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

J. Mol. Biol. (1997) 271, 545±565

A New Model for the Three-Dimensional Folding ofEscherichia coli 16 S Ribosomal RNA.II{. The RNA-Protein Interaction Data

Florian Mueller and Richard Brimacombe*

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

{ Paper I in this series is an accomMueller & Brimacombe (1997).

Abbreviations used: EM, electronimmuno EM.

0022±2836/97/340545±21 $25.00/0/mb9

The map of the mass centres of the 21 proteins from the Escherichia coli30 S ribosomal subunit, as determined by neutron scattering, was ®ttedto a cryoelectron microscopic (cryo-EM) model at a resolution of 20 AÊ of70 S ribosomes in the pre-translocational state, carrying tRNA moleculesat the A and P sites. The ®t to the 30 S moiety of the 70 S particles wasaccomplished with the help of the well-known distribution of the riboso-mal proteins in the head, body and side lobe regions of the 30 S subunit,as determined by immuno electron microscopy (IEM). Most of the pro-tein mass centres were found to lie close to the surface (or even outside)of the cryo-EM contour of the 30 S subunit, supporting the idea that theribosomal proteins are arranged peripherally around the rRNA. The ribo-somal protein distribution was then compared with the correspondingmodel for the 16 S rRNA, ®tted to the same EM contour (described in anaccompanying paper), in order to analyse the mutual compatibility of thearrangement of proteins and rRNA in terms of the available RNA-proteininteraction data. The information taken into account included the hy-droxyl radical and base foot-printing data from Noller's laboratory, andour own in situ cross-linking results. Proteins S1 and S14 were not con-sidered, due to the lack of RNA-protein data. Among the 19 proteinsanalysed, 12 (namely S2, S4, S5, S7, S8, S9, S10, S11, S12, S15, S17 andS21) showed a ®t to the rRNA model that varied from being excellent toat least acceptable. Of the remaining 7, S3 and S13 showed a rather poor®t, as did S18 (which is considered in combination with S6 in the foot-printing experiments). S16 was dif®cult to evaluate, as the foot-print datafor this protein cover a large area of the rRNA. S19 and S20 showed abad ®t in terms of the neutron map, but their foot-print and cross-linksites were clustered into compact groups in the rRNA model in thoseregions of the 30 S subunit where these proteins have respectively beenlocated by IEM studies.

# 1997 Academic Press Limited

Keywords: electron microscopic reconstruction; neutron scattering map;RNA-protein cross-linking; protein foot-printing; computer modelling
*Corresponding author
Introduction

Two-thirds of the mass of the bacterial ribosomeconsists of rRNA, and one-third corresponds to theribosomal proteins. In the case of the Escherichiacoli ribosome, a number of different techniqueshave been applied to investigate the spatialarrangement of the individual proteins, with par-

panying paper,

microscopy; IEM,

71211

ticular attention being given to the 21 proteins ofthe smaller (30 S) ribosomal subunit. The principalmethods used have included in situ protein-proteincross-linking (e.g. see Traut et al., 1980), immunoelectron microscopy (IEM; e.g. see StoÈ f¯er &StoÈ f¯er-Meilicke, 1986; Oakes et al., 1986), and neu-tron scattering (Capel et al., 1988). The cross-linkingapproach determines which pairs of proteins areneighbours within the ribosomal subunit, the IEMtechnique identi®es the locations of epitopes in theindividual proteins on the subunit surface, and theneutron scattering method measures the distancesbetween the centres of mass of selected pairs of

# 1997 Academic Press Limited

546 RNA-Protein Interactions in the 16 S rRNA Model

proteins inside the subunits. In general, the level ofagreement between the data sets obtained by thesethree approaches has been remarkably high,although there are a few signi®cant discrepancies(for discussion, see Brimacombe, 1995).

A corresponding amount of effort has also beenapplied to the study of RNA-protein interactionswithin the E. coli ribosomal subunits. Here, themethods used have included analysis of RNA-binding sites for the individual proteins (e.g. seeAllmang et al., 1994), in situ RNA-protein cross-linking (e.g. see Brimacombe, 1991), and foot-print-ing of the proteins on the 16 S rRNA either withbase-speci®c probes (Stern et al., 1988) or withhydroxyl radicals generated from Fe(II)-EDTA(Powers & Noller, 1995). These RNA-protein datahave been combined with those relating to thespatial distribution of the proteins, in order toderive models for the three-dimensional folding ofthe 16 S rRNA (Brimacombe et al., 1988; Stern et al.,1988; Malhotra & Harvey, 1994; Fink et al., 1996).Because of its quantitative nature, the neutron scat-tering map of the protein arrangement (Capel et al.1988) has played a particularly prominent role inthis type of model-building study. However,recently we have come to the conclusion (e.g. seeBrimacombe, 1995) that a model-building strategybased primarily on ®tting the 16 S rRNA directlyto the neutron map can lead to serious errors. Thereason for this can be described very simply; if oneconsiders a typical ribosomal protein to be a sphereof 30 AÊ diameter, and if this protein sphere is incontact with two different 16 S rRNA helices (eachwith a diameter of 20 AÊ ), then the two rRNA he-lices can either be in direct contact with each other,or their respective centres can be 50 AÊ apart (ifthey lie on opposite sides of the protein). Anyintermediate arrangement is also possible, with theresult that, in the absence of other constraints, alarge element of uncertainty is introduced into therRNA model, and errors can accumulate from onepart of the model to the next, via the interconnec-tivities in the rRNA secondary structure. The pro-blem is of course exacerbated by the lack ofknowledge of the exact shapes of the majority ofthe ribosomal proteins; protein L9 from the 50 Ssubunit, for example, is extremely elongated(Hoffman et al., 1994). Nevertheless, it is obviousthat at the same time any plausible model for thethree-dimensional folding of the 16 S rRNA, regard-less of how that model was derived, must be ableto account for the RNA-protein interaction data.

Here, we document how our model for the 16 SrRNA, ®tted to the cryo-electron microscopic (cryo-EM) reconstruction at 20 AÊ resolution of the E. coli70 S ribosome in the pre-translocational state (Starket al., 1997) as described in the preceding paper(Mueller & Brimacombe, 1997), can be correlatedwith the RNA-protein interactions. To do this, we®rst placed the neutron map of the 30 S ribosomalproteins (Capel et al., 1988) within the 70 S EMreconstruction, so as to give the best empirical ®tto the 30 S subunit moiety of the latter, using the

computer programme ERNA-3D (Mueller et al.,1995). Then, again with the help of ERNA-3D, theRNA-interaction data for each protein were exam-ined, by displaying the protein concerned withinthe EM contour, together with the correspondingRNA-protein cross-link sites (Brimacombe, 1991),and Fe(II)-EDTA (Powers & Noller, 1995) or base-speci®c (Stern et al., 1988) foot-print sites, in theirrespective helical or single-stranded regions of the16 S rRNA model. As in the preceding paper(Mueller & Brimacombe, 1997) we use an all-atom``wire-frame'' presentation to display these results,so that individual satisfactory ®ts and discrepan-cies can both be clearly seen. As before (Mueller &Brimacombe, 1997), we stress that the all-atompresentation is for this display purpose only, andshould not be over-interpreted at the current levelof resolution of the EM reconstruction and of therRNA model.

Results

The RNA-protein interaction data

The data sets that we have considered here aresummarized in Table 1 and Figure 1. The cross-link sites in Table 1 are those from the summaryby Brimacombe (1991), with the exception thathere we have not considered the cross-links fromthe extreme 50 terminus of the 16 S rRNA to pro-teins S3, S4, S5 and S8, or those from the extreme30 terminus to S7, S18 and S21; as previously dis-cussed (Brimacombe et al., 1988), this broad spec-trum of cross-links indicates that the relativelylong single-stranded terminal sequences (cf.Figure 1) of the 16 S rRNA are rather ¯exible, andin consequence the cross-links to these extreme ter-mini are not very helpful for the evaluation of therRNA model. In Table 1 the locations of the cross-links within the 16 S rRNA secondary structure aregiven by helix number (cf. Figure 1), and the pre-cise nucleotides concerned are shown in parenth-eses, as de®ned by the corresponding analysis foreach cross-link site in the original literature (seefootnotes to Table 1).

The foot-print data in Table 1 are also givenboth by helix number and nucleotide position.These foot-print data are taken from Powers &Noller (1995) in the case of the Fe(II)-EDTA foot-prints, and from the summary by Stern et al. (1988)in the case of the base-speci®c foot-prints. For thesake of simplicity, only the major foot-print sitesfrom both data sets are listed in Table 1. In thiscontext it is important to note that the minor foot-print sites, with only a very few exceptions, liewithin the same regions of the 16 S rRNA that arerepresented by the major sites from one of other ofthe two data sets (see Powers & Noller, 1995). Forexample, protein S3 has minor Fe(II)-EDTA foot-print sites in helix 31, and major base-speci®c sitesat similar positions in this helix; similarly, S20 hasminor Fe(II)-EDTA foot-prints in helix 13, andmajor base-speci®c sites in the same helix. The

Table 1. Summary of RNA-protein interaction data

Protein Cross-links Fe(II)-EDTA foot-prints Base-speci®c foot-prints

S2 ± h35 (1072±1073, 1102) h31 (962±964, 973±975); h34 (1053,1057, 1196, 1198, 1204, 1206); h36

(1081, 1084); h37 (1094)

S3 h39 (1155±1158)a h34 (1047±1057); h36±37 (1078±1086) h31 (962±964, 973±975); h34 (1053,1057, 1196, 1198, 1204, 1206); h37

(1094)

S4 h16 (413)b h16 (406±411, 427); h17 (437±440);h18 (500±504, 541±543)

h3 (31, 38); h17 (497±498); h18 (506,509±510); h23a (724±725, 727); h27

(893)

S5 h3 (559±561)b ± h3 (26)

S6 ± See S18 See S18

S7 h41 (1238±1240)c,d h43 (1377±1378)d h29 (938±939); h30 (946, 1235); h41(1236±1241, 1289±1290); h43 (1350±

1352, 1361±1364)

h28 (1382); h29 (935±940, 1346); h30(944±945, 949±951); h41 (1238, 1248±

1251, 1256, 1286±1288, 1299±1300);h42 (1302, 1316, 1331±1334); h43

(1349, 1360, 1362, 1365, 1374, 1377)

S8 h21 (593±597, 629±633, 651±654)d h21 (598±599, 642±643); h25 (820±828,872±875)

h20 (573, 575, 583); h24 (811); h26(858±859)

S9 h30 (954)a h39 (1130±1131)b h38 (1114±1118); h39 (1126±1130,1148±1150); h41 (1248±1252, 1279±

1280)

h39 (1126±1127, 1130, 1145±1146); h41(1279±1281)

S10 h39 (1140±1144)a h39 (1123±1124, 1149±1151) h39 (1150)

S11 h23 (702±705)a h23 (706±708) h23 (687, 694±695, 698, 703±705); h24(776, 778); h25 (819); h27 (890)

S12 ± h3 (33±35); h18 (517, 522±524, 536) h1 (21); h3 (558); h18 (521); h19 (563,881, 886±887); h27 (894)

S13 h29 (1337±1338)b h42 (1302±1303, 1307±1309, 1330) h30 (951, 953, 1227)

S14 ± ± h43 (1362)

S15 ± h20 (752±754); h22 (657) h22 (741±742); h23a (724, 727, 729±730)

S16 ± h1 (21±25); h3 (31±32); h7 (229±230);h12 (301±304); h15 (376±377); h17(449±452); h21 (607±612, 631±632)

h3 (31); h5 (51, 362±364); h6a (119,315); h12 (301±302); h13 (325); h21

(607±609, 619±622); h22a (718)

S17 h11 (278±280)a h21 (629±633)e h7 (234±235); h11 (254±255, 264±265,279)

h11 (246, 251, 262±266, 274, 279±281)

S18 h26 (845±851)a �S6f: h22 (736, 743); h23 (673±675);h26 (844±845)

�S6f: h22 (664); h23 (674±676, 687,694±695, 698, 703±705); h22a (715±718); h23a (728); h24 (778, 803, 812)

S19 h30 (1223±1231)b h30 (955); h31 (958±959, 976±978);h33 (1013); h34 (1046±1047); h42

(1312±1314)

h28 (1395±1398); h30 (1225±1227); h31(958±959, 976±980); h32 (983); h33

(993±994, 996, 1014, 1016, 1036); h34(1046, 1213); h42 (1316±1319); h43

(1361)

S20 ± h8 (172±174); h9 (184±187); h44(1433±1434)

h6a (108); h9 (194); h11 (246, 251, 274);h13 (329); h44 (1433±1434, 1446±1447)

S21 h23a (723±724)e ± h24 (800)

In each column the locations of the data points in the 16 S rRNA are shown with the helix number (cf. Figure 1) in bold face, andthe precise nucleotide(s) concerned in parentheses. The cross-link sites are taken from the references given in the footnotes. TheFe(II)-EDTA foot-print data are taken from Powers & Noller (1995), and the base-speci®c foot-print data from Stern et al. (1988).

a Cross-links with bis-(2-chloroethyl)-methylamine (Greuer et al., 1987).b Cross-links with methyl p-azidophenyl acetimidate (Osswald et al., 1987).c Cross-link induced by direct UV-irradiation (Zwieb & Brimacombe, 1979).d Cross-links with 2-iminothiolane (Wower & Brimacombe, 1983).e Cross-links with 2-iminothiolane (Kyriatsoulis et al., 1986).f The foot-print data are available only for proteins S6 � S18, together.

RNA-Protein Interactions in the 16 S rRNA Model 547

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reader should refer to the original literature(Powers & Noller, 1995) for the full details.

The data as presented in Table 1 show thespread of cross-linking or foot-printing data acrossthe 16 S rRNA sequence, for each protein. Conver-sely, in Figure 1 the same data, shown schemati-cally in the 16 S rRNA secondary structure (cf.Brimacombe, 1995), indicate which different pro-teins are involved in interactions with the sameregions of the 16 S molecule. For the most part, in-dividual helices contain sites for only one or twodifferent proteins that are close neighbours in theneutron map (Capel et al., 1988; and see Figure 3,later); for example, helix 39 (Figure 1) containscross-link sites and both types of foot-print site forS9 and S10. However, in the case of the base-speci®c foot-prints there are one or two locationsthat show data for proteins that are far apart in theneutron map; for example, helix 23a shows foot-print sites for S4, S15 and S6 � S18, which arewidely separated, and helix 27 shows foot-printsites for S11, S4 and S12, S11 being far from the lat-ter two proteins. This type of discrepancy is notunexpected, in view of the observation (Powers &Noller, 1995) that the base-speci®c foot-printdata are less ``concise'' than their Fe(II)-EDTAcounterparts. Nonetheless, it is clear that in suchcases the foot-print data cannot all be satis®ed by asingle rRNA model, and these (and other) pro-blems with the RNA-protein interaction data arediscussed, where they arise, for each individualprotein below. As with Table 1, Figure 1 is only asummary of the relevant data, and again the readershould refer to the original literature (e.g. seeBrimacombe, 1991; Powers & Noller, 1995) for thedetails.

Fitting the neutron map to theEM reconstruction

The same EM reconstruction of E. coli 70 S ribo-somes in the pre-translocational state (Stark et al.,1997) that was used to ®t the 16 S rRNA (Mueller& Brimacombe, 1997) was also used to ®t the neu-tron scattering map (Capel et al., 1988) for the masscentres of the 30 S ribosomal proteins. For this pur-pose, the 30 S subunit moiety was excised from the70 S reconstruction as before (Mueller &Brimacombe, 1997), and the neutron map wasmanouevred as a rigid entity, that is to say withoutin any way altering the positions of the proteinmass centres relative to one another, into the 30 SEM contour. The result is illustrated in Figures 2and 3. Figure 2 shows two stereo views of the pro-teins ®tted to the EM contour, whereas in Figure 3four ``mono'' views are shown, each rotated aboutthe vertical axis by 90�, with the protein numbersindicated. The latter four views correspond exactlyto those of Figure 4 of Mueller & Brimacombe(1997), and the P site tRNA is included (cf. Starket al., 1997) to orient the reader with respect to theposition of the 30 S:50 S subunit interface in eachcase. The proteins are coloured according to the

domain of the 16 S rRNA with which they interact,and the colour coding is the same as that used forthe corresponding domains of the 16 S rRNAmodel in the preceding paper (Figures 3 and 4 ofMueller & Brimacombe, 1997). Thus, proteins S2,S3, S7, S9, S10, S13, S14 and S19 (which interactwith the 30-domain of the rRNA; cf. Figure 1,Table 1) are light blue; proteins S6, S8, S11, S15,S18 and S21 (interacting with the central domain)are red; proteins S4, S5, S12, S16, S17 and S20 (in-teracting with the 50 domain) are dark blue. ProteinS1, which is rather large and for which there isvery little RNA-protein interaction information, isomitted from the Figures.

The distribution of the various rRNA domainsamong the principal features of the 30 S subunit(head, body and side lobe) are well de®ned in therRNA model; the 30-domain corresponds to thehead, most of the central domain is located in theside lobe, and the body comprises the 50-domain,most of the 30-minor domain and part of the centraldomain, as described (Mueller & Brimacombe,1997; also see Stern et al., 1988; Malhotra &Harvey, 1994; Fink et al., 1996). Accordingly, thebasic orientation of the neutron map within thesesame features of the 30 S subunit in relation bothto the IEM data for the proteins (StoÈ f¯er & StoÈ f¯er-Meilicke, 1986; Oakes et al., 1986) and to the 16 SrRNA models is a priori unambiguous. The detailed®t (Figures 2 and 3) was made simply by empiri-cally rotating and translating the neutron mapusing the programme ERNA-3D, so as to bring asmany of the protein mass centres as possible topositions within the EM reconstruction.

In Figures 2 and 3 (and in the subsequentFigures in this paper) the diameters of the proteinspheres have been reduced, for easier visibility, toabout two-thirds of the corresponding values dis-played by Capel et al. (1988), where the size of thespheres corresponded to the mass of the proteinsin each case. The proteins therefore occupy ca one-third of their actual volume in the diagrams.Nevertheless, many of the protein spheres still pro-trude outside the 30 S subunit surface envelope inFigures 2 and 3, and such protrusions have been``balanced'' as far as possible on opposite sides ofthe subunit (e.g. see proteins S3 and S13 inFigure 3(a) and (c), proteins S5 and S6 inFigure 3(b), S2 and S7 in Figure 3(b), or S8 andS18/S6 in Figure 3(d)).

In relation to our 16 S rRNA model (Mueller &Brimacombe, 1997), the fact that the neutron mapis slightly ``too large'' for the 30 S EM contour im-plies that, in general, the proteins must lie outsidethe rRNA, in agreement with earlier neutron scat-tering studies (e.g. see Stuhrmann et al., 1978) onthe respective radii of gyration of the protein andRNA moieties. Neutron scattering studies havefurther shown (Ramakrishnan, 1986) that the over-all centres of mass of protein and rRNA are dis-placed relative to one another, and this issupported by the relative protein-rRNA distri-bution in the different domains of the 30 S subunit.


As noted above, the head of the subunit containsproteins S2, S3, S7, S9, S10, S13, S14 and S19 (lightblue in Figures 2 and 3), and these have a com-bined molecular weight of 133 kDa (Wittmann-Liebold, 1986); the corresponding rRNA (helices 28to 43, Figure 1) comprises ca 470 nucleotides. Simi-larly, the side lobe region (proteins S6, S8, S11, S15,S18 and S21; red in Figures 2 and 3) contains70.8 kDa of protein and 250 nucleotides of rRNA(helices 19, 20, 22 to 26 and 45; Figure 1, and seeMueller & Brimacombe, 1997); the body region (S4,S5, S12, S16, S17 and S20; dark blue in Figures 2and 3) contains 82.5 kDa of protein and 820 nucleo-tides of rRNA (helices 1 to 18, 21, 27 and 44;

Figure 2. Stereo views of the neutron map of the 30 S riboreconstruction (Stark et al., 1997). The protein diameters areCapel et al. (1988). The proteins are coloured light blue, reddomain, the central domain or the 50-domain, respectively,transparent silhouette, and the P site tRNA is included for rthe solvent side of the 30 S subunit. (b) View at 90� to that o

Figure 1). From these values one can make thecrude calculation that the protein to rRNA ratio is0.28 kDa of protein per nucleotide in the head ofthe subunit, and is similarly 0.28 kDa/nt in theside lobe region. In contrast, the correspondingvalue for the body is only 0.10 kDa/nt, that is tosay the body region is richer in rRNA by a factorof almost 3. Thus, the protein distribution isskewed relative to that of the rRNA, rather thanforming a uniform ``blanket'' around the rRNAcore, as might be inferred from the earlier EMreconstruction of the 70 S ribosome by Frank et al(1991). As well as being qualitatively in agreementwith the neutron scattering result (Ramakrishnan,

somal proteins, ®tted to the 30 S moiety of the 70 S EM-reduced to two-thirds of their size in the original map byor dark blue, according to their interactions with the 30-of the 16 S rRNA. The EM contour is shown as a semi-eference as a green backbone tube model. (a) View fromf (a), with the subunit interface to the right.

Figure 3. Identi®cation of the individual 30 S ribosomal proteins. The proteins are coloured as in Figure 2, and the Spre®xes to their names are omitted for simplicity. The EM contour is shown as a semi-transparent silhouette as inFigure 2, and the P site tRNA is included for reference. The four views represent rotations by 90� about the verticalaxis. (a) View from the solvent side of the subunit (cf. Figure 2(a)). (b) View with the subunit interface to the left.(c) View from the interface side of the subunit. (d) View with the subunit interface to the right (cf. Figure 2(b)).



1986), this skewed distribution also concurs withthe greater packing density that we have alreadynoted for our 16 S rRNA model (Mueller &Brimacombe, 1997) in the body region of the EM

Figure 4. (a) Protein S7, with interaction sites in rRNAFigure 3(d). (b) Protein S9, with sites in helices 30, 38, 39 anS19, with sites in helices 28, 30, 31, 32, 33, 33a, 34.1, 42.2 and

contour, as opposed to the head and side lobeareas. Finally in this context, it should be notedthat protein S20 would appear to belong to thegroup of proteins occupying the subunit head

helices 28, 29, 30.1, 41, 42 and 43. Orientation as ford 41. Orientation similar to (a) or Figure 3(d). (c) Protein43. Orientation as for Figure 3(c). See the text for details.


(Figures 2 and 3), although the data of Figure 1and Table 1 suggest that it belongs to the 50-do-main and hence to the body; this discrepancy willbe dealt with below.

The RNA-protein interactions for theindividual proteins

The RNA-protein data for each protein are dis-played in stereo in Figures 4 to 10. EachFigure shows a single protein, colour-coded as inFigures 2 and 3, and each protein sphere has thesame reduced diameter as in the latter two Figures,for easier visualization of the data. In addition,each Figure shows the 16 S rRNA helices and con-necting single-stranded regions (Figure 1) that con-tain RNA-protein interaction information for theprotein concerned (Table 1). Within the rRNAelements the individual nucleotides correspondingto RNA-protein cross-link or foot-print sites arehigh-lighted as coloured ball-and-stick nucleotides;in these nucleotides the carbon atoms are white,and the non-carbon atoms are light blue for cross-link sites, yellow for Fe(II)-EDTA foot-print sites,and red for base-speci®c foot-print sites, respect-ively. In cases where the same nucleotide is both across-link and a foot-print site, then the cross-linkis given priority (i.e. that nucleotide is high-lightedin blue), and where a nucleotide is a site for bothtypes of foot-print the Fe(II)-EDTA data are givenpriority (i.e. that nucleotide is high-lighted in yel-low). In each case the data are displayed withinthe semi-transparent EM contour of the 30 S sub-unit, and the arrangement of the helices should becompared with their positions in the complete 16 SrRNA model, as depicted in Figures 3 and 4 ofMueller & Brimacombe (1997). Since Figure 1(above) is already very complex, the numbers ofthe sub-divisions of the 16 S rRNA helices withinthe secondary structure are not included in it. Thesub-divisions of the helices referred to in the fol-lowing sections are those de®ned in Figure 1 ofMueller & Brimacombe (1997), and they are quotedas in the latter publication (e.g. 3.1, 3.2 or, in thecomputer diagrams, h3d1, h3d2 for sub-divisionsof helix 3). Also as in the latter paper, the stereodiagrams in Figures 4 to 10 in the following sec-tions are intended to be viewed with a standard6 cm magnifying stereoscope.

Proteins in the subunit head

Protein S7 (Figure 4(a))

This protein is shown in Figure 4(a) togetherwith rRNA helices 28, 29, 30.1, 41, 42 and 43, in anorientation similar to that in Figure 3(d). Helix 28can be seen extending downwards towards thebottom of the Figure, with the distal region of helix41 (41.3 and 41.4) extending leftwards at the top.The foot-print sites form a compact mass aroundthe protein, with those in helices 30.1 and 42 lyingtowards the viewer, and those in helices 29, 41 and

43 behind; the sites in helix 43 are partly obscuredby the protein. Bearing in mind that, as notedabove, the protein spheres have been deliberatelyreduced in size, so that they only occupy aboutone-third of their actual volume, this situation rep-resents an ideal ®t. The two cross-link sites (lightblue) lie on opposite sides of the protein, the site inthe single-strand connecting to helix 41 (nucleo-tides 1238 to 1240) being above S7 in the Figure,and that in the single strand connecting helices 28and 43 (1377-1378) on the left. This is a goodexample of the dilemma described in the Introduc-tion, with regard to the modelling of multiplehelices of the rRNA around a single protein masscentre position; here the end result is clearly satis-factory. The S7-16 S rRNA interaction site asdepicted in Figure 4(a) furthermore agrees wellwith the engineered ``minimum binding site'' forthe protein described by Dragon & Brakier-Gingras(1993).

Protein S9 (Figure4(b))

The ®t of the RNA-protein data for S9 is alsogood. The 16 S rRNA helices concerned are 30, 38,39 and 41, and the orientation of the Figure is simi-lar to that in Figure 4(a). Helix 38 lies to the left ofthe protein, with helix 30 (30.1 and 30.2) on theright, and helices 39 and 41 lying parallel with oneanother in the upper part of the Figure. The foot-print sites are spread in an arc surrounding theprotein; most of these are shown as Fe(II)-EDTAfoot-prints (yellow), a large proportion of the base-speci®c foot-print sites (Table 1) being at identicalpositions. As with protein S7, the two cross-linksites are on opposite sides of the protein, the site atthe end of helix 30 (nucleotide 954) lying in frontof the protein on the right, and that in helix 39(1130-1131) lying behind and to the left.

Protein S19 (Figure 4(c))

The situation in the case of protein S19 is ratherdifferent. Here the 16 S rRNA helices displayed are28, 30, 31, 32, 33, 33a, 34.1, 42.2 and 43, and theview is similar to that of Figure 3(c). The foot-printsites, with the exception of the base-speci®c sites inhelices 28 and 43, lie in a compact cluster on theleft side of the Figure, and the cross-link site(which could only be localized to the nonanucleo-tide 1223-1231; Table 1) is in the middle of the clus-ter. This main group of foot-print sites partiallyoverlaps the corresponding sites for protein S7(Figures 1 and 4(a)), and the rRNA regions con-cerned agree precisely with the combined bindingsite (comprising helices 29 to 32, part of 41, 42 and43) for proteins S7 � S19 described by Wiener et al.(1988). However, the neutron position for proteinS19 is far to the back of the foot-print and cross-link site positions, as seen in Figure 4(c), and is along way from protein S7; the distance between S7and S19 in the neutron map can be seen mostclearly in Figure 3(d). In contrast, the most recent


IEM localization for S19 (Olson et al., 1988) placesit on the interface side of the 30 S subunit (close toS7; cf. Figure 3(c) and (d)), a location that is sup-ported by a number of site-directed cross-links toS19 from various positions in P or A site boundtRNA (Ofengand et al., 1986; Wower et al., 1990;Rosen et al., 1993; Osswald et al., 1995; Rinke-Appelet al., 1995). For these reasons, as discussed else-where (Brimacombe, 1995), we believe that proteinS19 is incorrectly placed in the neutron map. Themain cluster of sites in Figure 4(c) is in good agree-ment with the IEM location of S19, but the base-speci®c foot-print sites in helices 28 and 43 (lowerright in Figure 4(c)) cannot readily be accounted for.In particular, those at the base of helix 28 (nucleo-tides 1395 to 1398) are adjacent to position C-1400,the well known site of cross-linking to the antico-don loop of P site tRNA (Prince et al., 1982), andthese positions cannot be brought into the vicinityof the main group of foot-print sites in Figure 4(c)without violating important constraints in the de-coding area (cf. Mueller et al., 1997).

Figure 5. (a) Protein S10, with interaction sites in helix 39. Ohelices 29, 30 and 42. Orientation similar to Figure 3(b).


All of the data for S10 are concentrated in helix39, which is shown in Figure 5(a) in an orientationcorresponding to that in Figure 3(d). The base-speci®c foot-print is overlapped by the Fe(II)-EDTA sites, so only the latter can be seen. The neu-tron position for S10 lies slightly outside the EMcontour, with the result that the protein is unavoid-ably displaced somewhat to the right and in frontof the foot-print and cross-link sites in Figure 5(a).Nevertheless, the ®t is satisfactory.

Protein S13 (Figure 5(b)

The situation for S13 is similar to that of S10, inthat the neutron position for the protein lies out-side the EM contour, although in this case the dis-placement is greater. The rRNA helices displayed,29, 30 and 42, form a compact group in the model,with the foot-print and cross-link sites clusteredaround helices 30 and 42.1. The orientation in theFigure is similar to that in Figure 3(b). In the stereoview shown in Figure 5(b) the protein lies well in

rientation as for Figure 3(d). (b) Protein S13, with sites in

Figure 6. (a) Protein S2, with interaction sites in helices 31, 34, 35, 36 and 37. Orientation as for Figure 3(a). (b) Pro-tein S3, with sites in helices 31, 34, 36, 37, 39.1 and 40. Orientation similar to (a) or Figure 3(a).


front of the group of sites on the rRNA, a substan-tial part of this separating distance beingaccounted for by the gap between the mass centreof the protein and the surface of the EM contour.

Proteins S2 and S3 (Figure 6)

As has been pointed out by Malhotra & Harvey(1994), the base-speci®c foot-print data for S2 andS3 are virtually identical, and may therefore rep-resent allosteric effects; the two proteins are wellapart from each other in the neutron map(Figure 3(a)). Furthermore, the neutron positionsand the corresponding IEM locations of S2 and S3(StoÈ f¯er & StoÈ f¯er-Meilicke, 1986) are somewhat atvariance, so the two proteins, both of which arelarge, may have elongated or irregular shapes. Thedata for protein S2 are displayed in Figure 6(a),together with rRNA helices 31, 34, 35, 36 and 37,which form an extended structure in the rRNAmodel with the base-speci®c foot-print sites spreadalong it. The orientation in the Figure is similar tothat in Figure 3(a). There is no cross-link for S2,and the Fe(II)-EDTA foot-prints are concentrated in

helix 35, on the left in the Figure. As with S10 andS13, the neutron position of S2 lies outside the EMcontour, so that, making allowance for this, the ®tto the Fe(II)-EDTA sites in helix 35 is reasonablefor such a large protein.

In the case of S3 (Figure 6(b)), the helices dis-played are 31, 34, 36, 37, 39.1 and 40, and theorientation is similar to that of Figure 6(a), butrotated slightly around the horizontal axis. TheFe(II)-EDTA foot-print sites are in two distinctgroups, in helix 34 (on the right) and in helices 36and 37 (on the left), with the cross-link site in thesingle strand connecting helices 39 and 40 (nucleo-tides 1155 to 1158) close to the latter group. Theprotein, which again lies outside the EM contour,is satisfactorily close to the Fe(II)-EDTA foot-printsites in helix 34, but the ®t to the other group ofsites (on the left in Figure 6(b)) is poor, although itcould be explained, as just suggested, by anelongated con®guration of the protein. The pos-ition of S3 is quite close to the empty lobe of EMdensity (just to the left of the protein sphere inFigure 6(b)), and this lobe could conceivablyaccommodate part of the protein.


Protein S14

The only major foot-print site for S14 is a base-speci®c one at nucleotide 1362 in helix 43. There is

Figure 7. (a) Protein S8, with interaction sites in helices 19,(b) Protein S15, with sites in helices 20.2, 22 and 23a. Orienthelices 23, 24 and 25. Orientation as in (a) or Figure 3(a) (clo

no cross-link site for this protein, and the Fe(II)-EDTA data show only a minor site at nucleotide ca993 between helices 32 and 33 (Powers & Noller,1995). Because of the lack of data, no Figure is

20, 21, 24.1, 25, 26a and 26t. Orientation as for Figure 3(a).ation as in (a) or Figure 3(a). (c) Protein S11, with sites inse-up view).


shown for protein S14. It should, however, benoted that nucleotide 993 lies reasonably close tothe position of S14 as determined by IEM (StoÈ f¯er-Meilicke & StoÈ f¯er, 1990), although it is fartheraway from the neutron location (cf. Figure 3).Nucleotide 1362 is also far away from the neutronlocation of S14, as this nucleotide, at the loop endof helix 43, is in the ``S7 area'' (cf. Figures 1 and4(a)).

Proteins interacting with the central domain


The position of protein S8 relative to the 16 SrRNA model ®ts well to the cross-linking and foot-printing data, as well as to the established bindingsite (e.g. see Allmang et al., 1994) for the protein inhelix 21. Figure 7(a) shows helices 19, 20, 21, 24.1,25, 26a and 26t, in an orientation corresponding tothat in Figure 3(a). Helix 21, in which the threecross-link sites and some of the Fe(II)-EDTA foot-print sites are located, lies to the right of the pro-tein, with the cross-link site at nucleotides 651 to654 at the top, that to nucleotides 593 to 597 in themiddle, and that to nucleotides 629 to 633 atthe bottom. Bearing in mind the reduced size ofthe protein sphere in Figure 7(a), the prominentgroup of Fe(II)-EDTA foot-print sites in helix 25(lower centre) as well as the base-speci®c foot-printsites in the vicinity of helices 20 and 26a (centreleft) are also potentially in contact with the protein.

Protein S15 (Figure 7(b))

Protein S15 has a well-studied binding site at thethree-way junction of helices 20, 21 and 22 (e.g. seeBatey & Williamson, 1996). Figure 7(b) showshelices 20.2, 22, and 23a, which contain the foot-print data for S15, there being no cross-link site forthis protein. The orientation in the Figure is thesame as that in Figure 7(a), and the Fe(II)-EDTAfoot-print sites in helices 20.2 and 22.1 at the three-way junction (helix 21 is not displayed in thisFigure) can be seen just above the protein, with thebase-speci®c sites in helix 22.2 immediately adja-cent to these. The further set of base-speci®c foot-print sites (on the far left in Figure 7(b)) are inhelix 23a. As already noted above, the latter helixcontains base-speci®c foot-prints to the widely-sep-arated proteins S4 and S6-S18 as well as to S15 (cf.Figure 3(a) and (b)), and it is therefore highly un-likely that these foot-prints represent direct con-tacts to all of the proteins concerned.


The orientation in Figure 7(c), showing a close-up view of the data for S11, is the same as that inFigure 7(a) and (b). The Figure shows helices 23, 24and 25, with the cross-link site in helix 23 (nucleo-tides 702 to 705) and the Fe(II)-EDTA foot-printsites lying just beneath and behind the protein

sphere. The majority of the base-speci®c foot-print sites are also in helix 23, in the lower re-gion of the latter. Of the remaining base-speci®cfoot-print sites, those in helix 24 (Figure 7(c),lower centre) can be regarded as still being plau-sibly close to the protein, but there is anothersite in helix 25 (Figure 7(c), bottom right) that isfar away, as well as a site in helix 27 (seeTable 1) that is even more remote (outside ofFigure 7(c) to the right). As is the case with helix23a, mentioned above in connection with proteinS15, helix 27 similarly contains foot-prints to S4and S12 in addition to S11 (cf. Figure 3(a)), andagain we suggest that these foot-prints cannotrepresent direct contacts to all three proteins. Thelocations of protein S11 and helix 23 in the 16 SrRNA model are of particular importance in thecontext of the functional data (see Mueller et al.,1997).


The relatively limited amount of data relating toprotein S21 is illustrated in Figure 8(a), showinghelices 23a and part of helix 24 (24.2 and 24.3). Theorientation of this close-up view corresponds tothat in Figure 3(c). The cross-link site in helix 23a(nucleotides 723-724) is in front of the proteinsphere, and it is noteworthy that, although there isno major Fe(II)-EDTA foot-print site for S21(Table 1), there are minor sites at positions 670 to672 (Powers & Noller, 1995) in helix 22, very closeto this cross-link site (cf. Figure 1). There is onlyone base-speci®c foot-print site for S21, at position800 in helix 24 (Figure 8(a), bottom right); this isanother example where the site is rather remotefrom the protein sphere.

Proteins S18 and S6 (Figure 8(b))

In the foot-printing experiments of Stern et al.(1988) and of Powers & Noller (1995), proteins S6and S18 are considered together, and the data aredepicted in Figure 8(b). The orientation in thisFigure is similar to that in Figure 3(a) (protein S6is obscured behind S15 in the latter) and S6 iscoloured orange to distinguish it from S18. TherRNA helices shown are 22, 23, 23a, 24 and 26,with helix 22 extending across the upper part ofthe Figure, helices 23 and 23a on the left, andhelix 24 lying above helix 26 at the bottom. Thefoot-print sites of both types in helices 22, 22a,23, 23a, 24.1 and 24.2 are acceptably close to oneor other of the two proteins but, on the otherhand, the base-speci®c foot-prints at the extremeend of helix 24.1 (Figure 8(b), centre right), theFe(II)-EDTA foot-print site in helix 26 (lowercentre) and, particularly, the cross-link site to S18in helix 26 (nucleotides 845 to 851; Figure 8(b),lower centre) do not ®t well. It should be noted,however, that the mass centres of both S6 andS18 lie outside the EM contour (cf. Figure 3(d)),and that furthermore this part of the side lobe


area of the 30 S subunit is likely to be involvedin major conformational changes (Mueller et al.,1997).

Proteins in the body of the subunit


Protein S4 interacts with the very compactlyfolded region of the 50-domain of the 16 S rRNAlocated on the ``shoulder'' of the 30 S subunit body(Mueller & Brimacombe, 1997), and the interactionsites together with helices 3, 16, 17.2, 18, 18t, 23aand 27 are illustrated in Figure 9(a). The Figure isin an orientation corresponding to that inFigure 3(d). The majority of the foot-print sites(those in helices 3, 16 and 18) form a compact clus-ter just to the right of the protein, and the cross-link at nucleotide 413 in helix 16 is also within thiscluster. Further Fe(II)-EDTA foot-print sites can beseen extending downwards towards helix 17.2 inthe single strand connecting helix 16 to the latter(Figure 9(a), lower centre); these are still reason-ably close to the protein sphere, but it should beremembered that the location of helix 17.2 in the

Figure 8. (a) Protein S21, with interaction sites in helices 23S6 and S18, with sites in helices 22, 23a, 24 and 26. Protein S

rRNA model (and hence the location of this groupof foot-print sites) is only tentative (Mueller &Brimacombe, 1997). There are in addition twogroups of base-speci®c foot-print sites, in helices23a (centre right in Figure 9(a)) and 27 (lowercentre left). As mentioned above, foot-prints inthese helices are also shown by the very distantproteins S15 and S11, respectively, and are unlikelyto represent direct contacts.


Figure 9(b) is in approximately the same orien-tation as that in Figure 9(a), and shows helices 1, 3and 19, together with the data for S5, which consistof a single base-speci®c foot-print site at nucleotide26 (connecting helices 1 and 3) and the cross-linksite (nucleotides 559 to 561) in the single strandconnecting helices 3 and 19. Helix 1 is obscured be-hind helix 3.1 on the right of the protein, but thefoot-print and cross-link sites are neverthelessclearly visible, the single strand containing thecross-link leading downwards to helix 19 at thebottom of the Figure. There are no Fe(II)-EDTAfoot-print data for protein S5.

a, 24.2 and 24.3. Orientation as for Figure 3(c). (b) Proteins6 is orange. Orientation similar to Figure 3(b).



The orientation in Figure 9(c) is rotated slightlyabout the vertical axis in relation to that ofFigure 9(b). Helices 1, 2, 3, 18, 18t, 19 and 27 are

Figure 9. (a) Protein S4, with interaction sites in helicesFigure 3(d). (b) Protein S5, with sites in the region between(c) Protein S12, with sites in helices 1, 2, 3, 18, 19 and 27. Or

displayed, and the majority of the foot-print sites(in helices 1, 3, and 18) are clustered just aboveand to the right of the protein. The Fe(II)-EDTAfoot-print sites at nucleotides 33 to 35 (Table 1) inhelix 3.2 are obscured behind the protein, that at

3, 16, 17.2, 18, 18t, 23a and 27. Orientation similar tohelices 3 and 19. Orientation similar to (a) or Figure 3(d).ientation similar to Figure 3(d).


nucleotide 33 being just visible in the Figure. Otherbase-speci®c foot-print sites occur in helices 19 and27, and in the single strand connecting helices 3

Figure 10. (a) Protein S16, with interaction sites in helices 1as for Figure 3(c). (b) Protein S17, with sites in helices 7, 11 atein S20 in helices 6a, 8, 9, 11, 13 and 44. Orientation similathe text for an explanation.

and 19, in the lower part of Figure 9(c). The sites inhelices 19 and 27 are rather far from the proteinsphere, those in helix 27 once again being unlikely

, 3, 5, 6a, 7, 12, 13, 15, 17.2 17.3, 21.2 and 21.3. Orientationnd 21. Orientation similar to Figure 3(a). (c) Sites for pro-r to Figure 3(d). The protein shown (orange) is S17; see


to be direct contacts (see the discussion above onproteins S4 and S11). The protein sphere lies infront of the EM contour as viewed in Figure 9(c)(on the interface side of the 30 S subunit), but itshould be noted that the EM contour is quite thinin this area. Furthermore, IEM studies have loca-lized protein S12 at a similar level in the body ofthe 30 S subunit, but on the solvent side ratherthan on the interface side of the body (Oakes et al.,1986; StoÈ f¯er-Meilicke & StoÈ f¯er, 1990).


There are no cross-linking data for S16, but onthe other hand this protein has both Fe(II)-EDTAand base-speci®c foot-prints covering a large areaof the 16 S rRNA in the 50 and central domains.Figure 10(a) shows these data, in rRNA helices 1,3, 5, 6a, 7, 12, 13, 15, 17.2, 17.3, 21.2 and 21.3.There is a further base-speci®c site at position 718(Table 1), also in the central domain, which is notvisible in Figure 10(a) (out of the picture on theright). The orientation in the Figure is similar tothat in Figure 3(c). A relatively large proportion ofthe foot-print sites (those in helices 1, 3, 5, 12 and15) lie in an arc around the lower side of the pro-tein sphere in Figure 10(a), but other sites (those inhelices 6a, 7, 13, 17 and 21) extend downwardstowards the bottom of the subunit. Powers &Noller (1995) have suggested that the extensiveprotections by S16 may be due to assembly-in-duced conformational changes, and this contentionis supported by the fact that the foot-print sites inhelices 7 and 21 (Figure 10(a), bottom) are in rRNAregions containing protections and/or cross-linksto S8 and S17, proteins that are not very close toS16 (cf. Figure 3(c)).


In contrast to S16, the data for S17 are compactand are located in helices 7, 11 and 21, which areshown in Figure 10(b) in an orientation similar tothat of Figure 3(a). The foot-print sites are exclu-sively in helices 7 and 11, which lie just to the rightof the protein in Figure 10(b); the two helices areparallel with one another, with the lower part ofhelix 11 (11.3) being to the left of helix 7.3(Figure 10(b), lower centre; the helix number, 11.3,is obscured by the yellow ball-and-stick nucleo-tides). There are two cross-link sites to S17; the®rst (in helix 11, nucleotides 278 to 280) can beseen to the right of the protein, and the other (inthe single strand joining helices 21.2 and 21.3,nucleotides 629 to 633) is in front of the proteintowards the left. It is of interest that the second ofthe two cross-links has recently been re-con®rmedand the corresponding cross-link site within theprotein identi®ed (Urlaub et al., 1997). Further-more, the structure of S17 has been derived byNMR (Golden et al., 1993), and the cross-link site islocated in one of the two exposed loops of the pro-tein. The atomic structure of the protein can thus

be oriented with the main bulk of the protein incontact with helices 7 and 11, and the exposedloop extending towards the cross-link position inhelix 21; this detailed model will be presented else-where.


The ®nal protein, S20, has for some time posed aproblem (for discussion, see Brimacombe, 1995).While the neutron data (Figure 3) for this proteinwould appear to place it in the head of the subunit,the foot-print data are exclusively located in rRNAregions that the model (Mueller & Brimacombe,1997) places at the extreme lower end of the sub-unit body (e.g. some of the S20 foot-print sitesoverlap these of S17; Figure 1). IEM studies(Schwedler et al., 1993) have localized S20 in a pos-ition below S17 at the bottom of the subunit, andwe believe that, as with S19, the neutron locationof S20 is incorrect. Accordingly, in Figure 10(c), thefoot-print data for S20 (in helices 6a, 8, 9, 11, 13and 44) are shown together with protein S17,which is the nearest neighbour to S20 on the basisof the IEM data. The protein is coloured orange toemphasize that it is the ``wrong'' protein, and theorientation in the Figure is similar to that inFigure 3(d). The foot-print sites in helix 44 (44.7and 44.8) are on the right side of Figure 10(c), withthose in helices 8 and 9 lying below and in front ofthe S17 protein sphere at the bottom. The sites inhelix 11 are in front of the protein, and those inhelix 13 slightly above it. The remaining base-speci®c foot-print site in helix 6a is rather far away(Figure 10(c), left centre). By virtue of its identitywith protein L26 from the 50S subunit (Wittmann-Liebold, 1986), protein S20 must a priori lie at thesubunit interface, and remains attached to eitherthe 30 S or 50 S subunit when the 70 S ribosome isdissociated. This fact, together with the IEM result(Schwedler et al., 1993) suggests a location for S20at the interface in front and to the right of proteinS17 as viewed in Figure 10(c) (cf. Figure 3(c)). Thiswould be in acceptable agreement with the ma-jority of the foot-print positions, which are spreadover more than 1200 nucleotides in the primarystructure of the 16 S rRNA (Figure 1). There is nocross-link site for protein S20.

Discussion

As outlined in the preceding paper (Mueller &Brimacombe, 1997), our model for the 16 S rRNAwas built up around the functional centre of themolecule, using the secondary structure of the 16 SrRNA, the intra-RNA cross-linking data, and the®ne structure of the EM contour (Stark et al., 1997)as the primary constraints for folding the 16 SrRNA into three dimensions. The modelling pro-cedure is interactive and empirical, and in this pro-cess the positions of the mass centres of theribosomal proteins in the neutron map (Capel et al.,1988) were used as ``secondary'' rather than pri-


mary constraints; that is to say, the general locationof a protein (and hence the corresponding RNA-protein interaction data) was borne in mind duringthe construction of the model but, for the reasonsgiven in the Introduction, no attempt was made to``force'' the rRNA elements concerned towards thespeci®c neutron positions of the protein masscentres. In the later cycles of re®nement of themodel, some adjustments were made so as to bringthe different interaction sites on the rRNA for par-ticular proteins closer together, but only insofar asthis could be achieved without violating the 30 SEM contour.

The ®tting of the neutron map to the 30 S EMcontour (Figures 2 and 3) is, on the other hand, aprocess that is entirely independent of the detailsof the rRNA model. The basic orientation of theneutron map in relation to the principal features(head, body, etc.) of the EM contour is unambigu-ous, by comparisons with the IEM locations of theproteins (StoÈ f¯er & StoÈ f¯er-Meilicke, 1986; Oakeset al., 1986), as well as with the overall distributionof the rRNA domains in the head body and sidelobe regions (Mueller & Brimacombe, 1997). Hav-ing established this basic orientation, the precise ®tof the neutron map was then made, as described inResults, simply by empirically searching for thebest ®t to the EM contour (Figures 2 and 3). It fol-lows that the question of how well the RNA-pro-tein interaction data can be correlated with the®tted rRNA model on the one hand, and with the®tted neutron map on the other, is a very exactingtest.

We demonstrate here that for 12 out of the 19proteins considered (S1 and S14 were not takeninto account, because of the lack of experimentaldata), the ®t between the rRNA-protein interactiondata in the rRNA model and the neutron positionsof the corresponding proteins varies from beingexcellent to at least acceptable (Figures 4 to 10),bearing in mind the reduced size of the proteinspheres as displayed in these Figures. The 12 pro-teins are S2, S4, S5, S7, S8, S9, S10, S11, S12, S15,S17 and S21. Among the remaining seven proteins,S6 is dif®cult to evaluate, as the foot-print data(Stern et al., 1988; Powers & Noller, 1995) considerthis protein only in conjunction with S18(Figure 8(b)). S16 is also dif®cult to evaluate, inthis case because the protein shows a wide spec-trum of foot-print sites covering a large area of the16 S molecule (Figure 10(a)). For proteins S19(Figure 4(c)) and S20 (Figure 10(c)), the ®t of theRNA-protein interaction data is bad in relation tothe neutron positions of the proteins but, as dis-cussed, is in good agreement with their IEM place-ments (Olson et al., 1988; Schwedler et al., 1993)within the 30 S subunit. This leaves just proteins S3(Figure 6(b)), S13 (Figure 5(b)) and S18(Figure 8(b)), where in particular the ®t of theRNA-protein cross-links sites is not good. In thecases of S3 and S18, the cross-link site and the neu-tron position of the corresponding protein lie at ap-proximately the same level on the vertical axis of

the model, and the discrepancy concerns their rela-tive locations in the thin horizontal dimension ofthe EM contour. In the case of S13, the protein pos-ition lies out of contact with the rRNA model (seebelow, and cf. Mueller et al., 1997). With regard tothe RNA-protein foot-print data, there are a few in-stances (Figures 4 to 10) where individual datapoints cannot be brought into agreement with therRNA model. Almost all of these few discrepanciesinvolve the base-speci®c foot-print sites (Stern et al.,1988), and the majority of them furthermore con-cern helices (in particular helices 23a and 27) thatshow simultaneous foot-prints to proteins that arewidely separated in the neutron map. This sup-ports the contention by Powers & Noller (1995)that the Fe(II)-EDTA foot-print data are more con-cise than their base-speci®c counterparts.

In the context of the ®t of the RNA-protein datait is important to note that the same proteinswhich cause dif®culty in our model are equallyproblematic in other models (e.g. see Stern et al.,1988; Fink et al., 1996) where no account is taken ofan EM structure. For example, proteins S6 and S13,which lie outside the EM contour in our model(Figure 3) are out of contact with the rRNA in thelatest model by Fink et al. (1996); these authorsconsidered only the secondary structure of the 16 SrRNA, the Fe(II)-EDTA foot-print data, and theneutron map as modelling constraints. Similarly,the uncertainty relating to the positions of proteinsS19 and S20 is noted also by Fink et al. (1996), andhad the consequence that the long penultimatehelix (helix 44, Figure 1) was not included in theirmodel at all (cf. also Stern et al., 1988). Discrepan-cies of this nature between independent data sets,which are fortunately rare, place the model-builderin the dif®cult position of having to decidebetween the alternatives. With proteins S19 andS20 a decision in favour of the IEM placement wasfacilitated by the corroborative evidence fromother sources; namely, the site-speci®c cross-linksfrom tRNA in the case of S19, and the overlap ofthe foot-print sites with those of S17 in the case ofS20 (see Results). The discrepancy with the IEMdata here could possibly be explained by the factthat the neutron data of Capel et al. (1988) werecollected from reconstituted 30 S ribosomal sub-units carrying the deuterated proteins. In particularS20, which is either a 30 S or a 50 S protein in itsdual role as S20 or L26 (Wittmann-Liebold, 1986),is presumably not very tightly bound to either sub-unit, and its position may as a result be labile.Conformational differences between the respectiveribosome preparations, or between different dy-namic functional states of the ribosome (for discus-sion, see Mueller et al., 1997), could of courseaccount for other instances where the ®t betweenthe neutron map and the RNA-protein data(Figures 4 to 10) is less satisfactory; the situationshould become clearer as the resolution of the EMreconstructions improves.

The fact that the neutron map is ``larger'' thanthe EM contour (see Figure 3) means that, in gener-


al, the proteins lie outside the rRNA. It followsthat the distances between rRNA helices interact-ing with proteins may be considerably less that thecorresponding distances between the protein masscentres. This point can be illustrated by a simpleexample: if the centres of mass of two sphericalproteins each 30 AÊ in diameter are 70 AÊ apart, thenthe distance of closest approach between the twoproteins is 40 AÊ . If two rRNA helices (with diam-eter 20 AÊ ) are in contact with the innermost sidesof these proteins, one with each protein, then thereis just room to pack the two helices between theproteins, with the helices touching each other. Thistrivial-sounding consideration does in fact serve tooffer at least a partial solution to some controver-sies, in particular that concerning the distancebetween the ``530 loop'' (helix 18) and the ``1400 re-gion'' (helix 44). Here, Powers & Noller (1994)have maintained that these regions must be farapart, on the grounds that the neutron positions ofthe ``nearest'' proteins (S12 and S7, respectively)are separated by 112 AÊ . However, Alexander et al.(1994) attached an oligonucleotide carrying aphotoaf®nity label with a bridging distance of 24 AÊ

to the loop end of helix 18, and found that thislabel became cross-linked to S7. In our rRNAmodel the RNA-protein data for both S7(Figure 4(a)) and S12 (Figure 9(c)) agree acceptablywell with the respective neutron positions of theproteins (although as already noted there is a dis-crepancy between the IEM and neutron locationsfor S12), but nevertheless the loop end of helix 18(which contains foot-print sites to S12) is onlyabout 40 AÊ (instead of 112 AÊ ) from the nearestpoint on the S7 protein sphere (compare Figure 4(c)or (d) of the preceding paper (Mueller &Brimacombe, 1997) with Figure 3(c) and (d) here).Binding of the oligonucleotide probe (Alexanderet al., 1994) at the loop end of helix 18 must disruptthe pseudo-knot in this loop end (see Figure 1),thereby possibly giving the probe additional ¯exi-bility above the nominal 24 AÊ . The position of helix18 is discussed further in the accompanying paper(Mueller et al., 1997).

The actual shapes of the individual proteins willobviously in¯uence their ®nal locations relative tothe rRNA, and crystal or NMR structures are nowavailable for proteins S5 (Ramakrishnan & White,1992), S8 (Davies et al., 1996), S15 (Berglund et al.,1997) and S17 (Golden et al., 1993) from the 30 Ssubunit. Furthermore, speci®c sites within three ofthese protein structures have been identi®ed in anumber of contexts that are directly relevant to themodelling of the rRNA. Thus, RNA-protein cross-links have been identi®ed at the amino acid/nucleotide level with both S8 and S17 (Urlaub et al.,1997), and a protein-protein cross-link site betweenS5 and S8 was analysed at the amino acid levelsome time ago (Allen et al., 1979). Within S5 thereare sites causing resistance to antibiotics(Piepersberg et al., 1975) that can be correlatedwith corresponding antibiotic resistance sites onthe 16 S rRNA (discussed by Brimacombe, 1995).

Also with S5, an Fe(II)-EDTA probe has been at-tached at different amino acid positions in the pro-teins, and the resulting sites of hydroxyl radicalcleavage in the 16 S rRNA have been identi®ed(Heileck & Noller, 1996). The atomic structures ofS5, S8 and S17 have been incorporated into the ap-propriate regions of our rRNA model with thehelp of these data, and the results of this study willbe presented in due course.

In the preceding paper (Mueller &Brimacombe, 1997) we describe how the second-ary structure of the 16 S rRNA can be ®tted tothe 30 S EM contour, taking into account datadirectly related to the rRNA structure per se, suchas intra-RNA cross-links. Here, we show how theneutron map of the protein arrangement can be®tted to the same EM contour, and then be corre-lated with the rRNA model, via the various typesof RNA-protein interaction data. Since the 30 SEM contour was taken from an EM reconstruc-tion of 70 S ribosomes in an active con®guration(Stark et al., 1997), it follows that both the rRNAmodel and the neutron map are now physically``anchored'' in this 70 S structure. The next ques-tion to be considered is that of the ``functional''data; namely the cross-links and foot-prints tomRNA, tRNA, factors, antibiotics, etc. These in-teractions played a central role in the construc-tion of the 16 S rRNA model, and in the thirdpaper of this series (Mueller et al., 1997) weaddress the question of how well these functionaldata ®t into the completed 16 S rRNA model inthe context of the 70 S ribosome.

Materials and Methods

All of the relevant procedures are described in the pre-ceding paper (Mueller & Brimacombe, 1997).

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Edited by D. E. Draper

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

Note added in proof: A crystal structure is also available for protein S6 (Lindahl, M., Svensson, A.,Liljas, A., Sedelnikova, S. E., Eliseikina, A., Fomenkova, N. P., Nevskaya, N., Nikonov, S. V.,Garber, M. B., Muranova, T. A., Rykonova, A. I. & Amons, R. (1994). Crystal structure of the ribo-somal protein S6 from Thermus thermophilus. EMBO J. 13, 1249±1254).

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