The Tetrameric Form of Ribosomal Protein L7/L12 from Escherichia coli*

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 16, Issue of June 5, pp. 9210-9214, 1989 Printed in U.S.A.

The Tetrameric Form of Ribosomal Protein L7/L12 from Escherichia coli* (Received for publication, January 11, 1989)

Yannis GeorgalisSS,Jan Dijkll, HaraldLabischinskill, and Peter R. Wills** From the Slnstitut fur Kristallographie, Freie Universitat Berlin, Takustrasse 6, 1000 Berlin 33, Federal Republic of Germany, Wylvius Laboratoria, Der Rijksuniversiteit Leiden, Wasenaarseweg 72, Postbus 9503, 2300 RA Leiden, the Netherlands, IIRobert-Koch-Institutdes Bundesgesundheitsamtes, Nordufer 20, 1000 Berlin 65, Federal Republic of Germany, and the **Department of Physics, University of Auckland, Private Bag, Auckland, New Zealand

A tetrameric form of the ribosomal protein L7/L12 neutral pH confirms the existence of the suspected tetrameric has been prepared and its structure studied by using species detected by Osterberg et al. (1976) and suggests that hydrodynamic methods, photon correlation spectros- the four copiesof this molecule in thelarge ribosomal subunit copy, and small angle x-rayscattering. The tetrameric form a single complexstructure. nature of the protein preparation is confirmed by three Studies of the dimer of L7/L12 have all indicated an elonindependent determinations of its molecular weight, gated conformation, whereas the C-terminal fragment is comwith analysis of accurate sedimentation equilibrium pact and globular, at least in the crystal (Leijonmarck et al., data giving the most reliable estimate. The species has 1980). This leaves onlythe 52 residues of the N-terminal part a Stokes radius of 4.0 2 0.1 nm and an absolute fric- to confer any elongated shape on themonomer. We haveused tional ratio of 1.7. Taken together, the hydrodynamic measurements suggest the possibility of a flat struc- physicochemical methods to study the tetramerof L7/112 in we propose amodel for the arrangeture, and this is consistent with the x-ray scattering solution, and on this basis results. The molecule has a radius of gyration of 3.6 f ment of idealized monomers consisting of a compact C terextended N terminusattached.While 0.05 nm and a maximum dimension of 11-12 nm. A minus withalong geometric model consisting of four elongated mono- caution must be exercised in assigning significance to the details of models whichare built up this way, clear distinctions mers, arranged in a plane, is proposed. can be made between grossly different structures. Our data indicate that theL7/L12 tetramer has a flat structure rather than being rod-like. The complex of the ribosomal proteins L7 and L12 has been very thoroughly studied because of its importance in ribosome function. In a recent search of the literature (see Moller and Maassen, 1986 and references cited therein), we have found more than 60 studies dealing with the complex or its individual components. Despite sucha wealth of information there still exists amajorcontroversy concerning the structural studies; namely several investigators still claim a dimeric form for the L7/L12 complex, whereas others suggest a tetrameric form. By combiningseveral hydrodynamic techniques, we present concrete evidence for the tetrameric form of the protein and establish a plausible model for the shape of the L7/L12 complex in solution. The Escherichia coli ribosome contains four copies of the proteins L7 and L12 that differ only with respect to acetylation at theN terminus (Subramanian, 1975). Preparations of L7/L12 have been studied using a variety of techniques revealing the presence of a dimer in solution (Wong and Paradies, 1974; Osterberg et al., 1976; Luer and Wong, 1979). In view of the observed localization of L7/L12 on the ribosome (Strycharz et al. 1978) and the existence of a complex (L8) containing four copies of L7/L12 and one copy of L10 (Petterson e t al. 1976), it is of interest to know whether L7/L12 can exist in solution asa complex with physiological stoichiometry. Our finding that a stable tetramer of L7/L12 dissociatesfromthe ribosome at high saltconcentrationsand * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by Grant ST2J-0034-1/F from the European Community.

EXPERIMENTALPROCEDURES

Protein Preparation-Protein L7/L12 was isolated from the 50 S subunit of E. coli MREGOO by a purification procedure that avoided use of urea and exposure to other denaturing agents. The proteins were extracted from the subunits with 2 M NaCl in the presence of 5 mM M e a t pH 7.5. The extract was fractionated by chromatography on CM-Sephadex C-25 in 0.01 M phosphate buffer using a linear NaCl gradient. Proteins L7/L12 and the L7/L12-L10 complex were not absorbed on the column and were recovered by adsorption to DEAE-Sephadex A-25 at low ionic strength followed by elution with 2.5 M NaCl in 0.02 M phosphate buffer at pH7.0. Further separation was achieved by chromatography on phenyl-Sepharose CL-4B using a NaCl gradient of 2.5-0.5 M in phosphate buffer at pH7.0. Proteins L7/L12 were concentrated by adsorption at low ionic strength to a small DEAE-Sephadex column followedby elution with a small volume of high ionic strength buffer. The protein preparation was stored in 0.35 M NaCl and 0.02 M phosphate at pH 7.0. During the entire purification procedure and during further experimentation, the protease inhibitors phenylmethanesulfonyl fluoride and benzamidine were present at a concentration of 0.1 mM. The purity of the protein preparation was checked by slab gel electrophoresis in the presence of sodium dodecyl sulfate (Laemmli and Favre, 1973) and by twodimensional gel electrophoresis (Kaltschmidt and Wittmann, 1970). Concentration Determination-Protein concentrations were determined by amino acid analysis and by differential refractometry. Triplicate samples containing norleucine as an internal standard were analyzed by using a Durrum D500 amino acid analyzer, and the fringe displacement in the interference optical system of a Beckman model E analytical ultracentrifuge due to the protein was measured simultaneously. We thus obtained the relation c = 0.2355, where c is the concentration in g/ll and J is the fringe displacement relative to buffer dialysate in a 12-mm path length ultracentrifuge cell. Buffer-All physical measurements were made at 20‘C with the protein in 0.02 M sodium phosphate buffer at pH 7.0 with 0.35 M

9210

The abbreviation used is: 1, liter.

Tetrameric Formof Ribosomal ProteinL7/L12from E. coli NaC1. The density po of this buffer is 1.0152 g/ml, and the viscosity is 1.046 X lo-' P. Densities-The densities of several protein solutions were measured by using a precision density meter (DMA 60 and DMA 601 M) at 20.0 f 0.1 "C. A value of 0.77 f 0.02 ml/g was obtained for the partial specific volume of the L7/L12 tetramer. This is close to the value of 0.745 ml/g calculated from the amino acid composition and used by other workers (Luer and Wong, 1979, Gudkov et al., 1980). Because of the uncertainty in our measurements, we used the latter value in our calculations. Sedimentation-A Beckman Model E analytical ultracentrifuge equipped with schlieren and interference optics was used to conduct sedimentation velocity and equilibrium experiments. All experiments were conducted at 20.0 f 0.1 "C. For the sedimentation velocity experiments, a capillary type synthetic boundary cell was used in an An-D rotor spinning at 5.2 X lo4 rpm, and a single peak, symmetric within detectable limits, was observed by using the schlieren optical system. Sedimentation coefficients were determined by the second moment method. Two equilibrium experiments were conducted by using interference optics to obesrve the concentration profile of the protein in a 12-mm path length cell. The latter experiments were conducted in an An-H rotor. In the first experiment a column height of 5.57 mm and an angular speed of 1.2 X IO4 rpm were employed, and patternsrecorded after 69 and 101 h were found to be coincident within experimental error. In the second experiment a column height of 3.72 mm and an angular speed of 1.0 X 10' rpm wereemployed. Patterns recorded after 45 and 60 h were coincidental. The loading concentrations were determined by using a synthetic boundary cell and were found to be 2.97 fringes (0.698 g/l) and 14.69 fringes (3.452 g/l) for the first and second experiment, respectively. Patterns were recorded throughout on metallographic plates and measured by using a Leitz travelling microscope. After subtraction of the cell deviation, the measured concentration was integrated with respect to the square of the radial distance (r), and knowledge of the loading concentration allowed application of the "hinge point refinement" (Howlett et al., 1972) to determine the fringe displacement relative to the buffer dialysate that has been placed in the reference channel of the centrifuge cell. It was estimated that there was a final absolute error of 0.03 g/l in the concentrations, and thus weighting factors of (c/0.03)' were used for each point in linear least squares fitting when the logarithm of the concentration (c) (in g/l) was the dependent variable. Apparent molecular weights were calculated from sedimentation equilibrium data by using the equation

Mapp= {2RT/w2(1 - ipo)(dnc/dP)

(1)

where R is the universal gas constant andT the absolute temperature. The dependence of Mappon c was investigated by finding the slope of the best fit straight line to groups of five consecutive data points (Georgalis, 1983). Photon Correlation Spectroscopy-Diffusion coefficients were determined by using photon correlation spectroscopy. This technique has been proved to be extremely useful for studying the Brownian dynamics of macromolecular solutions (Chu, 1974; Berne and Pecora, 1976). Protein solutions were filtered through Millipore filters (0.25pm pore size) to standard fluorescence cuvettes. The optical arrangement and instrumental specifications have been described elsewhere (Doster et al., 1980). The 514.5-nm laser line was used and the light scattered at 90"was detected. A Malvern 96 channel single-clip digital correlator has been used for building up the intensity autocorrelation functions. All experiments were conducted at 20.0 & 0.1 "C. Correction of the refractive index of the protein solutions relative to the buffer value of 1.3367 was made by using a measured value of 0.22 ml/g for the refractive index increment of the protein. The normalized intensity autocorrelation functions were analyzed by using a simple cumulant fitting (Koppel, 1972) since polydispersity was found to be minimal. The first cumulant, which is proportional to the z-average diffusion coefficient, could be determined with a precision better than 2% (Georgalis et al., 1987). From the free particle diffusion coefficient (DO), the Stokes radius (Rs) of the protein was calculated by using the Stokes-Einstein relation Do =(2) kT/(6~9&s) where k is Boltzmann's constant and tois the viscosity of the solvent. Viscosity Meosurements-Viscosities of protein solutions were

9211

measured by using an Ostwald-type microcapillary viscometer whose flow time for water at 20.0 & 0.1 "C was 361.45 -C 0.05 s. An automatic data collection system (AVS/G, Schott Glass Co. Mainz) was used. Gel Chromatography-A column 1.0 X 145 cmwas packed with Sephadex G-75 superfine and equilibrated with a buffer at a flow rate of 4 ml/h. The elution volume (V.) of each protein was determined from the maximum of the peak obtained by continuous monitoring of the 230 nm absorbance of the eluate. The void volume ( VO) of the column was determined by using ferritin and found to be 41.6 ml. The totalincluded volume (V,) was found to be 109.2 ml, determined conductimetrically by using a 3 M NaCl solution. The distribution coefficient (Kd)for each protein was calculated by using the equation K d

=

(v- vO)/(vt- VO)

(3)

and a calibration curve was obtained by plotting the inverse error function, e r r ' (1 - KJ,against Stoke's radius (Ackers 1970) using the following proteins (Stoke's radii and elution volumes are shown inparentheses):rabbit muscle aldolase (4.6 nm 43.2 ml), bovine serum albumin (3.6 nm 47.4 ml), ovalbumin (3.0 nm 54.4 ml), bovine erythrocyte carbonic anhydrase (2.4 nm 62.3 ml), and bovine pancreas chymotrypsinogen A (2.2 nm, 64.4 ml). Protein L7/L12 eluted a t 44.3 ml. X-ray Scattering-A highly stabilized x-ray generator (Seifert Debyeflex 1500) with a copper target tube (50 kV, 50 mA) was used as source, and the scattered radiation was detected by using a Kratky camera. The protein solutions were placed in Mark capillaries (1-mm diameter) and kept at 20.0 & 0.1 "C during irradiation by using a temperature-stabilized cuvette (Anton Paar KG, Graz). The scattered intensities were recorded by a scintillation counter with pulse-height discrimination set to receive the copper lines K, and KO.Scattering to 1 X lo-' rad were set by an angles ranging from 3 X electronically programmed step scanning device (EFG GmbH, Berlin). A t least lo5 pulses were taken at each angle in order to get the relative statistical error down to 0.3%.The absolute intensities were determined by using a Lupolen sample (Kratky et al., 1966) which had been previously calibrated a t theInstitut fur Physikalische Chemie, Graz. Examination of the statistical reliability of the data and background scatter were determined by using a computer program written by Zipper (1972). Corrections for collimation effects were made by using the conventional methods (Glatter, 1974) or the indirect Fourier transform method developed by Glatter (1977). The experimental data were compared with theoretical scattering curves for various models of uniform electron density using tabulated values for simple triaxial bodies and a computer program which handles models built up from a series of surfaces of second degree (Labischinski and Bradaczek, 1977). RESULTS AND DISCUSSION

In Fig. 1 the reduced data from one of two sedimentation equilibrium experiments are displayed. This logarithmic plot of the concentration against the square of the radial distance shows slight curvature in the low concentration range where the effects of a reversible dissociation reaction wouldbe evident; however, this deviation from linearity is well within the bounds set by the systematic error in the determination of the absolute fringe displacement at the meniscus. A weighted linear least squares fit to this set of data gave a molecular weight of (5.03 k 0.15) x lo4 g/molby using Equation 1. Differentiation of these data and data from the other sedimentation equilibrium experiment allowed calculation of the apparent molecular weight of the protein in the range 0-6 g/l. Over this entire concentration range there is no significant systematic change in the apparent molecular weight, as would be expected for a dissociating or self-associating system. A slight tendency to lower values of the apparent molecular weight at higher concentrations was observed, but this could beattributed to theminor effect of thermodynamic nonideality. The measured molecular weight is, within error, exactly four times the amino acid sequence molecular weight of the L7/L12 monomer (1.22 X lo4 g/mol) and demonstrates that our preparation consists of a stable tetramer. The diffusion coefficient of the protein was determined from the mean linewidth (r)of the normalized autocorrelation

Ribosomal Protein L71L1.2 from E. coli

Tetrameric Form of

9212

the concentration range 0.2-1.90 g/l, where accurate diffusion measurements are difficult, were used. This method gave a value of 4.2 f 0.2 nm for Rs, corroborating previous indica1.5 tions that the tetramer L7/L12 does not dissociate to any appreciable extent under physiological conditions. The Stoke's radius may be compared with the minimum 1.0 radius (R,,,in) of 2.4 nm calculated assuming the tetramer to be a rigid compact sphere comprised of four monomers of 0.4 ; 2 0.5 molecular weight of 12,200 g/mol. We thus find that the absolute frictional ratio is 1.7 and conclude that themolecule 0.0 is highly assymetric or heavily hydrated, or both. The possi0.2 bility of a compact spherical shapeis excluded. The sedimentation coefficient (s) of the protein was meas0.5 m b ured at a number of concentrations. Results areshown in Fig. 1 I I I I I I I] 44 45 46 47 48 49 50 51 2b. Extrapolation tozero concentration and correction for the r'lem'l the free buffer viscosity gave a value of 3.0 f 0.1 S for FIG.1. Logarithm of thefringe displacement J versus particlesedimentation coefficient in water a t 20 "C. This square of the radial distancer for sedimentation equilibrium provided for a further estimate of the molecular weight by of ribosomal protein L7/L12.The position of the meniscus (m) using the Svedberg equation and base ( b ) of the ultracentrifuge cell are indicatedas is the scale of M = RTs"/D" (1 - GPO) (5) proteinconcentration (c). Errorbarsare of the length twice the expected standard error in J , and the straight line was obtained by weighted linear least squares fitting. The loading concentration of The value obtained of (5.56 f 0.25) x lo4 g/mol is quite close protein was 0.689 g/l. A column height of 5.57 mm and an angular to four times the molecular weight of the L7/L12 monomer speed of 1.2 X lo4 rpm were employed. when possible sources of systematic error in the determination of Do and so are considered. Theconcentrationdependence of the reducedviscosity (~d of ) the L7/L12 tetramer is shown Fig. in 2c. Extrapolation to zero concentration gave a value of 8.5 f 0.2 ml/g for the intrinsic viscosity [q]. This value should be compared with that of 28.0 ml/g obtained for the dimer of L7/L12 (Wong and Paradies, 1974; Luer & Wong, 1979). Although our value is outside the range (3.0-4.0 ml/g) expected fornearly globular I I I 1 proteins (Tanford, 1961), the extreme elongationascribed to 0 10 20 30 L7/L12 dimer (Luer and Wong, 1979) is not evident in the tetramer. An estimate of the shape of a protein in solution can be achieved on the basis of the measurement of hydrodynamic parameters (Scheraga and Mandelkern, 1953). The dimensionless Scheraga-Mandelkern P-parameter increases steeply above the minimumvalue of 2.12 X lo6 for spheres in the case of prolate ellipsoids of increasing asymmetry, but for oblate ellipsoids it reaches a value less than2% above the minimum when the ratioof semiaxes is 40. Using the equation 2.0

-

3

--

4

p

q

l

f r

0

,

2

4

I

,

6

8

=N

DDqo[q]%M"/(RT)

(6)

( N is Avogadro's number) and the experimentally determined values of Do, [SI, and M , we find a value of (2.13 f 0.4) x lo6 for P. This means that a relatively symmetric shape for the

I I 10

c (g/L)

FIG.2. Diffusion coefficient ( D ) ,sedimentation coefficient and reducedviscosity ( q d ) as a function of concentration (c) for ribosomal protein L7/L12 in 0.02 M phosphate buffer (pH 7.0)with 0.35 M NaCl at 20 OC. Typical experimental error bars are indicated, as are best fit straight lines to each set of data. (8).

n L

function according to therelation D = r/q2

v

n

(4)

where q = (47rn/X) sin(0/2) is the scattering vector. Here X denotes the wavelength and 0 the scattering angle. Results are shown in Fig. 2a. Extrapolation tozero concentration and I correction for the buffer viscositygave a value of (5.40 f 0.07) X cm'/s for D ~ o ,the ~ , free particle diffusioncoefficient 0 2 4 6 8 1 0 1 12 2 in water a t 20 "C. This corresponds toa Stoke's radius of 4.0 f 0.1 nm. The Stokes radius was also determined by using a FIG.3. Distance distribution function derived from small calibrated gel chromatography column. Protein solutions in angle x-rayscattering data for ribosomal protein L7/L12.

Tetrameric Formof Ribosomal Protein L7/L12 from E. coli molecule cannot be excluded on the basis of hydrodynamic measurements, but, more important, if the molecule is highly assymetric, then it is likely to have a flat structure than an elongated form such as the dimer of Osterberg et al. (1976) and Luer and Wong (1979). Small angle x-ray scatteringstudies indicated thatthe scattering curves, which were obtained using two dilute solutions of the protein, were within experimental error coincident, once normalized with respect to concentration. The Guinier plots from these experiments reproducibly gave estimates of 3.64 f. 0.05 nm for the radius of gyration. The data from higher concentration experiments, which showed lower statistical error, were analyzed further, assuming that it gave an accurate representation of the form of the single-particle scatter. Elegant approaches are available for deducing distance distribution functions from x-ray data (Glatter, 1980). In the present work the distance distribution function p ( r ) was calculated by using the indirect Fourier transform algorithm of Glatter (1977). The plot of p ( r ) versus r is shown in Fig. 3 where it can be seen that the largest diameter ( d ) of the L7/ L12 tetramer is between 11 and 12 nm. Values of 13 (Wong & Paradies, 1974), 18 (Osterberg et al., 1976), 19 (Luer and Wong, 1979) and 12 nm (Behlke 1982b) have been suggested for models of the L7/L12. By use of the relation Ri

= lh

idp(r)rzdr/idp(r)dr

(7)

numerical integration gave a value of 3.64 nm for R,, confirming the result obtained from the Guinier plots. An estimate of the molecular weight can be obtained from the magnitude of the intensity (lo) at zero angle (Kratky et al., 1959). In this way we found a molecular weight of (5.1 & 0.1) X lo‘ g/mol, where the error has been calculated from uncertainties in lo, and the capillary thickness only. This result is in excellent agreement with that obtained from the sedimentation equilibrium experiments, but it is subject to large uncertainty because of possible small errors in the concentrationdeterminationand the valueof the partial specific volume used in the calculation. The volume (V) of the protein in solution can also be calculated from the scattering curve by using the relation (Porod, 1952)

9213

-OD

-0.5

-

I

- 1.0

0

CD

0

-

1.5

- 2.0

-

2.5

log(h.r)

FIG. 5. Small angle x-ray scattering curve for ribosomal protein L7/L12.Data were collected by using a solution of concentration 12.1 mg/ml. Experimental data are shown as open circles, and fits to the dataobtained using a circular disc model (dotted line)and the tetrameric model (solid line) shown in Fig. 4 are indicated. The scattering vector ( h ) is defined in terms of the scattering angle (20) and the wavelength of the x-rays by h = (47r/X) sine.

In small angle scattering studies of proteins in solution, it is usually assumed that the protein molecule can be modeled as a rigid bodyof uniform electron density. By comparing our experimental scattering curve with those calculated for a series of simple triaxial bodies of uniform scattering density, we were unable to obtain a satisfactory fit to the datafor any elliptical model. On the other hand, a circular disc (height, 1.3 nm; radius, 5.2 nm) gave a reasonable representation of the experimental data, except for small deviations in the outer portion of the scattering curve where the assumption of homogeneous electron density breaks down and theexperimenV = 2.9 X Zo/Q (nm3) (8) tal errors are large. The simple circular discmodel for the L7/L12 tetramer where Q is the “invariant” defined by gives a satisfactory representation of the x-ray scattering data and is certainly consistent with what can be deducedfrom the Q = [I(@) (2@)*d(20). (9) hydrodynamic measurements, but it is difficult to reconcile with the other available information about the structure of We thus found a volume of 106 f 12 nm3 that maybe L7/L12. We therefore adopted the strategy of building a very compared with the minimum value of 61 nm3 for a compact elementary geometric model of the monomer and investigatprotein consisting of four L7/L12 monomers. This result ing which of four arrangements were consistent with the suggests that theL7/L12 has a ratherhigh degree of internal available data. The C-terminal fragment of the monomer, hydration 6 of 0.55 g of water/g of protein. whose structure has been determined by using x-ray crystallography (Leijonmarck et al., 1980), was represented as a 3.5 nrn short cylinder (diameter, 2.0 nm; length, 3.5 nm). The remaining 52 residues of the N terminus were represented as an attached coaxial cylinder (diameter, 1.4 nm; length, 6.3 IZ.Onrn nm). This is a possibility that was suggested by the tertiary structure predictions of Gudkov et al. (1977). The scattering .g nrn curves were calculated for structures consisting of four of these monomers packed together in various parallel and an11.4 nrn tiparallel configurations, with all four in a single plane or with two dimers on top of one another. Only two arrangements 6.3 nrn gave a satisfactory fit to theexperimental data. In both models all four monomers were in the same plane, FIG. 4. Scale drawing of the geometric model of the L7/L12 tetramer that gave the best fit to the small angle x-ray data. the first consisting of all four parallel to one another and the

-

eo 11

Tetrameric Form of

9214

Ribosomal Protein L7/L12 from E. coli TABLEI

Physical parameters describing the tetrameric form of ribosomal protein L7/L12from E. coli Parameter

Symbol

Partial specific volume

iJ

Molecular weight

Value

Units

0.754 0.77 f 0.02 (5.03 f 0.15) X (5.56 f 0.25) X

lo4 lo4

5.1 X

lo4

Method

Amino acid composition densitometry Sedimentation equilibrium Svedberg equation (Equation 4) small angle x-ray

Diffusion coefficient

5.4 f 0.07

Photon correlation

Stokes radius

4.0 & 0.1 4.2 f 0.2

Stokes-Einstein gel chromatography

Sedimentation coefficient

3.0 f 0.1

Sedimentation velocity

Frictional ratio

Calculated from Rs, M,, and 6 Viscometry

1.7

Intrinsic viscosity Scheraga Mandelkern P-parameter

8.5 f 0.2 (2.13 f 0.04) X

Radius of gyration

3.63 k 0.05 3.64 f 0.05

Maximum dimension Molecular volume

11.5 f 0.5

lo4

Calculated from Do,M,, and [q] Guinier plot (12.1 g/l) Guinier plot (3.8 g/l) Distance distribution function Small angle x-ray

106 f 12

Degree of internal hydration

Calculated from V , R,i,,

0.55

other, shown in Fig. 4, consisting of an alternating parallelantiparallel arrangement. The latter modelgave a slightly better fit to theexperimental data compared with the former model, and both were superior to the circular disc model. A comparison between the circular disc and the parallel-antiparallel arrangement of monomers is shown in Fig. 5. The radius of gyration of the favored model is 3.68 nm and the maximum dimension 12.4 nm; thus, both are in good agreement with the experimental values for the L7/L12 tetramer. It should be mentioned that the latter value of R, is 10% lower than the corresponding value reported by Behlke (1982a and b). A summary of the parameters characterizing the L7/L12 tetramer is provided in Table I. In view of the mild conditions under which this species have been isolated, our results indicate that it is an intact andstable fragment of the ribosome. The L7/L12 dimer, which may be prepared by using a different extraction procedure (Luer and Wong, 1979), does not apparently reassociate to form a more compact tetramer. Acknowledgments-We thank Professor H.-G. Wittmann for support and P. T. M. Hughes for expert technical assistance. P. R. Wills and Y. G. thank Professor B. Hess and Dr. R. Jones for the opportunity to perform photon-correlation experiments in Max-PlanckInstitut fur System und Ernahrungsphysiologieat Dortmund. REFERENCES Ackers, G. K. (1970) Adu. Protein Chem. 2 4 , 243-246 Behlke, J. (1982a) Stud. Biophys. 84, 111-114 Behlke, J. (198213) Stud. Biophys. 9 2 , 119-122 Berne, B. J. & Pecora, R. (1976) Dynamic Light Scattering: With Applications to Chemistry, Biology, & Physics, John Wiley & Sons, New York Chu, B. (1974) Laser Light Scattering, Academic Press, New York Doster, W., Hess, B., Watters, D. & Maelicke, A. (1980) FEBS Lett. 113,312-314 Georgalis, Y. (1983) Physico-Chemical Studies on Ribosomal Proteins from E. coli, Ph.D. thesis, University of Athens

and d

Georgalis, Y., Ruf, H. & Grell, E. (1987) in Topics in Molecular Pharmacology (Burgen A. S. V., Roberts, G. C. K. & Anner, B. M., eds) pp. 1-20, Elsevier Science Publishing Co., Amsterdam Glatter, 0.(1974) J. Appl. Crystallogr. 7 , 147-153 Glatter, 0.(1977) J. Appl. Crystallogr. 1 0 , 415-421 Glatter, 0.(1980) Acta Phys. Austr. 5 2 , 243-256 Gudkov, A. T.,Tumanova, L. G., Gongadze, G. M. & Bushuev, V. N. (1980) FEES Lett. 1 0 9 , 34-38 Gudkov. A. T., Behlke.. J.,. Vtiurin, N. N. & Lim, V. I. (1977) FEBS Lett. 82, 125-129 Howlett. G. J. P. D. & Nichol. L. W. (1972) J. Phvs. Chem. 76,777783 Kaltschmidt, E. & Wittmann, H.-G. (1970) Anal. Biochem. 3 6 , 401402 Koppel, D. E. (1972) J. Chem. Phys. 5 7 , 4814-4820 Kratky, O., Porod, G. & Kahovec, I. (1959) Elektrochim. Acta 6 5 , 53-59 Kratkv. 0.. Pilz. I. R. & Schmitz. P. J. (1966) J. Colloid Interface Sci. 2 1,24-34 Labischinski, H. & Bradaczek, H. (1977) J. Appl. Crystallogr. 1 0 , 363-364 Laemmli, U. K. & Favre, M. (1973) J. Mol. Biol. 80,575-599 Leijonmarck, M., Eriksson, S. & Liljas, A. (1980) Nature 2 8 6 , 824826 Luer, C. A. & Wong, K.-P. (1979) Biochemistry 18,2019-2027 Moller W. & Maassen J. A. (1985) in Structure, Function and Genetics of the Ribosomes (Hardesty, B. & Kramer, G., eds) pp. 309-325, Springer-Verlag, New York Osterberg, R., Sjoberg, B., Liljas, A. & Pettersson, 1. (1976) FEBS Lett. 6 6 , 48-51 Petterson, I., Hardy, S. J. S. & Liljas, A. (1976) FEBS Lett. 6 4 , 135138 Porod, G. (1952) Kolloidn. Zh. 1 2 5 , 51-122 Scheraga, H. A. & Mandelkern, L. (1953) J. Am. Chem. SOC.75,179184 Strycharz, W. A., Nomura, M. & Lake, J. A. (1978) J . Mol. Biol. 1 2 6 , 123-140 Subramanian, A. R. (1975) J. Mol. Biol. 9 5 , 1-8 Tanford, C. (1961) Physical Chemistry of Macromolecules, John Wiley & Sons, New York Wong, K.-P. & Paradies, H. H. (1974) Biochem. Biophys. Res. Commun. 61,178-184 Zipper, P. (1972) Acta Phys. Austr. 3 6 , 27-28 '