doi:10.1006/jmbi.2000.4289 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 305, 167±177

Localization of the Protein L2 in the 50 S Subunit andthe 70 S E. coli Ribosome

Regine Willumeit1*, Stefan Forthmann1, JoÈ rn Beckmann1,2

Gundo Diedrich2, Ralf Ratering3, Heinrich B. Stuhrmann1

and Knud H. Nierhaus2

1GKSS ForschungszentrumGeesthacht GmbH, Institut fuÈ rWerkstofforschung WFS, Max-Planck-Straûe, D-21502Geesthacht, Germany2Max-Planck-Institut fuÈ rMolekulare Genetik, AGRibosomen, Ihnestraûe 73D-14195, Berlin, Germany3GMD ForschungszentrumInformationstechnik GmbHInstitut fuÈ r AngewandteInformationstechnikD-53754, Sankt AugustinGermany

E-mail address of correspondingwillumeit@gkss.de

Present addresses: G. Diedrich, HMedical Institute, Yale University S310 Cedar Street FMB421, New HavH. B. Stuhrmann, Institut de BiologLCM, 41 Avenue des Martyrs, F-381, France.

Abbreviations used: superscript Dand an H protonated; for example,deuterated 50 S subunit that containprotein L2.

0022-2836/01/010167±11 $35.00/0

The protein L2 is found in all ribosomes and is one of the best conservedproteins of this mega-dalton complex. The protein was localized withinboth the isolated 50 S subunit and the 70 S ribosome of the Escherichiacoli bacteria with the neutron-scattering technique of spin-contrast vari-ation. L2 is elongated, exposing one end of the protein to the surface ofthe intersubunit interface of the 50 S subunit. The protein changes its con-formation slightly when the 50 S subunit reassociates with the 30 S sub-unit to form a 70 S ribosome, becoming more elongated and movingapproximately 30 AÊ into the 50 S matrix. The results support a recentobservation that L2 is essential for the association of the ribosomal sub-units and might participate in the binding and translocation of thetRNAs.

# 2001 Academic Press

Keywords: protein biosynthesis; neutron scattering; ribosomal protein L2;spin-contrast variation

*Corresponding author

Introduction

In recent years remarkable progress has beenmade in the shape and ®ne structure of the ribo-some with the help of cryo-electron microscopy1,2

and breakthroughs in X-ray crystallography.3-6

Important functional centers have been localized:identi®cation of the decoding center on the smallribosomal subunit via the detection of the tRNAanticodon region on the ribosome at the A andP sites4,7-9 and the peptidyl transferase center byidentifying the CCA-end region of the tRNA in theP site.4,8

author:

oward Hugheschool of Medicine,en, CT 06510, USA;

ie Structurale, IBS-027 Grenoble Cedex

means deuterated,D50 S[HL2] is as a protonated

The peptidyl transferase center is the site of thecentral enzymatic activity of the ribosome. It isthere that the nascent peptide chain is prolongedby one amino acid via a peptide bond. The twocentral questions concerning this center are: (i)what is the molecular mechanism;10 and (ii) whichribosomal components are involved?

Both questions can now be discussed seriouslywith the help of the 2.4 AÊ crystallographic struc-ture of the 50 S subunit of Haloarcula maritima.11

When the crystals were soaked with a transition-state analog of the peptidly-transferase center thestructure of the large ribosomal subunit could beresolved to 3.2 AÊ . The region of the peptidyl trans-ferase center is free of ribosomal proteins within adistance of about 20 AÊ , suggesting that the ribo-some is a ribozyme.12 However, one should takeinto account that isolated 50 S subunits have aninactive peptidyl transferase center under con-ditions that allow signi®cant protein synthesis. The50 S subunits need either 30 % alcohol13 or theassociation with the small subunit. The latterrequires an activation energy of about 80 kJ/mol(Nils Burkhardt & K. H. N., unpublished results).Both activation procedures indicate that the iso-lated 50 S subunit has to undergo a conformational

# 2001 Academic Press

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change in order to activate the peptidyl transferase(PTF) center. Such a conformational change mightbring a protein into the center for activation. Fur-thermore, the crystal was grown at pH 5.8, atwhich even 70 S ribosomes are inactive in peptide-bond formation. Nevertheless, evidence has beenpresented that 23 S rRNA is essential for peptidyltransferase activity, although it has not beenshown that isolated 23 S rRNA is active in peptide-bond formation 14-17 (and references therein). Inaddition to the 23 S rRNA the ribosomal proteinsL2, L3 and L4 are candidates.18-21 Of the proteins,L2 is the most likely candidate, since it is founduniversally in all ribosomes and is one of the mostconserved proteins of the large subunit.22 L2 alsoseems to be an essential element of a bridgebetween the ribosomal subunits, and is importantfor tRNA binding.23

We determined the position of L2 within boththe 50 S subunit and the 70 S ribosomes bymeans of the neutron-scattering method proton-spin contrast-variation. This technique gives thebest signal-to-noise ratio of all of the small-angleneutron-scattering techniques. Small-angle neu-tron-scattering has been successfully employed todetermine the positions of ribosomal proteinswithin both subunits24,25 and also the location oftRNAs in pre and post-translocational states ofthe ribosome.26 A remarkable achievement wasthe localization of ``protein islands'' within the70 S ribosome.27 Here, we analyze two aspects.(i) Is the position of L2 within reach of thepeptidyl transferase center? (ii) Does the positionof L2 move upon association of the ribosomalsubunits? The results presented here indicatethat both answers are yes. Furthermore, theyprovide evidence that L2 plays an active roleduring the association process.

Figure 1. Protein content of particles and protein mixtureparticles. (b) L2 region of the DTP50-L2 preparation; no traceof HL2 before the reconstitution.

Results

Preparation of specifically labeled subunitsand ribosomes

Three ribosomal fractions have to be preparedfor the reconstitution of a deuterated 50 S particlecontaining a protonated L2: (i) a protonatedpuri®ed L2 (HL2); (ii) a deuterated total proteinfraction of the 50 S subunit lacking the protein L2(DTP50-L2;); and (iii) a deuterated rRNA fractioncontaining 23 S and 5 S rRNA.

Protonated L2 was isolated in a two-step pro-cedure from HTP5028 (see Materials and Methods),and DTP50-L2 was prepared from DTP50 similarly,but here the fractions not containing L2 were com-bined. Figure 1(b) demonstrates that the DTP50-L2fractions did not contain any traces of L2, whereasnormal amounts of L2 are seen after the additionof stoichiometric amounts of protonated L2 beforethe reconstitution (Figure 1(c)). The protein patternof the ®nally reconstituted particle D50 S[HL2] isshown in Figure 1(a): all proteins are present innormal amounts.

The deuterated rRNA fraction was isolated fromD70 S ribosomes, since the presence of 16 S rRNAdoes not in¯uence or impair the reconstitution of50 S subunits.29 Two advantages are seen in theuse of total rRNA from 70 S ribosomes for thereconstitution of 50 S subunits. (a) There is no needto isolate 50 S subunits, and (b) the 23 S rRNA iscompletely intact in contrast to isolated 50 S sub-units that can contain up to 30 % degraded 23 SrRNA, even under very careful isolation con-ditions. Three successive phenol extractions of pro-tonated 50 S subunits usually yield protein-free23S rRNA. To our surprise the same procedureyielded 23 S rRNA that contained signi®cantamounts of various L-proteins when the rRNA,was derived from deuterated 70 S ribosomes. Even

s by two-dimensional gel-electrophoresis.41 (a) D50S[HL2]s of L2 can be detected. (c) Same as (b) but after addition

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after six phenol extractions, up to 25 % of L2 wasobserved and similar amounts of 13 other deuter-ated proteins as shown by 2D gel electrophoresis.The proteins still present on the 23 S rRNA afterone phenol extraction are shown in Figure 2. Forthe reconstitution of the 50 S[DL2] particles, deuter-ated rRNA which was subjected to six phenolextractions was used. Therefore, the occupancy ofthe deuterated reconstituted particles, with proto-nated L2 is about 75 %. This has to be consideredin the processing of the spectra of the scatteredneutrons in a way that the remaining 25 %of the ribosomes are occupied with deuteratedL2 (� 25 % scattering contribution of unlabeledribosomes). Controls of the rRNA of the reconsti-tuted particles demonstrated that the 23 S rRNAwas intact to greater than 95 %, the activity inpoly(Phe) synthesis after complementing with pro-tonated 30 S subunits was about 68 %, a very goodvalue if one takes into consideration that nativedeuterated 50 S particles show a correspondingactivity of about 80 % as compared to protonatednative subunits (R. Lietzke & K. H. N.,unpublished results).

A portion of the preparation of D50 S[HL2]particles was associated with native D30 S andthe resulting D70 S[HL2] ribosomes puri®ed viazonal centrifugation (see Materials and Methods).Analysis of the rRNA content revealed about40 % intact 23 S rRNA corresponding to about

Figure 2. Protein content of DrRNA derived from D70 S afextracted 70 S rRNA were treated with 5 % (w/v) trichloroatate the proteins. These were resolved in an SDS-containingtrophoresis.41 Only L-proteins were detected, namely L2, L3,L2 content was estimated to be 40 % of that of 10 A260 unitamount was reduced to 25 %. This DrRNA was used for thefrom protonated ribosomes is completely devoid of proteins

40 % activity in poly(Phe) synthesis as comparedto deuterated 70 S ribosomes. It follows that theD70 S[HL2] ribosome preparation is inferior tothe corresponding D50 S[HL2] subunit prep-aration. The reason for the increased fragmenta-tion of the 23 S rRNA is the manipulationnecessary to form reassociated 70 S ribosomes;the manipulation comprises several incubationand centrifugation steps (see Materials andMethods). Nevertheless, we subjected the spectraof both particles to the data processing pro-cedure; the consistent results obtained justi®edthe same treatment of both 50 S subunits and70 S ribosomes (see the next section).

Localization and in situ structuredetermination of L2: some remarks onthe method

The aim of these investigations was to determinethe in situ position of protein L2 in the 50 S sub-units and 70 S ribosomes and to explore whetheror not structural changes occur in the protein dueto the reassociation of the subunits. The chemicalcomposition of the samples D50 S[HL2] andD70 S[HL2] used for analysis by nuclear spin-con-trast variation is summarized in Table 1. The scat-tering spectra were measured at the SANS1instrument at the GKSS research center. Spectrawere taken for the unpolarized target and with

ter one phenol extraction. A total of 10 A260 units of thecetic acid at 90 �C to hydrolyse the RNA and to precipi-sample buffer and subjected to two-dimensional gel elec-L4, L16, L18, L21, L22, L23, L25, L27/L28 and L30. The

s of native particles. After six phenol extractions the L2reconstitution of the D50 S[HL2] particles. rRNA derived

after three phenol extractions.

Table 1. Chemical composition of the samplesD50 S[HL2] and D70 S[L2] prepared for the nuclear spincontrast variation (all weights in mg)

Sample D50 S[HL2] D70 S[HL2]

Concentration 450 A260 478 A260

Ribosomes 11.46 12.552H2O 632.14 633.11H2O 1.27 1.272H-Glycerol 818.03 824.14H-Glycerol 10.77 10.86Cr(V) 13.17 12.65Imidazole, MgCl2,KCl2 (total) 18.94 18.33

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either parallel or antiparallel aligned proton spinsrelative to the spins of the incoming neutrons.The collected data were processed according tostandard corrections and deconvoluted into thethree basic scattering functions U(q)2, Re[U(q)V(q)]and V(q)2. These scattering functions were ®tted bytheoretical basic scattering functions obtained froman electron microscopy structure of the 50 S sub-unit and 70 S ribosomes30,31 into which possiblepositions of the L2 protein were inserted and thecoordinates of this position were changed. In a ®rststep, the radius of gyration RG and the coordinatesof the center of mass of the protein were deter-mined. For the calculation of the coordinates thesimplest case of a spherical shape for the proteinL2 was assumed. From the comparison of themeasured RG with a theoretical RG calculated fromthe mass of the protein one can deduce the overallmolecular dimensions and its state of elongation.When the in situ structure of the protein was mod-eled it was assumed that a chain of spheres coulddescribe the L2 structure. The spheres can overlap,be shifted linearly and vary in their size. The onlyrestriction applied was that the RG value of thisnew structure was of the same size as the RG deter-mined for L2. The simpli®ed description of a labelas a set of spheres is suf®cient, as it was shownfor model calculations of tRNA molecules in theribosome.26

L2 in the 50 S subunit

Assuming that L2 is a densely packed proteinsphere within the 50 S subunit a calculation of theradius of gyration yields the theoretical RG,theo

value of 16 AÊ . Calculations of the RG of the struc-ture determined for the RNA binding site of pro-tein L2, which is approximately half the protein L2(residues 60-19532), using the program CRYSOL(by D. Svergun & M. Kozin, EMBL) gave anRG,Crystl value of 14 AÊ . Our neutron-scatteringmeasurement resulted in a radius of gyrationRG,50 S � 22(�1) AÊ that indicates an elongated insitu structure of L2. In excellent agreement withthese data is the radius of gyration that we calcu-lated from the recently published crystallographicdata11, namely an RG,Crystl value of 22 AÊ .

The position of the center of mass was deter-mined. Polar coordinates (r, f, y) are used todescribe the location of the protonated L2 withinthe ribosomal matrix. The coordinates of the cen-ter of mass of the protonated label in eitherstate were varied stepwise over the total volumeof the ribosome and its near vicinity during the®tting procedure. The root-mean-square deviationR (see Materials and Methods) of the theoreticaland experimental basic scattering functions wascalculated. The parameter set with the smallestvalue of R represents the center of mass of L2within the ribosome in the state under consider-ation.

The mass center of gravity for L2 was found onthe side of the L1 stalk, parts of the protein beingexposed to the 50 S intersubunit interface(R-value � 2.259). The distance r of the protein L2from the center of mass of the ribosomal subunit,which is the origin of our coordinates system,was 79 AÊ , the angle values were j � 3.087 andy � 1.044. A comparison with the crystallographicstructure and position of the protein L2 at 2.4 AÊ

resolution11 showed that L2 indeed exposes half ofits structure on the surface of the intersubunitinterface but that our position is shifted byapproximately 50 AÊ towards L1.

Since the radius of gyration indicated anelongated L2 structure, this feature was taken intoaccount in additional ®tting routines. The R-valuewas improved by 4 % (R-value � 2.166) giving theposition r � 79 AÊ , f � 3.041 and y � 1.159. Theposition changed by only 10 AÊ , surprisingly to theresult. Figure 3(a) demonstrates the quality of the®t to the three characteristic functions.

The critical ®t parameters are the angles f and ywhich sometimes cannot be determined unambigu-ously. To test the stability of the results, i.e.whether there are local minima around the absol-ute minimum or whether one global minimumexists, all possible combination of r/f/y werecalculated and are presented in the in a f/y mapfor a distinct r-value. Figure 3(b) demonstratesadditional possible protein positions with R-valuesup to 5 % higher than optimal. Therefore, the pos-ition of the center of gravity of L2 seems to beunequivocal.

The modeling of the protein in an elongatedshape revealed that L2 has dimensions of41(�2) AÊ � 61(�2) AÊ (main axes of a prolateellipsoid of revolution). This is in good agreementwith the crystal structure of L211 where the dimen-sions are approximately 37 AÊ � 62 AÊ .

In Figure 3(c) and (d), the positions and theshape of L2 in the 50 S subunit as obtainedfrom a single-sphere modeling and an elongated-shape modeling are compared. It can be seenthat on the surface of the intersubunit interfacealmost half the mass of L2 is exposed, while aminor part extends into the ribosomal matrix.This corresponds well with the in situ structureof L2 from crystallography, where a compact L2part was found embedded in the surface of the

Figure 3. Neutron-scattering results from the D50 S[HL2] particles. (a) The basic scattering functions for the in situstructure determination of the protein L2 in the 50 S subunit. The elongated shape of the protein was taken intoaccount. The function Re[U(q)V(q)] was multiplied by (ÿ1) for a better display. The corresponding lines represent theoptimal ®t of the data. (b) Possible protein positions with R-values of up to 5 % higher than the best value of 2.259are taken into account. Red sphere, position corresponding to the best R-value (2.259); yellow spheres, positions with3 % higher R-values; green spheres, positions with 5 % higher R-values. Note that the 50 S subunit is rotated 45 �, sothat the central protuberance comes towards the viewer. (c) and (d) Positions of protein L2 in the 50 S subunit deter-mined with the assumption of a spherical shape (red sphere) or an elongated shape of the protein (green ellipsoid).Front view of the 50 S subunit in (c) and side view with the L1 protuberance in front in (d). The ribosome model isfrom Malhotra and co-workers.8 Due to perspective distortion in this 50 S view, L2 appears more spherical than it isin the model (ratio of the main axis of the corresponding ellipsoid 41/61).

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50 S subunit, while extended loops of the proteinintrude into the subunit. From the neutron-scat-tering data obtained, it was not possible todetermine unequivocally a single direction of the

elongated protein. In Table 2 coordinates of thecenter of mass of protein L2 with respect to the50 S subunit and the focal points of an ellipsoidare presented.

Table 2. Position of protein L2 determined by spin-contrast variation in the 50 S subunit and the 70 S ribosome

L2 in the 50 S subunit L2 in the 70 S ribosome

RG (AÊ ) 22 � 1 26 � 1Assumption: one sphereDistance to CoM (AÊ ) 79 � 5 55 � 5/31 � 5 a

Coordinates L2 CoM (X/Y/Z) 37 � 5/ÿ63 � 5/32 � 5 37 � 5/ÿ36 � 5/19 � 5Assumption: elongatedDistance to CoM (AÊ ) 79 � 5 58 � 2/34 � 2 a

Dimensions of L2 (AÊ ) (41 � 2) � (61 � 2) (33 � 1) � (68 � 1)Coordinates L2 33 � 5/ÿ53 � 5/29 � 5 40 � 5/ÿ72 � 5/34 � 5Ellipsoid (X/Y/Z) 36 � 5/ÿ54 � 5/20 � 5 39 � 5/ÿ19 � 5/18 � 5

The center of this coordinate system is the center of mass of the 50 S subunit.The original data analysis procedure took place in two different coordinate systems: The coordinates of protein L2 in the 50 S sub-

unit are calculated with respect to the ribosome structure described30, the coordinates of L2 in 70 S are calculated with respect to thestructure from.31 In this Table all 70 S coordinates are transferred into the 50 S coordinate system. In addition to a translational shiftof (21/ÿ25/ÿ10) AÊ the 70 S coordinate system had to be rotated. CoM, center of mass. Note that from the distance to CoM aloneone cannot calculate the shift of the protein position because the rotation of the different coordinate systems has to be taken intoaccount. Errors are standard deviations.

aDistance to the 70 S center of mass.

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L2 in the 70 S ribosome

The data of the sample for L2 within the 70 Sribosome were treated as described in the preced-ing section for the 50 S subunit. The ®nal resultsfor the data processing with the modeling of anelongated protein are shown for the basic scatter-ing functions in Figure 4(a). The radius of gyrationwas slightly larger than the that of L2 in the 50 Ssubunit, namely RG,70 S � 26(�1) AÊ resultingin a dimension of L2 in the 70 S ribosome of33(�2) AÊ � 66(�2) AÊ . The center of mass of theprotein L2 is 31 � 2 AÊ away from the center ofmass of the 70 S ribosome, viz. the origin of thecoordinate system.

A circumscribed region of the minima calculatedfrom r/f/y (with R-values deviating up to 3 %from the optimum) de®nes a narrow volumeelement of 10 AÊ � 46 AÊ � 54 AÊ . of possible pos-itions of the center of mass of L2 (Figure 4(b)).There are two reasons for the fact that the result isnot as de®ned as in the case of the 50 S subunit.(i) An almost spherical structure like the 70 S ribo-some makes it more dif®cult to identify an internalposition than the 50 S structure with its asym-metric features. (ii) The structure of the 70 S wasmore ``loose'' than that of the 50 S subunit, as indi-cated by the partially fragmented 23 S rRNAwithin the 70 S ribosome (see above). L2 was loca-lized near the intersubunit interface (Figure 5(a),(b) and (c)).

Discussion

The ribosomal protein L2 is essential for a num-ber of important ribosomal functions, such as theassociation of ribosomal subunits, tRNA bindingand peptide bond formation.23,16,33 Whether thehighly conserved His229 of L2 is involved in thechemical catalysis is still a matter of debate.23

By means of immuno-electron microscopy, twostudies have identi®ed slightly different sites of L2

on the intersubunit-surface of the 50 S subunitbetween the L1 and the central protuberances,possibly due to hitting different epitopes of L2.34,35

The location of these epitopes agrees with theelongated structure demonstrated here. The radiusof gyration of RG = 22(�1 AÊ ) is in excellent agree-ment with other neutron scattering investigations,which gave an RG value of 22(�2) AÊ ,36 and thetheoretical calculations for L2 within the recentlypublished complete crystallographic structure.11

However, we note a signi®cant difference in theposition of this protein, since the position found inneutron scattering does not coincide with that ofthe crystal structure, but rather touches the lowertip of the ellipsoid ®tted in our calculation of theprotein L2 in the 70 S ribosome.

The position of L2 within the 70 S ribosome wasfound at a similar place to that within the 50 S sub-unit, but the center of mass of the protein shiftedtowards the ribosomal subunit center of mass by30 AÊ . It is more exposed on the intersubunit sur-face in the 50 S and seems to move into the 50 Ssubunit upon association with the 30 S subunit(Figure 5(b)). This movement is signi®cantly largerthan that observed with proteins of the smallsubunit applying cryo-electron microscopy: TheS-proteins were found to move for 5 to 10 AÊ

from the surface into the ribosomal matrix uponassociation.1

Another striking feature is the change of theradius of gyration upon 70 S formation. The RG

value is 1.2 times higher in the 70 S than that inthe 50 S. Together with the already mentionedimportance for the association of the ribosomalsubunits the following scenario can be postulated.The exposed L2 on the isolated 50 S subunits facili-tates the docking of the 30 S subunit. If the speci®ccontacts have been established, then L2 moves intothe 50 S matrix thus tightening and stabilizing theassociation of the subunits. The observation thatthe protein is more elongated in the 70 S ribosomecan be explained by a conformational change of

Figure 4. L2 position in the 70 S ribosome. (a) The basic scattering functions for the in situ structure determinationof the protein L2 in the 70 S ribosome. The elongated shape of the protein was taken into account. The functionRe[U(q)V(q)] was multiplied by (ÿ1) for a better display. The lines represent the optimal ®t. (b) The ``minimum map''for positions of protein L2 in the 70 S ribosome. For three different distances between the mass centers of gravity ofthe ribosome and L2 the R-values obtained from the ®tting procedure are shown. The best R-value was 2.402; pos-itions with R-values up to 5 % larger are also shown. Most of the positions with R-values larger than the optimumare near the absolute minimum, indicating the unambiguous character of the ®nal result.

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the 50 S subunit upon association with the 30 Ssubunit, as suggested by the activation energyrequired for the association reaction (see above).

To our best knowledge this is the ®rst time thatthe position of L2 has been determined in the 70 Sribosome. The fact that L2 is found near to theintersubunit space ®ts the observation that L2 isessential for the association of the subunits and forthe binding of tRNA, in addition to its likely invol-vement in peptide bond formation.23 It is possiblethat L2 is a component of a family of intersubunitconnectors that belong to the bridge 2 family,according to the nomenclature of Frank and co-workers (bridge B2e).1

Materials and Methods

Deuterated cells of Escherichia coli strain MRE600rifwere purchased from CDN Ltd, Tallinn, Estonia(cdnest@hotmail.com), L-[U-14C]Phe from Amersham-Buchler, Braunschweig, chemicals for gel preparationfrom BioRad, Richmond, poly(U)mRNA from Boehrin-ger, Mannheim, Hepes from Calbiochem, Frankfurt, andall other chemicals including 2H2O and d8-glycerol werepurchased from Merck, Darmstadt.

Isolation of ribosomal components

The protonated E. coli MRE600rif cells were grown ina 100 l batch fermentation.37

Figure 5. Comparison of the positions of the protein L2 in the 50 S subunit and in the 70 S ribosome. (a) Frontview of the 50 S subunit. (b) Side view of the 50 S subunit with the L1 protuberance in front. (c) Side view of the70 S ribosome with the L1 protuberance in front; the 30 S subunit is in yellow. The ribosome model is from Malhotraand co-workers.8 the subunits in the 70 S model were identi®ed by the Frank group by cutting the 70 S ribosomethrough the ``bridges'' that connect both subunits.

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The isolation of deuterated and protonated crude 70 Sribosomes, ribosomal subunits, rRNA and total proteinsfrom the 50 S subunits followed published procedures.38

The TP50 were dialyzed overnight against Rec4-6Ubuffer containing 20 mM Hepes-KOH (pH 7.6 at 0 �C),4 mM MgAc2, 400 mM NH4Cl, 4 mM 2-mercaptoetha-nol, 0.2 mM EDTA and 6 M urea. After removing theurea via dialysis against Rec4 buffer (the same as Rec4-6U except urea) the proteins were shock-frozen in liquidnitrogen and stored at ÿ80 �C.

Since protein L2 and other proteins are bound tighterto deuterated rRNA than protonated rRNA, the standardprotocol for phenol extraction for rRNA38 was modi®edslightly. After three phenol extractions the water phasewas extracted twice with phenol/chloroform/isoamylalcohol, followed by a wash-step with chloroform/isoa-myl alcohol. The pellets of rRNA were lyophilized andresuspended in a buffer containing 20 mM Hepes-KOH(pH 7.6 at 0 �C) and 4 mM MgAc2.

During the preparation extreme care had to be takento prevent contamination from ribonucleases. Thereforeonly glass tubes baked at 180 �C for at least two hours orautoclaved plastic tubes and pipette tips were used and

all preparations including the column runs were per-formed at 4 �C.

L2 and the fraction TP50(-L2) were isolated and pre-pared as described.28 The protein fractions were storedin Rec4 buffer at ÿ80 �C.

Preparation of specifically protonated 50 S subunitsand 70 S ribosomes

For the preparation of 70 S ribosomes and the 50 Ssubunits the published procedure38 was modi®edslightly in order to increase the yield of active reconsti-tuted particles. The ®rst reconstitution step lasted onehour at 44 �C. The Mg2 � concentration was raised to20 mM and a second incubation step followed for fourhours at 50 �C.

The reconstituted D50 S[HL2] subunits were isolatedby a sucrose gradient centrifugation (22 hours,19,000 rpm, 10 %-40 % (w/v) sucrose, Beckman SW27rotor) followed by ultra-centrifugation (20 hours,35,000 rpm, Beckman 45Ti rotor). The pellet was resus-pended in a 2H2O buffer I100M20K400 containing 100 mMimidazole/2HCl (p2H 7.6 at 0 �C), 20 mM MgCl2 and400 mM KCl and extensively dialyzed against the same

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buffer to remove exchangeable protons from the sample.The imidazole, MgCl2 and KCl in the deuterated bufferwere lyophilized and dissolved in 2H2O three timesbefore being used in buffers, to get rid of exchangeableprotons or protonated crystal water.

For the preparation of D70S[HL2] the describedD50 S[HL2] sample was dialyzed against a buffer contain-ing 20 mM Hepes-KOH (pH 7.4), 20 mM MgCl2, 20 mMKCl and 4 mM 2-mercaptoethanol.39 D30 S were addedto the reconstituted particles in a 2: 1 molar ratio andincubated for one hour at 40 �C. The reassociatedD70 S[HL2] were puri®ed by means of a sucrose gradientcentrifugation (17 hours, 19,000 rpm, Beckman Ti15zonal rotor, 0 %-40 % sucrose) followed by ultra-centrifu-gation (27 hours, 20,000 rpm, Beckman 45Ti rotor). Thepellet was resuspended in a 2H2O buffer containing100 mM imidazole/2HCl (pH 7.6 at 0 �C), 20 mM MgCl2and 30 mM KCl. The resuspension was incubated forone hour at 40 �C to ensure reassociation of the subunitsafter centrifugation. Finally the ribosomes were dialyzedagainst I100M20K400.

Analysis of the rRNA, proteins, subunits and the70 S ribosomes

All preparation steps were monitored with differentanalytical methods to ensure the quality of the prep-aration. The particle homogeneity of the isolated sub-units obtained from the cells was tested with ananalytical sucrose gradient and the biological activitywas checked with an in vitro protein synthesis assay(poly(Phe) synthesis as described).40

The protein preparation was controlled by means of2D-polyacrylamide gel electrophoresis.41 The RNAintactness was tested by polyacrylamide gel electro-phoresis following a published protocol.42 The bio-logical activity of the ®nal D50 S[HL2]samples was68 % of that of the native 50 S subunit as tested inpoly([14C]Phe) synthesis after complementation withnative 30 S subunits.40

Polarization-dependent small-angleneutron-scattering

General principles

The scattering of thermal or cold neutrons by atoms ina sample is mainly due to the interaction between theincident neutron and the nucleus. The scattering is pro-portional to the scattering length b0, which varies withthe isotope and its spin state, the most prominentexample of which is the scattering lengths for the twohydrogen isotopes: 1H (proton), b0 � ÿ0.374 � 10ÿ12 cmand 2H (deuteron), b0 � � 0.667 � 10ÿ12 cm. The differ-ence in the scattering length density is called contrastwhich can be created by isotopic substitution (substi-tution of 1H by 2H) either in the solvent (exchange ofH2O by 2H2O) or, if possible, in the macromolecule itself.

Usually these isotopically labeled structures have theadvantage that their biological activity is not severelyin¯uenced and thus can be considered as nativeparticles. Ionization effects can be neglected and there-fore radiation damage is not a problem with neutronscattering.

The neutron as well as the hydrogen nucleus doeshave spins resulting in scattering lengths that are depen-dent on the degree of polarization. A polarized neutronbeam and a polarized 1H target have two possible rela-

tive spin orientations, viz. parallel ("") or antiparallel("#). The scattering lengths differ for these two possibili-ties: it is �1.082 � 10ÿ12 cm when the spins are parallel("") or ÿ1.83 � 10ÿ12 cm when they are antiparallel("#). Thus, the polarization-dependent scattering canvary over a wide range of different contrasts within onefrozen sample.

The other nuclei that are present in biological sampleseither show very small changes in the scattering lengthwith spin polarization, or are not affected by the polariz-ation process at all.

Measurement and data treatment

In order to perform the nuclear spin-contrastvariation, a frozen sample plate containing paramagneticimpurities is needed. We use the chromium(V)-EHBAcomplex (®nal concentration 0.85 %, v/w). A matrixd8-glycerol (®nal concentration 54 %, v/w) in 2H2O wasused. The sample preparation followed the proceduredescribed43 and the ®nal sample composition can befound in Table 1. The data were collected at the small-angle scattering instrument SANS1 at the research reac-tor FRG1, GKSS research center44 using the technique ofdynamic nuclear polarization (DNP).45,46

The measurements for D50 S[HL2], D70 S[HL2] and therespective solvents were performed as follows. Thewavelength l was 8.5 AÊ with �l/l � 10 %. The sample-detector distances were 0.7, 1.8 and 4.5 m, respectivelyresulting in a q-range from 0.01 to 0.25 AÊ ÿ1 (q is the scat-tering vector). All samples were measured in the unpo-larized state (PH � PD � 0, one-two days) and as protonspin targets (PH 6� 0, PD � 0, three-four days) with paral-lel or antiparallel orientations of neutron and hydrogenspins. The measuring time for a single measurement was1000 seconds resulting in about 320 measurements persample over the entire ®ve-day period of measurements.The proton polarization which could be obtained afterDNP were PH � ÿ 69 % for D50 S[HL2] and PH � ÿ 71 %for D70 S[HL2]. They remained stable during the datacollection. The solvent polarization varied from PH �ÿ 68 % to PH � ÿ 67 % and from PH � ÿ 80 % to PH �ÿ 74 % for the 50 S and the 70 S particles, respectively

The data treatment followed the procedure asdescribed47,48 resulting in a ®tting routine for the basicscattering functions U(q)2, Re[U(q)V(q)] and V(q)2 basedon electron microscopy models.45 For the 50 S subunit a40 AÊ resolution structure30 and information about theprotein distribution49, were taken into account. Themodel calculations for 70 S exploited the 25 AÊ electronmicroscopy structure.31 Even though higher resolutionribosomal structures are available, for example a 15 AÊ

resolution structure,8 a calculation of scattering curveswith the 15 AÊ structure did not give a better represen-tation of our measured ribosome data.

To determine the position of the label (e.g. of a pro-tein) the scattering contributions of the three parts of theribosome (RNA, TP and label) to the scattering ampli-tudes U and V are calculated. While the scattering contri-butions of the RNA and TP are known, the scatteringcontribution of the label consisting of position and struc-ture of the label is varied, giving different scatteringamplitudes for the label. From these scatteringamplitudes, the theoretical scattering intensity I(q)calculated

is calculated and compared with the experimental scat-tering intensity I(q)measured using a least-squares ®t rou-tine to minimize the parameter R:

176 Author to supply

R � 1

N

XN

i�1

�I�q�measured;i ÿ I�q�calculated;i�2si

( )1=2

�Minimum

for N � 40 intervals of q in 0.01 < q < 0.2 AÊ ÿ1 for all basicscattering functions. si is the standard deviation of themeasured data. When the position of the label issearched ®rst, the coordinates represented in polar coor-dinates R, y and are varied in a step-width of 1-5 AÊ and0.1-0.5 rad. When a minimum is reached the step-widthbecomes smaller, usually 0.1 AÊ and 0.01 rad. To checkwhether this is the best possible minimum and thereforean optimal position of the label with respect of the start-ing conditions, a minimum map is calculated. If thisminimum map shows a better R-value the correspondingcoordinates are used as starting parameters for the next®tting procedure. When a position is established and anelongated form can be deduced from RG, the label isassumed to be composed of more than one sphere. Inthe case of L2 the best ®tting procedure was with twoand three overlapping spheres for 50 S subunits and 70 Sribosomes, respectively.

Acknowledgments

We thank Roisin M. Owens for help and discussions.

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Edited by D. E. Draper

(Received 28 July 2000; received in revised form 25 October 2000; accepted 29 October 2000)