit, the30-domain to the head, and the central domain pre-dominantly to the side lobe (or platform). Parts ofthe central and 30-minor domains also contributeto the body of the subunit, where the packing ofthe rRNA is much more dense than in the headand side lobe areas. In fact, a simple calculation ofthe amount of ribosomal protein mass associatedwith the various rRNA domains (Mueller &Brimacombe, 1997) indicates that the protein torRNA ratio in the head and side lobe domains isapproximately three times greater than that in thebody. However, as noted above, the areas ofthe subunit that are shown devoid of rRNA in thehead and side lobe regions (Figure 2(b) and (c))were variable in the different EM reconstructions,and it is therefore likely that material other thanribosomal proteins (e.g. factors etc.) may contributeto the EM density here. The view in Figure 2(c) isfrom the interface side of the 30 S subunit, and theareas of density forming connections to the 50 Ssubunit (as well as the density due to the A and Psite tRNA molecules; cf. Figure 2(a)) can be seenprotruding towards the reader in the Figure; the

precise cut-off points within these intersubunit con-nections where we have excised the 30 S subunitfrom the 70 S reconstruction are unavoidablysomewhat arbitrary.

In order to enable the reader to identify the pos-itions of the individual helices, the helical regionsof the 16 S rRNA model are illustrated in the formof cylinders (cf. Brimacombe et al., 1988) inFigures 3 and 4. The colour coding is according tothe rRNA domain structure, and is the same asthat in Figure 2(c). Figure 3 shows two stereoviews of this cylinder representation within thesemi-transparent EM contour, one from the solventside of the 30 S subunit and the other with the30 S:50 S interface to the left, respectively, whereasFigure 4 shows four ``mono'' views without theEM contour, each rotated about the vertical axis by90�. In Figure 4 the helices are numbered as inFigure 1; e.g. 41.1, 41.2, 41.3 and 41.4 are the foursections of helix 41 in the 30 domain. The P sitetRNA molecule is also included in Figures 3 and 4as a backbone tube model (cf. Figure 2(a)), to indi-cate the orientation of each diagram with respectto the position of the 50 S subunit. It can further-more be seen that the decoding area of the 30 Ssubunit, that is to say the region surrounding theanticodon loop of the P site tRNA (and that of theneighbouring A site tRNA, not shown in Figures 3and 4), lies at the junction of the four differentcoloured domains. This is in agreement with thebiochemical evidence and, as noted in the Intro-duction, our 16 S model has been built up aroundthe decoding area. However, in view of the confor-mational heterogeneity already mentioned in theIntroduction in connection with the functionaldata, it is simpler to describe the model in terms ofthe domain distribution of the 16 S rRNA in thecompleted structure; a description of the functionalcentre is given in the second accompanying paper(Mueller et al., 1997). The following section showssome details of the ®tting of the 16 S rRNA to theEM contour, with emphasis on the locations ofnucleotides involved in intra-RNA cross-links (seeFigure 1 and Table 1). Details of other parts of thestructure can be found in the accompanying paperthat deals with the RNA-protein interaction data(Mueller & Brimacombe, 1997).

Arrangement of rRNA in the 30 S subunit body

As in most previous models (Brimacombe et al.,1988; Stern et al., 1988; Malhotra & Harvey, 1994),the 50-terminal helix 1 and the pseudoknot helix 2(Figure 1) lie at the top of the 30 S subunit body,and are central to the entire 16 S rRNA structure,as they form connections to all four rRNA do-mains; namely, to the 50-domain via helix 3, to thecentral domain via helices 19 and 27, to the 30-do-main via helix 28, and to the 30-minor domain viahelix 44. These connections can best be seen in thecentre of Figure 4(a), helices 1 and 2 being mod-elled as a single stacked helix, with helices 19 and27 lying somewhat lower down in the model, by

Figure 3. Simpli®ed view of the helix packing in the 30 S subunit, with 16 S rRNA helices displayed as cylinders. TherRNA domains are colour coded as in Figure 2(c), 50 dark blue, central red, 30 light blue, 30-minor yellow. The P sitetRNA is included as a green tube model for reference. (a) Stereo view from the solvent side of the 30 S subunit.(b) Stereo view with the 30 S:50 S subunit interface on the left. (Note: the small black areas on the cylinders occurwhere polygons in the computer rendering of the cylinders coincide with those of the EM contour.)


virtue of the relatively long single strands connect-ing them to helices 3 and 2, respectively (Figure 1).An all-atom stereo presentation of the arrangementof helices 1, 2, 3 and 28, together with helix 4, isshown in Figure 5(a). Here, and in the Figures 6 to8, the colour code is the same as that of Figure 2(a);namely, white for helical regions, red for inter-heli-cal single strands, green for intra-helical loops andblue for hairpin loop-ends. The helices are num-bered in red, with the numbers of the terminalnucleotides of each strand of the helices marked inblue. The orientation of the structure in Figure 5(a)corresponds approximately to that of Figure 4(c),and here helices 1 and 2 are partly obscured be-

hind helix 3.1 (h3d1). The main purpose ofFigure 5(a) is to illustrate the intra-RNA cross-linkI (cf. Figure 1, Table 1), which is a UV-inducedzero-length cross-link; the cross-linked nucleotidesare indicated here (and in the following Figures)by ball-and-stick nucleotides, with the 50-com-ponent of the cross-link site in purple and the 30-component in pale blue. Cross-link I forces helix 4to fold back towards helix 3, and this is a crucialfeature of the close-packed arrangement of the he-lices in this part of the subunit body, which deter-mines the placement of helix 16 and of thefunctionally important helix 18 (cf. Figure 1, andsee Figure 4(c), centre left).

Figure 4. Identi®cation of helices within the 16 S rRNA model (cf. Figure 1). The domains are colour coded as inFigure 3, and the P site tRNA is included for reference. (a) View from the solvent side of the subunit (cf. Figure 3(a)).(b) View with the subunit interface on the left (cf. Figure 3(b)). (c) View from the interface side. (d) View with thesubunit interface on the right. Each view is rotated by 90� with respect to the next one. (Note: the white versus blacknumbering has no signi®cance other than to achieve maximum visibility.)

Figure 5. Some details in stereo of the 50-domain of the 16 S rRNA model. (a) Helices 1, 2, 3, 4 and 28 together withcross-link I (cf. Figure 1). The orientation is similar to that in Figure 2(c), and the colour coding and numbering aredescribed in the text. The cross-linked nucleotides 31 and 48 are denoted by purple and light blue ball-and-sticknucleotides, respectively. (b) Helices 3, 4, 6a, 6b, 12 and 15, together with cross-links II and III. The orientation issimilar to that in (a). Cross-link II (nucleotides 31:306) is denoted by the large ball-and-stick nucleotides, cross-link III(nucleotides 366 to 369:397 to 399) by smaller ones. (c) The same as (b), but slightly rotated about the vertical axis,and with the semi-transparent EM contour superimposed.



The same nucleotide (G-31) that forms cross-linkI in helix 3 is involved in cross-link II (Figure 1).Cross-link II is illustrated in Figure 5(b), whichshows helices 3, 4, 6a, 6b, 12 and 15, in an orien-tation similar to that in Figures 5(a) and 4(c).Cross-link II is indicated by the larger ball-and-stick nucleotides, whereas cross-link III (which alsolies in this group of helices; Figure 1) is denoted bythe smaller ball-and-stick nucleotides (with whiterather than black sticks). The same group of he-lices, rotated slightly about the vertical axis, isshown within the EM contour in Figure 5(c), whichdemonstrates how helix 15, together with helix 4,®lls out the ``shoulder'' of the EM contour in thispart of the model. Cross-link II was formed by ni-trogen mustard (Table 1), a reagent with a span-ning distance of ca 7 AÊ , and it can be seen fromFigure 5(b) and (c) that the nucleotides involved inthis cross-link, G-31 (purple) and A-306 (pale blue),are slightly too far apart (by about 5 AÊ ). The separ-ation could be shortened by moving helix 12 closerto helix 3, but with the consequence that helix 12then protrudes partly outside the EM contour,which is quite thin in the plane of Figure 5(c) (cf.Figure 4(d)) in this region. In the case of cross-linkIII, the nucleotides concerned could not be pre-cisely pinpointed (Table 1), but nevertheless theoligonucleotide sequences identi®ed (366 to 369;397 to 399) are reasonably close together in themodel.

The involvement of helix 12 in cross-link II(Figure 5(c)) determines the location of the adjacenthelices 6a and 6b (Figure 1), which are visible atthe bottom left corner of Figure 5(b) and (c). Thepositions of the helices lying between helix 6a andhelix 15, namely helices 5, 13 and 14 (Figure 1), canbe seen in Figure 4(c), below helices 16 and 18.More important is the fact that helices 6a and 6bform the direct connection to helices 7 to 11, whichoccupy the lower part of the subunit body. Helix 6(lying between helices 5 and 6a; Figure 1), togetherwith helices 21 and 27 from the central domain ofthe 16 S rRNA and helix 44 from the 30-minor do-main, also lie in the lower part of the body. Someviews of these helices are illustrated in Figure 6.Figure 6(a) shows helices 6, 7, 8 and 10 within theEM contour, in an orientation similar to that inFigure 4(d). Helix 7 lies approximately vertically,connecting to helix 6b at its upper end (not shownin Figure 6(a)), and to helix 8 at its lower end. Thelatter helix precisely ®lls the prominent toe featureat the bottom of the subunit (cf. Figures 3(a) and4(a)). The lower left-hand curve of the EM contourin Figure 6(a) is ®lled by helices 6 and 10, andhelix 9, which connects to helices 8 and 10, facestowards the 50 S subunit (see Figure 4(b), bottomleft).

Helix 11 lies parallel with helix 7, and must lie(relative to the latter) on the side proximal to thecentral domain of the rRNA, because of the intra-RNA cross-link IV between helices 11 and 27(Figure 1). The general arrangement of these he-lices can be seen in Figure 4(a) (lower centre), and

a detailed stereo view of helices 7, 11 and 27, in-cluding cross-link IV, is shown in Figure 6(b). Herethe orientation of the diagram corresponds roughlyto that of Figure 4(b), i.e. at 90� to that ofFigure 4(a), so that helix 7 lies behind and to theleft of helix 11, with helix 27 also behind helix 11at the top right corner of the Figure. The 30-com-ponent of the cross-link site (nucleotides 891 to894, Table 1) is in a single-stranded region (lightblue in Figure 6(b)), and there is clearly suf®cient¯exibility here to enable this sequence to contactthe 50-component of the cross-link site (nucleotides243-244). In Figure 6(c), helices 11, 21 and thelower part of 44 (44.5 to 44.8) are shown, withinthe EM contour; helix 44 ®lls out the contour onthe left of the Figure, with helix 11 in the middleand helix 21 on the right towards the back, so as toenable it to connect with the other helices of thecentral domain. The orientation in Figure 6(c) isapproximately that in Figure 4(b).

Helix 44 is oriented towards the 50 S subunit,consistent with the protection studies by Gornickiet al. (1989), and moreover the upper region of thehelix (44.4) has been cross-linked to helix 69 of 23 SrRNA (Mitchell et al., 1992; see Figure 1); this partof the helix lies immediately adjacent to the princi-pal inter-subunit bridge connecting the 30 S and50 S subunits (Stark et al., 1995, 1997; cf. Figure 2(a)and (c)), and is just below the anticodon loop ofthe P site tRNA (cf. Figures 3(b) and 4(d)). Thelower end of helix 44 (44.7 and 44.8) projects outfrom the rest of the structure (Figure 4(b) and (d)),and it is noteworthy that in another EM recon-struction (of ribosomes in the post-translocationalstate; Stark et al., 1997) the corresponding densitywas less bulged out, enabling this part of helix 44to be packed snugly against the main part of therRNA at the lower end of the body. The very irre-gular upper part of helix 44, containing the single-base-pair ``helices'' 44.1 and 44.2 and the two-base-pair sub-helix 44.3 (Figure 1), has to be directedback towards helix 28, which forms the connectionto the head of the 30 S subunit (Figures 1, 4(c) and(d)). This necessitates a sharp bend in the helix be-tween sub-helices 44.3 and 44.4 (cf. Mueller et al.,1997), and it is interesting to note that in the recentNMR analysis of an analogue of this rRNA regionby Fourmy et al. (1996) there does indeed appearto be a bend in the structure at this point.

The only remaining helix in the subunit body ishelix 17, which is dif®cult to locate because of boththe paucity of biochemical data and the large ¯ex-ible intra-helical loops in the central region of thehelix. As already noted above, the termini of he-lices 16 and 18 are too far apart (see Figure 4(c)) toallow the formation of base-pairs between nucleo-tides 437 to 440 and 494 to 497 (Figure 1), and wetentatively place helix 17, by default, runningdown to an area of vacant density at the base ofthe subunit body; helix 17.2 is visible in Figure 4(b),lower centre, and helices 17.3 and 17.4 inFigure 4(a), bottom left.

Figure 6. Stereo views of the lower part of the subunit body. (a) Helices 6, 7, 8 and 10 in the EM contour. The orien-tation is similar to that in Figure 4(d). (b) Helices 7, 11 and 27, together with cross-link IV, the latter (nucleotides 243-244:891 to 894) being indicated by the ball-and-stick nucleotides. The orientation is similar to that in Figure 4(c).(c) Helices 11, 21 and 44.5 to 44.8 in the EM contour, in an orientation similar to that in Figure 4(b).



Arrangement of rRNA in the side lobe

The general arrangement of the helices in thecentral domain of the 16 S rRNA (helices 19 to 27,Figure 1) can clearly be seen in Figure 4(a) to (c).As described in the previous section, helices 21and 27 lie in the subunit body, whereas the re-maining helices occupy the side lobe area, which,as discussed in connection with Figure 2(b) above,is a region where the EM reconstruction has a par-ticularly detailed and at the same time stable ®nestructure. Figure 7(a) illustrates the arrangement ofhelices 19, 20, 25, 26, 26a and the pseudoknot helix26t (Figure 1) in this part of the 30 S subunit; theorientation of the diagram is the same as that ofFigure 4(a). Crucial to the model is the placementof helix 20 in the narrow bridge in the centre ofFigure 7(a). This helix is connected on the upperright-hand side directly to helices 21 and 22 (cf.Figure 4(a)), forming the three-way junction(Figure 1). On its lower side in Figure 7(a), helix 20

Figure 7. Stereo views of details of the central domain ofEM contour. The orientation is the same as that in FigureV (Figure 1) in the EM contour. The cross-link (nucleotidetides. The orientation is similar to that in (a).

is connected to the complex and tightly constrainedstructure involving helices 19, 26a and 26t. Helix19 connects back into the subunit body via the longsingle strands (Figure 1) joining it to helices 3 and27 (cf. the foregoing discussion, and Figure 4(a)),and at the same time is connected to helices 25 and26, which ®ll out the lower part of the EM contourin Figure 7(a).

A further detailed view of part of the central do-main is shown in Figure 7(b), with helices 20, 22,23 and 24. The purpose of this Figure is to demon-strate how these helices form a four-sided struc-ture, connected by the intra-RNA cross-link V(Figure 1). The orientation of the structure here isthe same as that of Figure 7(a). Helix 22 extendsfrom its connecting point to the upper end of helix20 (Figure 7(b), right), and ®lls out the ®ne struc-ture deep within the EM contour, to connect withhelix 23 on the left side of the diagram. Helix 24similarly extends leftwards from the lower end of

the 16 S rRNA. (a) Helices 19, 20, 25, 26, 26a and 26t in the4(a). (b) Helices 20, 22, 23 and 24, together with cross-links 693 to 696:794) is denoted by the ball-and-stick nucleo-


helix 20, so that its loop end meets that of helix 23in the lower left corner of Figure 7(b). Cross-link V,between nucleotides 693 to 696 and 794 (Table 1),is a nitrogen mustard cross-link, and the two com-ponents of this cross-link involve the respectiveloop ends of helices 23 and 24, which are clearly inclose proximity in the model. (An alternative ver-sion of cross-link V involved nucleotide G-799 in-stead of A-794 (Atmadja et al., 1986), and this siteis also within range of the ¯exible six-memberedloop containing nucleotides 693 to 696.) Thus, he-lices 20, 22, 23 and 24 lie in a quadrilateral arrange-ment similar to that in previous models(Brimacombe et al., 1988; Stern et al., 1988; Oakeset al., 1990), but with the important difference thatthe loop end of helix 23 is directed downwardsrather than upwards. This has the effect of placinghelices 22a and 23a (Figure 1) in the upper regionof the side lobe (see Figure 4(c), centre right), andhelix 24 together with cross-link V in the lower re-gion, as just discussed. The loop end of helix 24has been implicated in 30 S-50 S subunit associ-ation (Herr et al., 1979), and the placement of helix24 in this position (Figure 7(b)) does indeed bringit very close to a part of the 50 S subunit just belowthe L1 protuberance (not shown, but see Figure 6Bof Stark et al., 1997). An equivalent arrangementhas recently been proposed by Lata et al. (1996) onsimilar grounds.

Helix 45, in the 30-minor domain, should also bementioned at this point. Helix 45 connects to theupper part of helix 44 (Figure 1), and we tenta-tively place it in an element of density in the upperregion of the side lobe that can be seen inFigure 2(c) (cf. Figure 4(c)). The loop end ofhelix 45 has been cross-linked to the same sitein helix 69 of the 23 S rRNA as was cross-linked tohelix 44.4 (Figure 1, Table 1; Mitchell et al., 1992).The respective areas of helices 44 and 45 are some-what too far apart in the model to satisfy this sim-ultaneous constraint (see Figure 4(c), centre), but itshould be noted that in the EM reconstruction ofribosomes in the post-translocational state (Starket al., 1997), the element of EM density where weplace helix 45 was closer to the inter-subunit bridgeand hence to helix 44.4, underneath the anticodonloop of the P site tRNA (data not shown).

Arrangement of rRNA in the head ofthe subunit

The 30-domain of the 16 S rRNA comprises he-lices 28 to 43 (Figure 1), and their arrangement inthe head of the 30 S subunit can be seen in the var-ious views of Figure 4; some details are illustratedin Figure 8. A notable feature of the EM recon-struction of the subunit head is the hole in itscentre, around which the rRNA model is built up.The hole is visible in Figure 8(a), together with he-lices 28, 34 and 35, in an orientation similar to thatin Figure 4(a). (This hole should not be confusedwith the hole at the junction between the head andthe body of the subunit, through which the mRNA

passes (Stark et al., 1995; Frank et al., 1995); thelatter hole is located lower down in the subunit,approximately at right-angles to the hole inFigure 8(a), and is discussed in connection with themRNA path by Mueller et al., 1997.) On the lowerside of the hole (Figure 8(a)) there is a narrowbridge of density in the EM contour, which carrieshelix 34 in an analogous manner to the placementof helix 20 in the corresponding bridge in the cen-tral region of the subunit (cf. Figure 7(a)). Helix 34is a crucial element in this part of the rRNA, as itseparates helices 30 to 33 on the one side from he-lices 35 to 40 on the other (Figure 1). Helix 35 isconnected directly to helix 34 (on the left inFigure 8(a)), and ®lls out the contour on the sol-vent side of the neck of the subunit. Helix 28,which connects the head of the subunit to the body(cf. Figure 1), is included for reference inFigure 8(a); helices 28 and 34 are both functionallyimportant (Mueller et al., 1997), helix 28 lying closebehind helix 34 in the Figure.

More detail of the solvent side of the subunithead is given in Figure 8(b). Here the structure hasbeen rotated slightly around the vertical axis in re-lation to the view in Figure 8(a), and shows the ar-rangement of helices 34.3, 35 to 38 and 40, togetherwith intra-RNA cross-link VI (Figure 1). Helices34.3 and 35 are visible in the lower part of theFigure, with helix 35 connected to helices 36 and37 above it, the 50-component of the cross-link site(nucleotides 1090 to 1094, Table 1) being located inthe loop end of helix 37. Helix 34.3 connects tohelix 38 (centre right in Figure 8(b)), which is inturn connected by a rather long inter-helical singlestrand to helix 40 (upper left). Helix 40 containsthe 30-component of cross-link VI (nucleotides 1161to 1164), which lie within comfortable reach of thecorresponding 50-component in the ¯exible loopend of helix 37. There is a further intra-RNA cross-link in the 30-domain of the 16 S rRNA, cross-linkVII (Figure 1), which connects helices 39 and 41.These helices lie antiparallel to one another in theupper part of the subunit head, and are shown, to-gether with cross-link VII, in Figure 8(c). The viewin this case is from ``above'' the subunit, as ifFigure 4(a) were to be rotated 90� towards thereader, out of the plane of the paper around thehorizontal axis. Helix 41 is on the left in Figure 8(c)with its hairpin loop pointing downwards, andhelix 39 is on the right with its loop end pointingupwards. The cross-link, which joins nucleotides1125 to 1127 in helix 39 with 1280-1281 in helix 41(Table 1), can clearly be seen in the centre of theFigure.

The locations of the remaining helices from the30-domain of the 16 S rRNA are evident fromFigure 4. Helix 28 connects to helix 29 (Figure 4(b),upper centre), and this helix connects in turn tohelices 30 and 32, with helix 31 in between(Figure 4(c), upper left). More details of the ar-rangement of these helices are given in the accom-panying papers (Mueller & Brimacombe, 1997;Mueller et al., 1997). Helices 42 and 43 (cf. Figure 1)

Figure 8. Stereo views of details of the 30-domain of the 16 S rRNA. (a) Helices 28, 34 and 35 in the EM contour. Theorientation is similar to that in Figure 4(a). (b) Helices 34.3, 35, 36, 37, 38 and 40, together with cross-link VI, in theEM contour. The view is rotated slightly about the vertical axis in relation to (a). The cross-linked nucleotides (1090to 1094:1161 to 1164) are denoted by the ball-and-stick nucleotides. (c) Helices 39 and 41 in the EM contour, togetherwith cross-link VII (nucleotides 1125 to 1127:1280 to 1281) denoted by the ball-and-stick nucleotides. The view isfrom the top of the subunit, with the 30 S:50 S subunit interface at the upper edge of the picture.


are also visible in Figure 4(c), and, ®nally, helices33.1, 33.2 and 33a can be seen in Figure 4(a) and(d). The placement of the helix 33 group is tenta-tive, due to the lack of biochemical data in this sec-tion of the rRNA, as well as to the large amount ofavailable density in the EM contour at this cornerof the subunit head (cf. Figure 2(b)).

Locations of nucleotides identified by IEM, andclustering of modified bases

A number of nucleotides in the 16 S rRNA havebeen located on the surface of the 30 S subunit inolder IEM studies. These include the 50-terminal re-sidue (Mochalova et al., 1982), the methylated G re-sidue at position 527 in helix 18 (Trempe et al.,1982), the C residue at position 1400 in helix 44(Gornicki et al., 1984), the di-methylated A residuesat positions 1518 and 1519 in helix 45 (Politz &Glitz, 1977), and the 30-terminal residue (e.g. seeLuÈ hrmann et al., 1981). The locations of these resi-dues in our rRNA model, viewed from the inter-face side of the subunit (cf. Figure 4(c)) are shownin Figure 9(a). From left to right across the Figure,the 50 terminus is yellow, mG-527 red, C-1400green, m2A-1518/1519 purple, and the 30 terminusblue. Although it is dif®cult to make a detailedcomparison between our EM reconstruction (Starket al., 1997) and the older EM structures used to de-scribe the IEM sites, it is nonetheless clear that thelocations of the sites in our model (Figure 9(a)) arecompatible with their counterparts in the ``classi-cal'' structures (e.g. see Gornicki et al., 1984 for asummary); in the latter, the 50 terminus lies half-way down the body on the left side (as viewed inFigure 9(a)), mG-527 lies in the curve of the neckon the left, C-1400 and m2A-1518/1519 lie on theright side of the neck at the base of the so-calledcleft, and the 30 terminus lies on the right at the topof the ``platform''. The locations of these nucleo-tides in the model are particularly important in re-lation to the much-discussed question (e.g. seeBrimacombe, 1995) of the distance between the 530loop and C-1400 areas of the 16 S rRNA (seebelow).

A further question of interest is the distributionof the post-transcriptionally modi®ed bases in the16 S rRNA. These are located in helices 18, 31, 34,44 and 45 (Carbon et al., 1979; Van Charldorp et al.,1981; Bakin et al., 1994; cf. Figure 1), but despitethis wide scatter over the primary and secondarystructure, the modi®ed nucleotides form a distinctcluster in three dimensions. This is illustrated inFigure 9(b), which shows the A and P site tRNAsand a section of mRNA as backbone tube struc-tures, together with the modi®ed nucleotides intheir respective helices as CPK nucleotides. It canbe seen that the latter form a compact ``cage'' sur-rounding the anticodon stem±loops of the tRNAmolecules in the tRNA-mRNA complex, as hasbeen noted (Brimacombe et al., 1993). The view inFigure 9(b) is the same as that of Figure 4(d), andit is noteworthy that in this orientation the position

of mG-527 (red), which is located in the loop endof helix 18, lies to the left of the pseudouridine-516(black), which is within helix 18.2; this is a conse-quence of the pseudoknot helix 18t (Figure 1),which draws the loop end of helix 18 back towardsthe looped residues 506 to 508 joining helices 18.1and 18.2. The clustering of the modi®ed basesaround the functional complex is no surprise, sincethe regions of the 16 S rRNA carrying these modi-®ed bases have also been implicated by variouscross-linking and foot-printing studies as being inthe decoding area; the data concerned, in particularthose relating to helix 18 and the C-1400 region,are described in an accompanying paper (Muelleret al., 1997).

Discussion

The cryo-EM reconstruction at ca 20 AÊ resolutionby Stark et al. (1997) of E. coli ribosomes in the pre-translocational state carrying A and P site tRNAsoffers the most detailed representation of a riboso-mal structure that has so far been achieved. Thistype of EM reconstruction shows the surfacecharacteristics of the object being studied, andgives the internal density distribution, and boththese properties have been exploited in our ®ttingof the 16 S rRNA molecule. It is clear that bothrRNA and ribosomal protein will each contributeto the observed EM density. However, since two-thirds of the bacterial ribosome is rRNA and atleast 60% of the latter is present in double-strandedhelices (cf. Figure 1), then it follows that at least40% of the total mass consists of double-helicalRNA. These rRNA elements have the highest den-sity and can therefore be expected to dominate theEM density distribution. In this context it is note-worthy that many ®ne structural features in the re-construction by Stark et al. (1997) are very stablewith respect to the thresholding level used to com-pute the density, and do not expand or contractrapidly when the threshold is changed. (Thethreshold chosen in the latter publication corre-sponded to ca 10% more than the expected mass ofthe ribosome). In this respect, the data differ sig-ni®cantly from the earlier reconstruction by Franket al. (1991), where a density distribution was ob-served that was quite sensitive to the thresholdinglevel, and which the authors interpreted in termsof a hard core of density due to the rRNA sur-rounded by a softer envelope of medium density,which they attributed to ribosomal protein.

The amount of ®ne structural detail observed inthe new EM reconstruction (Stark et al., 1997) issuch that, even without taking into account thebiochemical constraints relating to the three-dimen-sional folding of the rRNA, the ®tting of the 16 Ssecondary structure to the EM data is by no meanstrivial. In fact it has turned out that, in general, the®tted 16 S structure satis®es the biochemical con-straints more thoroughly than the preceding non-®tted versions of the model (cf. Brimacombe, 1995),

Figure 9. Position of IEM-located nucleotides and modi®ed bases in the 16 S rRNA model. (a) The complete 16 SrRNA (black) in the EM contour, showing the IEM-located nucleotides in CPK format. The view is from the interfaceside of the subunit (cf. Figure 4(c)). The 50-terminal nucleotide is yellow, mG-527 red, C-1400 green, m2A-1518/1519purple, and the 30-terminal nucleotide blue. (b) Distribution of modi®ed bases. The functional complex is shown intube format, with A site tRNA light blue, P site tRNA green and mRNA white. The orientation corresponds to thatin Figure 4(d). Helices 18.2, 18t, 28, 29, 31, 34.1, 34.2, 44.1 to 44.4 and 45 are shown (black) in the EM contour. Themodi®ed bases are in CPK format, colour coded pseudo-U-516 black, mG-527 red, mG-966 and mC-967 orange, mG-1207 yellow, mC-1402 green, mC-1407 light blue, mU-1498 dark blue, and mG-1516 and m2A-1518/1519 purple. Seethe text for references.


which gives us added con®dence in our structure.Some of the 16 S rRNA helices could be placedrather precisely in speci®c features of the recon-struction, such as the ``bridges'' carrying helix 20(Figure 7(a)) or helix 34 (Figure 8(a)), or the toe(Figure 6(a)) containing helix 8. Other helices, in-cluding 4 and 15 (Figure 5(c)), 6 (Figure 6(a)), 44(Figure 6(c)), 25 and 26 (Figure 7(a)), 23(Figure 7(b)), 35 and 40 (Figure 8(b)), were ®tted toappropriate curves on the surface of the EM con-tour. The remaining helices were added, makinguse of the intervening EM density and of the con-

nectivities within the secondary structure. Only avery few helices, in particular 17 and 33 (Figure 4),had to be placed in the contour ``by default''. Themodel was built within these restrictions, applyingthe additional criterion that inter-helical distancesshould be kept as short as possible, so as to avoidhaving to use the inter-helical single strands tobridge large gaps in the model. Only in the casesof helices 3 and 19, and of helices 2 and 27 (cf.Figures 1 and 4(a)), was it necessary to stretch thecorresponding inter-helical strands to almost theirmaximum length; these two strands were also ex-


tended in our previous published model(Brimacombe et al., 1988). With regard to helices 2and 27, we have already noted above that the po-tential base-pairs (Lodmell et al., 1995) betweennucleotides 885 to 887 and 910 to 912 (the latterbeing part of the connecting strand between thetwo helices; Figure 1), can be formed only if thepseudoknot helix 2 becomes unpaired; a number ofdynamic models have been proposed (e.g. seeLeclerc & Brakier-Gingras, 1991) suggesting justsuch a process.

The question of how to display the 16 S rRNAmodel ®tted to an EM reconstruction at 20 AÊ resol-ution poses a problem. Clearly the use of cylinders(cf. Brimacombe et al., 1988; Malhotra & Harvey,1994; Fink et al., 1996) to display the helices,although this is useful for giving an overview ofthe structure as in Figures 3 and 4, is no longeradequate to describe the ®ne structure of themodel; the entry and exit sites of the helical strandson the cylinders cannot be visualized, and in thismode the all-important single strands are not dis-played at all. On the other hand, the resolution ofthe model is still obviously too low to justify a rep-resentation at the atomic level. Nevertheless, aftermuch experimentation, we elected to use the all-atom presentation, with sites of interest high-lighted as ball-and-stick or CPK nucleotides, asbeing visually the most satisfactory method for dis-playing selected regions (in particular the singlestrands) of the 16 S model within the EM contour.We have already emphasized that this presentationis purely for visual purposes, and should not beoverinterpreted.

Here, we have dealt only with those aspects ofthe model that are directly related to the 16 SrRNA itself; that is to say, the secondary structure,the intra-RNA cross-links, the modi®ed bases, andthe IEM locations of individual nucleotides. Withregard to the intra-RNA cross-linking data, it is im-portant to note that, as in our previous model(Brimacombe et al., 1988), we have considered onlycross-links formed in situ in intact ribosomal sub-units or in vivo (Table 1), but not those formed inisolated rRNA or those where the identi®cation ofthe cross-link sites was made only to an accuracyof �20 nucleotides by inspection of electron micro-graphs of the cross-linked rRNA (Wollenzien et al.,1985; cf. Hubbard & Hearst, 1991). In those caseswhere an intra-RNA cross-link site could be loca-lized only to within two to four bases (Table 1), theoligonucleotide sequence concerned is displayed inthe corresponding Figures (5 to 8), so as to give avisual indication of the level of uncertainty in themodel that is implicit in these data.

The model of the 16 S rRNA that we have pre-sented here is not the ®nal one, and modi®cationswill certainly be necessary as new biochemical in-formation becomes available and as the resolutionof the EM reconstructions improves. As noted inthe Introduction, there is no theoretical limit to theresolution that might be achieved by the single-particle EM approach (Henderson, 1995). We are

convinced, however, that already at the currentlevel of resolution the EM data provide a physicalframework of shapes and distances that can be di-rectly related to the three-dimensional folding ofthe 16 S rRNA, as we have demonstrated here. Upto now the only precise distance measurementsavailable have been those from the neutron scatter-ing map of the 30 S ribosomal proteins (Capel et al.,1988). The neutron data de®ne the relative pos-itions of the mass centres of the proteins, but, asdiscussed previously (Brimacombe, 1995), this in-formation is in our opinion too indirect to be usedas a primary criterion for modelling the 16 SrRNA. Nevertheless, it is obviously of crucial im-portance to show how well the neutron map canbe combined with the EM-®tted 16 S rRNA model,so as to account for the large amount of RNA-pro-tein interaction data that has been accumulated bycross-linking (Brimacombe, 1991) and foot-printing(Stern et al., 1988; Powers & Noller, 1995) studies.An accompanying paper (Mueller & Brimacombe,1997) deals exclusively with this topic.

Materials and Methods

Ribosomal RNA model generation

The programme ERNA-3D (Editor for RNA in 3-D)was developed in our laboratory (cf. Mueller et al., 1995).ERNA-3D is installed on a Silicon Graphics Indigo2-Extreme workstation (R4000, 100 MHz, 96MB memory,5GB HD, IRIX 5.1) and is entirely written in computerlanguage C. The source code of ERNA-3D amounts toover 100,000 programme lines, and the programme of-fers a ¯exible molecular modelling system with whichthe creation and manipulation of molecular represen-tations at atomic resolution can be performed. Only theprimary sequence and secondary structure of RNA mol-ecules are needed for the generation of 3D structuressuch as A-form RNA helices and single-stranded regions.There are almost no restrictions on the number of atomsthat can be handled with this software. The 16 S rRNAmodel generated consists of over 33,000 atoms, and ad-ditional structures, including the 23 S rRNA, 5 S rRNA,mRNA and tRNA, have been loaded without signi®-cantly slowing the rendering process. All atomic struc-tures can be saved as PDB ®les (in Brookhaven NationalLaboratory Protein Data Bank File format), or as mem-ory dump ®les (for faster loading times).

The user can interactively modify the atomic model,with the aid of stereo shutter glasses, which allow 3Dviewing. ERNA-3D allows the free positioning of RNAdouble helices together with the dynamic pulling of in-terhelical single strands, which is accomplished by calcu-lating appropriate reiterated rotations along the sugar-phosphate backbone. For this purpose, a small spatialcursor (called the selector) is provided, which acts as theuser0s ``hand'' in the model. The user can select any de-sired nucleotide and then pull the single strand in the re-quired direction. Hairpin loops or intrahelical bulges andloops are handled in precisely the same manner. If oneof the double helices is moved or rotated, the connectinginterhelical strands remain attached (provided that themaximum possible interhelical distance is not exceeded),and follow the helix being moved. In the case of ro-tations, the x, y and z axes remain orthogonal to the


viewer, regardless of the orientation of the model that iscurrently being displayed. Translations and rotations canalso be made by direct command, rather than using theselector, and here again the axes remain orthogonal tothe viewer.

ERNA-3D accepts the de®nition of selections, wherebysmaller regions of the molecule can be displayed andmanipulated at will. Colouring of individual residues,and the creation of backbone-tube, ball-and-stick or CPKrepresentations can be achieved in a similar way. Proteinmolecules can be displayed either as atomic structuresor, in the case of the display of the neutron scatteringmap of the positions of the 30 S ribosomal proteins(Capel et al., 1988), as spherical polygon structures. Animportant property of the SGI workstations in combi-nation with ERNA-3D is the rendering of semi-transpar-ent polygon structures simultaneously with wire-frameatomic structures. This feature was used to ®t the rRNAmodel to the EM contour (Stark et al., 1997).

Transformation of EM-derived densities

The cryo-EM reconstruction of the 70 S ribosome inthe pre-translocational state was that by Stark et al.(1997). The transformation of electron density data tocontour data was performed by the commercial softwarepackage IMSL/IDL (available from IMSL & RSI), whichis specialized in the visualization of scienti®c data. Sub-sequently, ®les transformed by IMSL/IDL had to betranslated into the polygon ®le format of ERNA-3D; thistranslation was accomplished by a small auxiliary pro-gramme.

In general, such converted ®les have the incorrect sizein relation to the actual dimensions of the atomic struc-ture. Accordingly, after loading into ERNA-3D, it isnecessary to measure the contour and change its scale byan appropriate factor. ERNA-3D allows the measure-ment of distances between contour vertices to be madevery simply, with the help of the selector, and it is easyto determine the diameters of objects. After adjusting thescale of the structure, translations and rotations weremade to adjust the contour to the coordinate system ofthe model. We used a coordinate system based on thecontour of a 70 S ribosome, where the small subunit lieson the negative and the large subunit on the positiveside of the x-axis. The origin of the coordinate system islocated in the inter-subunit cavity.

Data availability

Since the 16 S rRNA model has not been subjected toenergy minimization (see the text), the coordinates of thestructure have not been deposited in the BrookhavenData Bank. The coordinates will, however, be providedon request. ERNA-3D will shortly be available commer-cially.

Acknowledgements

We are most grateful to Dr Holger Stark for his helpin transferring the EM data to our computer.

References

Agrawal, R. K., Penczek, P., Grassucci, R. A., Li, Y.,Leith, A., Nierhaus, K. H. & Frank, J. (1996). Direct

visualization of A-, P-, and E-site transfer RNAs inthe E. coli ribosome. Science, 271, 1000±1002.

Atmadja, J., Stiege, W., Zobawa, M., Greuer, B.,Osswald, M. & Brimacombe, R. (1986). The tertiaryfolding of E. coli 16 S RNA, as studied by in situintra-RNA cross-linking of 30 S ribosomal subunitswith bis-(2-chloroethyl)-methylamine. Nucl. AcidsRes. 14, 659±673.

Bakin, A., Kowalak, J. A., McCloskey, J. A. & Ofengand,J. (1994). The single pseudouridine residue in E. coli16 S RNA is located at position 516. Nucl. Acids Res.22, 3681±3684.

Brimacombe, R. (1991). RNA-protein interactions in theE. coli ribosome. Biochimie, 73, 927±936.

Brimacombe, R. (1995). The structure of ribosomal RNA;a three-dimensional jigsaw puzzle. Eur. J. Biochem.230, 365±383.

Brimacombe, R., Atmadja, J., Stiege, W. & SchuÈ ler, D.(1988). A detailed model of the three-dimensionalstructure of E. coli 16 S ribosomal RNA in situ inthe 30 S subunit. J. Mol. Biol. 199, 115±136.

Brimacombe, R., Mitchell, P., Osswald, M., Stade, K. &Bochkariov, D. (1993). Clustering of modi®ednucleotides at the functional center of bacterial ribo-somal RNA. FASEB J. 7, 161±167.

Capel, M. S., Kjeldgaard, M., Engelman, D. M. & Moore,P. B. (1988). Positions of S2, S13, S16, S17, S19 andS21 in the 30 S ribosomal subunit of E. coli. J. Mol.Biol. 200, 65±87.

Carbon, P., Ehresmann, C., Ehresmann, B. & Ebel, J. P.(1979). The complete nucleotide sequence of theribosomal 16 S RNA from E. coli; experimentaldetails and cistron heterogeneities. Eur. J. Biochem.100, 399±410.

Expert-BezancËon, A. & Wollenzien, P. L. (1985). Three-dimensional arrangement of the E. coli 16 S riboso-mal RNA. J. Mol. Biol. 184, 53±66.

Fink, D. L., Chen, R. O., Noller, H. F. & Altman,R. B. (1996). Computational methods for de®ningthe allowed conformational space of 16 S rRNAbased on chemical foot-printing data. RNA, 2,851±866.

Fourmy, D., Recht, M. I., Blanchard, S. C. & Puglisi, J. D.(1996). Structure of the A site of E. coli 16 S riboso-mal RNA complexed with an aminoglycosideantibiotic. Science, 274, 1367±1371.

Frank, J., Penczek, P., Grassucci, R. & Srivastava, S.(1991). Three-dimensional reconstruction of the 70 SE. coli ribosome in ice; the distribution of ribosomalRNA. J. Cell Biol. 115, 597±605.

Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S.,Verschoor, A., Radermacher, M., Grassucci, R.,Lata, R. K. & Agrawal, R. K. (1995). A model ofprotein synthesis based on cryo-electron microscopyof the E. coli ribosome. Nature, 376, 441±444.

Gornicki, P., Nurse, K., Hellmann, W., Boublik, M. &Ofengand, J. (1984). High resolution localization ofthe tRNA anticodon interaction site on the E. coli30 S ribosomal subunit. J. Biol. Chem. 259, 10493±10498.

Gornicki, P., Baudin, F., Romby, P., Wiewiorowski, M.,Kryzosiak, W., Ebel, J. P., Ehresmann, C. &Ehresmann, B. (1989). Use of lead (II) to probe thestructure of large RNAs; conformation of the 30-terminal domain of E. coli 16 S rRNA and its invol-vement in building the tRNA binding sites. J. Biomol.Struct. Dynam. 6, 971±984.


Haselman, T., Camp, D. G. & Fox, G. E. (1989). Phyloge-netic evidence for tertiary interactions in 16 S-likeribosomal RNA. Nucl. Acids Res. 17, 2215±2221.

Henderson, R. (1995). The potential and limitations ofneutrons, electrons and X-rays for atomic resolutionmicroscopy of unstained biological molecules.Quart. Rev. Biophys. 28, 171±193.

Herr, W., Chapman, N. M. & Noller, H. F. (1979).Mechanism of ribosomal subunit association;discrimination of speci®c sites in 16 S RNA essen-tial for association activity. J. Mol. Biol. 130, 433±449.

Hubbard, J. M. & Hearst, J. E. (1991). Computermodelling 16 S ribosomal RNA. J. Mol. Biol. 221,889±907.

Lata, K. R., Agrawal, R. J., Penczek, P., Grassucci, R.,Zhu, J. & Frank, J. (1996). Three-dimensional recon-struction of the E. coli 30 S ribosomal subunit in ice.J. Mol. Biol. 262, 43±52.

Leclerc, D. & Brakier-Gingras, L. (1991). A confor-mational switch involving the 915 region of E. coli16 S ribosomal RNA. FEBS Letters, 279, 171±174.

Lodmell, J. S., Gutell, R. R. & Dahlberg, A. E. (1995).Genetic and comparative analyses reveal an alterna-tive secondary structure in the region of nt 912 ofE. coli 16 S rRNA. Proc. Natl Acad. Sci. USA, 92,10555±10559.

LuÈ hrmann, R., StoÈ f¯er-Meilicke, M. & StoÈ f¯er, G. (1981).Localization of the 30 end of 16 S rRNA in E. coli30 S ribosomal subunits by immuno electronmicroscopy. Mol. Gen. Genet. 182, 369±376.

Malhotra, A. & Harvey, S. C. (1994). A quantitativemodel of the E. coli 16 S RNA in the 30 S ribosomalsubunit. J. Mol. Biol. 240, 308±340.

Mitchell, P., Osswald, M., SchuÈ ler, D. & Brimacombe, R.(1990). Selective isolation and detailed analysis ofintra-RNA cross-links induced in the large riboso-mal subunit of E. coli; a model for the tertiary struc-ture of the tRNA binding domain in 23S RNA.Nucl. Acids Res. 18, 4325±4333.

Mitchell, P., Osswald, M. & Brimacombe, R. (1992).Identi®cation of intermolecular cross-links at thesubunit interface of the E. coli ribosome. Biochemis-try, 31, 3004±3011.

Moazed, D. & Noller, H. F. (1990). Binding of tRNA tothe ribosomal A and P sites protects two distinctsets of nucleotides in 16 S rRNA. J. Mol. Biol. 211,135±145.

Moazed, D., Stern, S. & Noller, H. F. (1986). Rapidchemical probing of conformation in 16 S ribosomalRNA and 30 S ribosomal subunits using primerextension. J. Mol. Biol. 187, 399±416.

Mochalova, L. V., Shatsky, I. N., Bogdanov, A. A. &Vasiliev, V. D. (1982). Topography of RNA in theribosome; localization of the 16 S RNA 50 end byimmune electron microscopy. J. Mol. Biol. 159, 637±650.

Mueller, F. & Brimacombe, R. (1997). A new model forthe three-dimensional folding of Escherichia coli 16 Sribosomal RNA. II. The RNA-protein interactiondata. J. Mol. Biol. 271, 545±565.

Mueller, F., DoÈring, T., Erdemir, T., Greuer, B., JuÈ nke,N., Osswald, M., Rinke-Appel, J., Stade, K.,Thamm, S. & Brimacombe, R. (1995). Getting closerto an understanding of the three-dimensional struc-ture of ribosomal RNA. Biochem. Cell Biol. 73, 767±773.

Mueller, F., Stark, H., van Heel, M., Rinke-Appel, J. &Brimacombe, R. (1997). A new model for the three-

dimensional folding of Escherichia coli 16 S riboso-mal RNA. III. The topography of the functionalcentre. J. Mol. Biol. 271, 566±587.

Nagano, K., Harel, M. & Takezawa, M. (1988). Predic-tion of three-dimensional structure of E. coli riboso-mal RNA. J. Theoret. Biol. 134, 199±256.

Noller, H. F., Green, R., Heilek, G., Hoffarth, V.,HuÈ ttenhofer, A., Joseph, S., Lee, I., Lieberman, K.,Mankin, A., Merryman, C., Powers, T., Puglisi, E. V.,Samaha, R. R. & Weiser, B. (1995). Structure andfunction of ribosomal RNA. Biochem. Cell Biol. 73,997±1009.

Oakes, M. I., Kahan, L. & Lake, J. A. (1990). DNA-hy-bridization electron microscopy; tertiary structure of16 S rRNA. J. Mol. Biol. 211, 907±918.

Politz, S. M. & Glitz, D. G. (1977). Ribosome structure;localization of N6,N6-dimethyladenosine by electronmicroscopy of a ribosome-antibody complex. Proc.Natl Acad. Sci. USA, 74, 1468±1472.

Powers, T. & Noller, H. F. (1995). Hydroxyl radical foot-printing of ribosomal proteins on 16 S rRNA. RNA,1, 194±209.

Serysheva, I. I., Orlova, E. V., Chiu, W., Sherman, M. B.,Hamilton, S. L. & van Heel, M. (1995). Electroncryomicroscopy and angular reconstitution used tovisualize the skeletal muscle calcium releasechannel. Nature Struct. Biol. 2, 18±24.

Stark, H., Mueller, F., Orlova, E. V., Schatz, M., Dube,P., Erdemir, T., Zemlin, F., Brimacombe, R. & vanHeel, M. (1995). The 70 S E. coli ribosome at 23 AÊ

resolution; ®tting the ribosomal RNA. Structure, 3,815±821.

Stark, H., Orlova, E. V., Rinke-Appel, J., JuÈ nke, N.,Mueller, F., Rodnina, M., Wintermeyer, W.,Brimacombe, R. & van Heel, M. (1997). Arrange-ment of tRNAs in pre- and post-translocationalribosomes revealed by electron cryomicroscopy.Cell, 88, 19±28.

Stern, S., Weiser, B. & Noller, H. F. (1988). Model forthe three-dimensional folding of 16 S ribosomalRNA. J. Mol. Biol. 204, 447±481.

Stiege, W., Atmadja, J., Zobawa, M. & Brimacombe, R.(1986). Investigation of the tertiary folding of E. coliribosomal RNA by intra-RNA cross-linking in vivo.J. Mol. Biol. 191, 135±138.

Stiege, W., Kosack, M., Stade, K. & Brimacombe, R.(1988). Intra-RNA cross-linking in E. coli 30 S ribo-somal subunits; selective isolation of cross-linkedproducts by hybridization to speci®c cDNAfragments. Nucl. Acids Res. 16, 4315±4329.

Trempe, M. R., Ohgi, K. & Glitz, D. G. (1982). Ribosomestructure; localization of 7-methylguanosine in thesmall subunits of E. coli and chloroplast ribosomesby immunoelectron microscopy. J. Biol. Chem. 257,9822±9829.

Van Charldorp, R., Heus, H. A. & van Knippenberg,P. H. (1981). 16 S ribosomal RNA of E. colicontains a N2-methylguanosine residue at 27nucleotides from the 30 end. Nucl. Acids Res. 9,2717±2725.

Van Heel, M. & StoÈ f¯er-Meilicke, M. (1985). Character-istic views of E. coli and B. stearothermophilus 30 Sribosomal subunits in the electron microscope.EMBO J. 4, 2389±2395.

Verschoor, A., Frank, J., Radermacher, M.,Wagenknecht, T. & Boublik, M. (1984). Three-dimensional reconstruction of the 30 S ribosomalsubunit from randomly oriented particles. J. Mol.Biol. 178, 677±698.


Von BoÈhlen, K., Makowski, I., Hanse, H. A. S., Bartels,H., Berkovitch-Yellin, Z., Zaytzev-Bashan, A.,Meyer, S., Paulke, C., Franceschi, F. & Yonath, A.(1991). Characterization and preliminary attemptsfor derivation of crystals of large ribosomal sub-units from H. marismortui diffracting to 3 AÊ

resolution. J. Mol. Biol. 222, 11±15.

Wollenzien, P. L., Murphy, R. F., Cantor, C. R., Expert-BezancËon, A. & Hayes, D. H. (1985). Structure ofthe E. coli 16 S ribosomal RNA; psoralen cross-links and N-acetyl-N0-(p-glyoxylylbenzoyl)cystaminecross-links detected by electron microscopy. J. Mol.Biol. 184, 67±80.

Edited by D. E. Draper

(Received 20 March 1997; received in revised form 18 June 1997; accepted 18 June 1997)

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. fitting the...

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