Volume 3 no.1 2 December 1 976
Nucleic Acids Research
Nucleic
Acids
Research
Implications of electrostatic potentials on ribosomal proteins
J.S.Kliber, G.Hui Bon Hoa, P.Douzou, M.Graffe and M.Grunberg-Manago* Groupe U 128, INSERM BP 5051, 34033 Montpellier, France and Equipe 262, CNRS, Institut de Biologie Physico-Chimique, Paris 75005, France Received 26 October 1976
ABSTRACT Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developped by the 16S and 23S rRNA fractions. Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations. The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed
accordingly. INTRODUCTION The association equilibrium of ribosomes has been intensively studied in recent years as a function of Mg2+ concentration (1, 2, 3,4,) and the possible roles of the different forces eventually contributing to the interactions between the robosomal subunits have been discussed (2,5,6) but no definite answer has been so far obtained. It has been assumed that Mg2+ ions form ionic bridges between the RNA chains of the subunits (1,2) ; participation of peptide bonds as well as intervention of the sulfhydryl groups of the ribosomal proteins has been suggested for binding the subunits (6,7,8,9), while some observations were compatible with hydrogen bonds holding the subunits
together (5,10,11,12). However no precise molecular mechanism of the role of Mg2+ on ribosome association has been proposed. Thus we do not as yet fully comprehend the physico-chemical basis of the interplay of divalent and univalent cations on ribosomal equilibrium. Attempts have been made, by Walters and Van Os for instance (13), to link the association to a sufficient neutralization of the total charge of ribosomal subunits
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3423
Nucleic Acids Research allowing short-range attractive forces to operate; more recently, Wishnia et al. (14) endeavored to demonstrate that the primary effects of Mg2+ is to decrease the contribution of electrostatic repulsion to the free-energy of activation, the effect being possibly modulated by interactions of this cation. These modulations of the strongly negative, locally cylindrical ), developped by the polyphosphate backelectrostatic potential ( bone of RNA, should have important implications at the level of the RNA and (or) ribosomal proteins as a consequence of the polyelectrolyte theory. Thus RNA as a polyanion provides a microenvironment in which physical chemical properties, such as local pH and the pKs of any ionizing group behaving as a weak electrolyte, could be widely varied through modulations of the electrostatic potential. Consequently proton -exchanges can be expected between is modulated ribosomal subunits and their suspension medium when by changes in ionic strength. The experiments show that increase in divalent cation concentrations favoring the association of ribosomal subunits determines a large reduction of the net proton charge of ribosomal proteins, as a direct consequence of the decrease in the electrostatic potential developped by the RNA. These results validate the polyelectrolyte model, and along with the observation that reduction of the net proton charge by lowering temperature similarly shifts the association equilibrium, they show the ti3ht interdependence of ribosomal RNA and proteins in the subunit interaction and provide new informationsand experimental suggestions about the molecular and "phase" mechanisms involved. EXPERIMENTAL
Preparation of ribosomes. E. coli ribosomes were prepared by alumina grinding and then centrifuged at low and high speed to eliminate alumina and cell debris, according to the procedure of Dondon et al. (16, 17,18). The pellet obtained was then washed twice in high-concentration salt buffer 3424
Nucleic Acids Research yielding B-type ribosomes with which most experiments were performed. The crude ribosomes thus obtained were purified by zonal centrifugation in Mg-Ac2 , 5mM ; NH4 Cl, lOOmM ; and Tris-HCI (pH 7.5), 10 mM, yielding a ribosome preparation corresponding to what has been called the "A type" or "tight couples " (19). The 30S and 50S ribosomal subunits were prepcWed by dissociation of the purified 70S ribosomes and separated by zonal centrifugation as described by Dondon et al. (18). At 1 mM Mg the ribosomes correspond to a mixture of 30S and 50S and at lOmM Mg2+ to 70S ribosomes .
All the ribosomal particles were then suspended, in a final buffer which contained for the 70S ribosomes: Tris-HCI (pH 7.5), 1OmM; NH4 Cl, 60mM; Mg-Ac2 a 1OmM ; and j6-mercaptoethanol, 7 mM; for the 30S and 50S particles NH4CI and Mg-Ac2 were respectively raised to 100 mM and 20 mM and kept at -80°C. Before use, each ribosomal solution was reactivated by heating 15 min at 370C, then centfifuged 45 min at 12000 g at 50C to eliminate dust or precipitates, and kept at OOC no longer than 20 hours. The ribosome concentrations (1 O6 M to 4 x 10 6 M) were determined by measuring absorbancy at 260 nm (A260), using the following extinction coefficients: E70S = 4.15 x 107; C30S = 1.4 x TO0; 650S = 2.75 x 107.
Chemicals. Magnesium solutions were prepared in bidistilled water, free of C02, from Merck's cristallized MgCI2 ; the concentrations were measured by the Eriochrome Black T method (20). Potassium chloride and ammonium chloride solutions were prepared in the same way from Merck's and Prolabo's cristallized KCI and NH4CI. The titrants were NaOH and HCI obtained from Merck's Titrisol 1N. Defined sodium hydroxide solutions were standardized by potentiometric titration against standard Merck's HCI solutions. All solutions were freshly prepared before use. Potentiometric titrations. Titration curves and pH - stat measurements were carried out -3425
Nucleic Acids Research with Tacussel Instruments: automatic titrator TT 100 and pH-meter TT 200 ; the potentiometric recorder EPLIB type was equipped with a TV 11 GD module ; the autoburette used was the Electroburex model with digital read-out and 5ml syringe equipped with hermetically fitting PTFE plungers and stopcocks. The resolution of the reagent delivery was 1
,ul.
A semi -micro-combined electrode was used (Radiometer GK 2321C ). Radiometer standard buffers (pH 7.00 ; pH 4.01 ; and pH 9.18, at 250C) were used to calibrate the pH-meter. Titrants (NaOH solutions) were stored in Pyrex bottles equipped with a siphon and protected by traps containing "Ascarite", a solid Co2 absorbent from Arthur H. Thomas, Co, Philadelphia. Hermetic connexions between Pyrex bottles, syringe and Pyrex vial were ensured by using high pressure connectors and pipes developped for liquid chromatography "IC system". A 2 ml Pyrex vial containing a magnetic stirrer was used ; this vial was enclosed in a cylindrical glass jacket through which water was circulated from a constant-temperature bath. This jacket forms a Faraday cage around the titration vial, so that electrostatic effects on electrodes O.10C are negligible. The precision of the measurements obtained was for temperature and + 0.01 unit for pH. In experiments with alkaline solutions, the vial was continuously flushed with nitrogen previously washed in a NaOH solution and water. This is of critical importance in order to prevent some pH drift due to the eventual presence of atmospheric CO2. In the pH-stat experiments, the pH was kept constant during the stepwise addition of Mg2+ and K +by the delivery of a precise volume of the titrant (NaOH 5 x 10-4N) controlled by the TT 100 titrator;. stock soluions of 0.1 M MgCI2 and 4 M KCI were used. The ratio (H+ (Rb) was calculated from the determination of the volume of the titrant after each addition of Mg2+ or K+ . Dilution effect was taken into account by using a. programmable calculator Tektronic 31. The accuracy
3426
of proton release measurements as a function of Mg 2+ or K +
Nucleic Acids Research was 10 % and depends of the ribosomal preparations. Continuous titration curves were recorded in the presence of different Mg2 and K concentrations. The initial pH (arbitrarily chosen as pH 8.5) was adjusted by addition of concentrated NaOH. Ribosome solutions were titrated from pH 8.5 to pH 6 by addition of 10o2 N HCI ; al the curves were reversibly titrated from pH 6 to pH 8.5 by addition of 10i2 N NaOH, and were calculated by substracting the blank curve from the experimental curve. The relative position of the titration curves can be obtained from the results of the pH-stat experiments.
RESULTS Protons released from ribosomal particles titrated by the pH-stat
method. Protons released from 70S ribosomes upon addition of Mg2 at selected concentrations of K , as well as upon addition of K at selected concentrations of Mg (arrow in Fig.1) were titrated by determining the amount of NaOH necessary to restore the initial pH of unbuffered solution. Fig. 1 shows the curves obtained at 25°C + 0.1 °, pH 7 + 0.01 and expressed as ( H+ ) / (Rb) versus (Mg2+). They were obtained with B type ribosomal particles. The curves are quite different from the association curves of the same particles (see insert in Fig. 1 showing association curves of B type ribosomes at the same Mg concentration range, i.e. 1.5 to 8 mM). As can be seen when the concentration of K+ is increased, the release of protons upon addition of Mg2 is proportionally decreased and reciprocally: (Hi) / (Rb) = 68 at 1 mM (K ) (curve 1); 36 at 100 mM (K ) (curve 3) and Mg concentrations between 1.5 and 8 mM; 50 at 1.5 mM (Mg ) (curve 2') ; and 10 at 8 mM (Mg ) (curve 5') and K+ concentration between 1 and 100 mM. much more efficient than K+ for determining proton release : it can be seen, comparing curves 1 and 1' that the same number of protons (36) is obtained at 1 .5 mM (Mg ) by increasing (K ) from
Mg2+
is
3427
Nucleic Acids Research [H
16-2
2
-(12
1.%.f
_a
%#
K[LMg--JmM
Figure 1 Protons released by ribosomes upon addition of Mg2+ or K measured by pH-stat method at 25°C, pH 7. Ribosomal solutions (from 40 to 50 A260 / ml) contained 7.3 mM ,8 -mercaptoethanol, 3 mM and 0.5 mM Tris. Each experiment depends on Mg2+ 1 mM and K+ concentration. Curves 1, 2, 3, 4 were obtained at fixed potassium concentration and stepwise increases of magnesium. Arrows 1', 2', 3', 4' were obtained at fixed Mg2+ concentration: a = 1 .5 mM ; b = 8 mM and stepwise additions of K+ to final concentrations of: 1 mM= 1' 50 mM =2'; 100 mM =3' ;and 500 mM =4'. The insert is a log log plot of the 70S association equilibrium as a function of magnesium concentration determined by relative scattering measurement at pH 7, 250C, 50 mM cacodylate buffer, 50 mM NH4 , ribosome concentration 4.3 : A260 / ml. "Rb" stands for ribosomes in molar concentrations, and 260P" for phosphate.
NH+
3428
AcJ
Nucleic Acids Research
r~~~~~~~~~~~2 1 to 50 mM, and at 1 mM (K ) by increasing (Mg )
only from 1.5 to 3.3 mM. Such a drastic difference is obtained with all the curves of Fig. 1. With isolated 30S and 50S particles and with their stoichiometric mixture, at the same temperature and pH as above, in the presence of 50 mM K (Fig. 2), the shape of the curves obtained is similar to that of the curves in Fig. 1.
Moreover, it can be seen that each subunit released approximately the same number of protons, and twice this amount for the stoichiometric mixtures. Comparison of Curve 4 in Fig. 2 and Curve 2 in Fig. 1 shows that two different ribosomal preparations do release a similar number of protons under the same conditions of medium. Fg. 3 shows the curves obtained with 70S ribosomes at 40C + 0.1 OC (dashed lines) compared to the curves obtained under similar conditions, i.e. between 1 and 8 mM Mg2+, at 250C.
As can be seen, protons released per particle [(H +) / (Rb)] are fewer when temperature is dropped from 250C to 40C. At pH 7.0, the number of protons decreased from about 45 to 30 whereas the difference is only of 5 protons at pH 6.0 between 1 and 8 mM Mg2.
Titration curves. The titration measurements of isolated subunits, of their mixture and of 70S ribosomes were carried out between pH 6 and 8.5 where ribosomes are stable, at 1 and 10 mM Mg2+ (Fig. 4 and 5) and varying the KCI concentration from 1 to 500 mM. In the pH range chosen, the charged groups of 16S and 23S RNA do not titrate and, according to previous results of Walter and Van Os (13), the change in pH only alters the charge of the ribosomal proteins. For direct comparison between the different curves obtained, the origin was arbitrarily chosen at pH 8.5, in the absence of possible determination of iso-electric points. All these titration curves are fully reversible between pH 6.0 and pH 8.5. They cannot show of course any resolution of pKl . It can be seen that they shift towards acidic values upon addition of K+ and of Mg2+, a behaviour indicating a decrease in the plk of 3429
Nucleic Acids Research [HXRb]
Fiqure 2 Protons released by ribosomal subunits upon addition of Mg2+ (pH-stat method)at pH 7, 251C in the presence of 7.3 mM , --mercaptoethanol ; 5 mM NH4 ; 2 mM Ac ; 0.5 mM Tris ; 50 mM K Final sibunit concentration was 10-6 M. Curve 1 = 30S alone ; curve 2 = 50S alone ; curve 3 = curve 1 + curve 2 ; curve 4 = stoichiometric mixture of 30S and 50S. .
0H11
10
rII
' tL 4 *C
12
2
20
I
I I I I t
30
!t 25-C
40
5 11
50
v
60
1 1
8 70 6.5
7
Figure 3: Protons released by ribosomes upon addition of Mg2 (p Hmethodjat 40C and 25°C as a function of pH. Ribosomal solutions (from 40 to 50 A260 / ml) contained 7.3 mM 0 -mercaptoethanol, 3 mM NH4+, 1 mM Ac- , 0.5 mM-Tris and 50 mM K+. The origin of (H+)/(Rb) = 0 was obtained from samples containing 1 mM Mg2 at each
stat
temperature. 3430
Nucleic Acids Research
[H/ 1
EH!/[PI
C
10012001 .5 10
30014001-
500F
KCI 500mM
1o-0
[Mg -1 mM
6001700k
1.5 10-1
8.5
7
5.5
pH
Figure 4: Titration curves of a mixture of 30S and 50 S particles in the presence of 1 mM Mg2+ ; temperature 25°C + 0.1. The titration mixture contained: 7.3 mM # -mercaptoethanol ; 3 mM NH4+; 1 mM Ac-; 0.5 mM Tris and KCI as indicated. The dilution of ribosome solution (60 A260 / ml) was about 20% after the experiment.
(Ht
[HI/
bl
_tP]
C
100F
.
QSiO
200 .0.5101
3004001
KCI 50rmM
500-
KCI It
[Mgj z10mM
6001
10-1
7001-
1.5 7
la,
pH
Figure 5: Titration curves of 70S ribosomes in the presence of 10 mM Mg The otFer conditions were the some as in Fig.4.
2+ .
3431
Nucleic Acids Research ionizing groups ; moreover, the net proton charge decreases markedly under these conditions. It has also been shown that at concentrations of 500 mM K , the curves obtained in the presence of 1 mM and 10 mM Mg2+ almost coincide, indicating that the net proton charge, Z, does not vary any further.
Titration curves were then performed at two different temperatures (Fig.6). We checked once again that at 25°C as well as at 40C, the 70S particles (10 mM Mg ) were fully reversible between pH 6.0 and 8.5. As can be seen (Fig. 6): 1) there are less titrable groups at 40C than at 250C, and 2) the net proton charge recorded at 40C is less sensitive to the addition of Mg than what is observed at 250C Which agrees with the results reported in Fig.3. Both experiments clearly show a somewhat un expected temperature effect on the potentiometric behaviour of ribosomal particles.
pH
Figure 6 : Titration curves of ribosomes in the presence of 50 mM K+; 7.3 mM 13-mercaptoethanol ; 3 mM NH +; 1 mM Ac- ; 0.5 mM - Tris and two concentrations of Mg2+: 1 and 10 mi. Ribosomes , 60 A260 / ml , dilution about 20%. Tempe rature 40C and 250C. 3432
Nucleic Acids Research Association equilibria as a function of temperature. The magnesium ion concentration-dependence of ribosome association equilibrium investigated by the above technique has been examined (Insert, Fig.1). Additional studies of such an equilibrium at selected temperatures were carried out to check any possible variation in (Mg ) 1/2, as a test for the kind of bond involved between the subunits. The results obtained between 370C and 120C are shown in Fig. 7. It can be clearly seen that (Mg2+) 1/2 decreases as a function of 1/T, from 4 mM at 37°C to2.5mM atl12C. DISCUSSION K+) or (Mg 2+ ) in Figures 1 and Rbvru versus (K From the plots of HH+/ Rb 2, it can be seen that these uni- and divalent cations chase protons from the ribosomal subunits. At fixed potassium concentration (50 mM (K+) ),
40 protons were released by 70S particles when (Mg2+ ) increased from 1.5 to 8 mM. Protons can also be released at fixed (Mg ) and increasing (K+), a result indicating that the number of H +released by subunits solely depends on the initial and final states of both uni- and divalent cations. From these different observations, we assume that proton release obtained by addition of Mg2+ or K is the consequence of a decrease in the electrostatic potential value of ribosomes favoring the association to E
j'5
[70S] 50%
4 'o
2
10
25a
40 1
32 3
34
3.5
TEMPERATURES in C
is+; (xio')
Evolution of (Mg2+) 1/2 of the asociation equilibrium of riboFigure 7 somes as a function of temperature. The ribosomes used for this experiment were of the A-type. 3433
Nucleic Acids Research 70S particles. If we accept that the association equilibrium is controlled by changes in the electrical charge of ribosomal subunits, we may consider the respective effects of uni- and divalent cations on such a charge and on the equilibrium. M92 is much more efficient than K in the release of H (Fig.1) which confirms that Mg 2+ is involved in a stronger interaction with ribosomes ; this interaction has been called "condensation" to point out that these cations concentrate in the immediate vicinity of the polyanion charged groups rather than at a few specific binding sites (24). The present results are in good agreement with the conclusions of Wishnia et al. (14) according to whom "the primary effect of Mg is to decrease the contribution of electrostatic repulsion to the free energy of activation". These authors postulate that interactions of divalent cations such as Mg with ribosomal substructures possibly modulate this effect, a supposition that can be translated in terms of proton release and of the polyelectrolyte theory. According to this theory,. the strongly negative and locally cylindrical electrostatic potential developped by the polyanionic backbone of RNA at low ionic strength determines an abnormal concentration in pro+ + + e I tons ( (H )in = (H )out exp / kT, where (H )out is the proton concentration in the aqueous medium, £ the charge unit and + the electrostatic potential) ; moreover, the pK of the ionizing groups (in fact weak electrolytes) is increased and pK4, = pKq,=o - 0.43 £/ kT, where pK k=0 is the pK value when 4t becomes zero. Thus protons released upon addition of cations could come both from the immediate vicinity of phosphate groups and from changes in pKs at the level of ionizing groups of nucleic bases and (or) amino-acid residues on protein surface. Calculations using data reported by Wishnia et al. (14) indicate that about 10 proton per particle would be released upon addition of K+, whereas 40 protons presence off550 mM rtn MK,wees4 tepeec 1 .5 to 7 mM Mg 2+ iin the are released under these conditions (Curve 2 of Fig.1). Since the protons recorded do not come from the immediate vicinity of phosphate groups, one could postulate that all or part of them 3434
Nucleic Acids Research originate from protonated bases such as cytosine, as a result of the decrease of the electrostatic potential I lowering their pK. However results reported with synthetic polynucleotides (15) and showing that protons were released by cytosine were obtained at pH 5.0. At pH 7.0, the number of protonated bases in the RNA fractions should be negligible even at low ionic strength. Local pH values (termed PH in and linked to pH out in the polyelectrolyte theory by the relationship pH in = pH out + 0.43 P-*/kT) should be lower than 5.5 since the pK of cytosine is 5.2 at low ionic strength. While it is known that cytosine content of the 50S particles is approximately twice that of the 30S, both ribosomal subunits release approximately the same number of protons upon addition of Mg (8 mM) (Fig. 2), further indicating that the contribution of protonated bases is ne-
gligible. Moreover Choi and Carr (21) have shown that the amount of Mg binding to each separate 30S and 50S subunit is essentially identical ; this suggests that the electrostatic potential of these subunits is practically the same for a given Mg2 concentration, and would imply that approximately half the phosphate of the RNA of 50S subunits is "neutralized" at low Mg2 ( 1 mM ). This was already suggested by theoretical calculations by Walters and Van Os (22) using a uniformly charged sphere as a model for the subunit. Figures 4 and 5 showing H + titrations within the range of pH 6.0 to pH 8.5 indicate drastic changes in the net proton charge upon addition an Mgg2+ . Changing the pH in of increasing concentrations of both KK+ and the same range, at low ionic strength, Walters and Van Os (13) found that only the charge of the ribosomal proteins was altered, and showed that the contribution of this charge to the total charge is no more than 10 per cent as compared to the contribution of the RNA phosphate groups. These authors assumed that there are no ionized groups on the ribosomal RNA, except the primary phosphate groups which bear no H + within this pH range. Therefore only the charge of ribosomal proteins is altered by pH changes. This conclusion can be accepted without restriction since the contribution of bases is negligible. 3435
Nucleic Acids Research It is thus evident that some proteins titrate "abnormally" at low ionic strength, which agrees with the foreseeable implications of the lyelectrolyte theory, and that these proteins undergo profound changes in their net proton charge Z when the electrostatic potential is modulated by changes in ionic strength. Such changes might have important quences when protein surfaces derive their polar character from the ionized groups more than from the un-ionized groups. In this case changes in ionization usually produce large changes in protein geometry (volume and surface area), which might be related to the well-known changes in volume of the ribosomal particles during association. po-
conse-
Two additional results deserve special attention and discussion.
These are: 1) The decrease of the number of protons released when the temperature is lowered from 25°C to 40C (Figures 3 and 6), and 2) The decrease of (Mg )1/2 under these conditions (Figure 7). It is granted that the dissociation constants of strong electrolytes such as neutral sals RNA phosphate groups, acidic and basic residues titrating at extreme pH values, are unaltered by temperature changes in the pH range explored (6.0 - 8.0). The observed "retention" of protons when temperature is dropped would result from changes in the dissociation constants of ionizing groups of ribosomal proteins with pK values located in the interval of the pH's explored. Under these conditions, lowering the temperature increases the pKs of the ,
groups:
B
+
H+-
BH+
and
A
+
H
+
-
AH.
The fact that (Mg )1/2 is then markedly lowered underlines once more the importance of the electrical state of ribosomal proteins in the association equilibrium of the sub-paoiticles and clearly indicates that such a state can be modified both through direct effect on the ionizing groups and through indirect effect, via changes in the electrostatic potential. and AH groups Increase in pK values with the concomitant increase in BH might influence a number of protein - protein, protein - RNA, RNA - RNA interactions, in addition to the foreseeable effects on protein geometry and ,
+
subsequent phase variations. 3436
Nucleic Acids Research These preliminary findings show the validity of the polyelectrolyte model applied to ribosomal sub-particles and open new conceptual and experimental perspectives now under examination in these laboratories. ACKNOWLEDGEMENTS: The authors are most grateful to Dr.A. Wishnia for fruitful discussions. This work was supported by the Institut Natienal de la Sant6 et de la Recherche M6dicale ( U 128 ) and the Centre National de la Recherche Scientifique(6quipe 262 and G.R.18), the D6l6gation G6n6rale 6 la Recherche Scientifique et Technique (Convention N°74.7.0356), the Fondation pour la Recherche M6dicale Frangaise ; the Ligue Nationale Frongaise contre le Cancer; N.A.T.O. Research grant N0894.
*Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France REFERENCES (1) Petermann,M.L. (1964) The physical and chemical properties of ribo somes (Elsevier, Amsterdam) pp 108 (2) Goldberg, A. (1966) J. Mol. Biol. 15, 663-673. (3) Spirin, A.S., Sabo,B. and Kovalenko,V.A. (1971) Febs Letters 15, 197-200 (4) Spirin,A.S. (1972) Febs Symposium 23, 197-228 (5) Watson,J.D. (1964) Bull. Soc. Chim. Biol. 46, 1399-1425 (6) Tamaoki, T. and Miyazawa, F. (1967) J. Mol. Biol. 23, 35-46 (7) Morgan,R.S., Greenspan, C. and Cunningham, B. (1963) Biochim. Biophys. Acta 68, 642-644 (8) Zak,R., Nair, K.G. and Rabinowitz, M. (1966) Nature 210, 169-172 (9) Miyazawa,F. and Tamaoki,T. (1967) J. Mol. Biol. 24, 485-489 (10) Marcot-Queiroz, J. and Monier,R. (1965) J. Mol. Biol. 14, 490-505 (11) Moore,P.B. (1966) J.Mol.Biol. 22, 145-163 (12) Petermann, M.L. and Pavlovec,A. (1967) Biochemistry 6, 2950-2958 (13) Walters, J.A.L.I. and Van Os, G.A.J. (1970) Biochim. Biophys. Acta 199, 453-463 (14) Wishnia, A., Boussert,A., Graffe,M., Dessen,Ph. and GrunbergManago,M. (1975) J.Mol.Biol. 93, 499-515 (15) Aronssohn, G. and Travers,F. (1976) Nucleic Acids Research 3, 1373-1385. (16) Godefroy-Colburn, Th., Wolfe,A.D., Dondon,J., GrunbergManago,M., Dessen,Ph. and Pantaloni,D. (1975) J.Mol.Biol. 94, 461-478 3437
Nucleic Acids Research (17) Debey,P., Hui Bon Hoa, G. Douzou,P., Godefroy-Colburn,Th., Graffe,M., and Grunberg-Manago,M. (1975) Biochemistry 14, 1553-1559 (18) Dondon,J., Godefroy-Colburn,Th., Graffe,M., and GrunbergManago, M., (1974) Febs Letters 45, 82-87 (19) Noll,H., Noll,M., Hapke,B. and Van Dielen,G., (1973) in 24th Colloquium der Gesellschaft fOr Biologische Chimie 26-28 april in Mosbach/Baden, Springer Verlag, Heilelberg, pp. 257-311 (20) Harvey, A.E., Komarmy,J.M. and Wyatt,G.M. (1953) Anal. Chem. 25, 498-500 (21) Yong Sung Choi and Carr,C.W. (1967) J.Mol.Biol. 25, 331-345 (22) Walters,J.A.L.I. and Van Os,G.A.J. (1971) Biopolymers 10, 11, 20
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Nucleic
Acids
Research
Implications of electrostatic potentials on ribosomal proteins
J.S.Kliber, G.Hui Bon Hoa, P.Douzou, M.Graffe and M.Grunberg-Manago* Groupe U 128, INSERM BP 5051, 34033 Montpellier, France and Equipe 262, CNRS, Institut de Biologie Physico-Chimique, Paris 75005, France Received 26 October 1976
ABSTRACT Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developped by the 16S and 23S rRNA fractions. Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations. The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed
accordingly. INTRODUCTION The association equilibrium of ribosomes has been intensively studied in recent years as a function of Mg2+ concentration (1, 2, 3,4,) and the possible roles of the different forces eventually contributing to the interactions between the robosomal subunits have been discussed (2,5,6) but no definite answer has been so far obtained. It has been assumed that Mg2+ ions form ionic bridges between the RNA chains of the subunits (1,2) ; participation of peptide bonds as well as intervention of the sulfhydryl groups of the ribosomal proteins has been suggested for binding the subunits (6,7,8,9), while some observations were compatible with hydrogen bonds holding the subunits
together (5,10,11,12). However no precise molecular mechanism of the role of Mg2+ on ribosome association has been proposed. Thus we do not as yet fully comprehend the physico-chemical basis of the interplay of divalent and univalent cations on ribosomal equilibrium. Attempts have been made, by Walters and Van Os for instance (13), to link the association to a sufficient neutralization of the total charge of ribosomal subunits
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3423
Nucleic Acids Research allowing short-range attractive forces to operate; more recently, Wishnia et al. (14) endeavored to demonstrate that the primary effects of Mg2+ is to decrease the contribution of electrostatic repulsion to the free-energy of activation, the effect being possibly modulated by interactions of this cation. These modulations of the strongly negative, locally cylindrical ), developped by the polyphosphate backelectrostatic potential ( bone of RNA, should have important implications at the level of the RNA and (or) ribosomal proteins as a consequence of the polyelectrolyte theory. Thus RNA as a polyanion provides a microenvironment in which physical chemical properties, such as local pH and the pKs of any ionizing group behaving as a weak electrolyte, could be widely varied through modulations of the electrostatic potential. Consequently proton -exchanges can be expected between is modulated ribosomal subunits and their suspension medium when by changes in ionic strength. The experiments show that increase in divalent cation concentrations favoring the association of ribosomal subunits determines a large reduction of the net proton charge of ribosomal proteins, as a direct consequence of the decrease in the electrostatic potential developped by the RNA. These results validate the polyelectrolyte model, and along with the observation that reduction of the net proton charge by lowering temperature similarly shifts the association equilibrium, they show the ti3ht interdependence of ribosomal RNA and proteins in the subunit interaction and provide new informationsand experimental suggestions about the molecular and "phase" mechanisms involved. EXPERIMENTAL
Preparation of ribosomes. E. coli ribosomes were prepared by alumina grinding and then centrifuged at low and high speed to eliminate alumina and cell debris, according to the procedure of Dondon et al. (16, 17,18). The pellet obtained was then washed twice in high-concentration salt buffer 3424
Nucleic Acids Research yielding B-type ribosomes with which most experiments were performed. The crude ribosomes thus obtained were purified by zonal centrifugation in Mg-Ac2 , 5mM ; NH4 Cl, lOOmM ; and Tris-HCI (pH 7.5), 10 mM, yielding a ribosome preparation corresponding to what has been called the "A type" or "tight couples " (19). The 30S and 50S ribosomal subunits were prepcWed by dissociation of the purified 70S ribosomes and separated by zonal centrifugation as described by Dondon et al. (18). At 1 mM Mg the ribosomes correspond to a mixture of 30S and 50S and at lOmM Mg2+ to 70S ribosomes .
All the ribosomal particles were then suspended, in a final buffer which contained for the 70S ribosomes: Tris-HCI (pH 7.5), 1OmM; NH4 Cl, 60mM; Mg-Ac2 a 1OmM ; and j6-mercaptoethanol, 7 mM; for the 30S and 50S particles NH4CI and Mg-Ac2 were respectively raised to 100 mM and 20 mM and kept at -80°C. Before use, each ribosomal solution was reactivated by heating 15 min at 370C, then centfifuged 45 min at 12000 g at 50C to eliminate dust or precipitates, and kept at OOC no longer than 20 hours. The ribosome concentrations (1 O6 M to 4 x 10 6 M) were determined by measuring absorbancy at 260 nm (A260), using the following extinction coefficients: E70S = 4.15 x 107; C30S = 1.4 x TO0; 650S = 2.75 x 107.
Chemicals. Magnesium solutions were prepared in bidistilled water, free of C02, from Merck's cristallized MgCI2 ; the concentrations were measured by the Eriochrome Black T method (20). Potassium chloride and ammonium chloride solutions were prepared in the same way from Merck's and Prolabo's cristallized KCI and NH4CI. The titrants were NaOH and HCI obtained from Merck's Titrisol 1N. Defined sodium hydroxide solutions were standardized by potentiometric titration against standard Merck's HCI solutions. All solutions were freshly prepared before use. Potentiometric titrations. Titration curves and pH - stat measurements were carried out -3425
Nucleic Acids Research with Tacussel Instruments: automatic titrator TT 100 and pH-meter TT 200 ; the potentiometric recorder EPLIB type was equipped with a TV 11 GD module ; the autoburette used was the Electroburex model with digital read-out and 5ml syringe equipped with hermetically fitting PTFE plungers and stopcocks. The resolution of the reagent delivery was 1
,ul.
A semi -micro-combined electrode was used (Radiometer GK 2321C ). Radiometer standard buffers (pH 7.00 ; pH 4.01 ; and pH 9.18, at 250C) were used to calibrate the pH-meter. Titrants (NaOH solutions) were stored in Pyrex bottles equipped with a siphon and protected by traps containing "Ascarite", a solid Co2 absorbent from Arthur H. Thomas, Co, Philadelphia. Hermetic connexions between Pyrex bottles, syringe and Pyrex vial were ensured by using high pressure connectors and pipes developped for liquid chromatography "IC system". A 2 ml Pyrex vial containing a magnetic stirrer was used ; this vial was enclosed in a cylindrical glass jacket through which water was circulated from a constant-temperature bath. This jacket forms a Faraday cage around the titration vial, so that electrostatic effects on electrodes O.10C are negligible. The precision of the measurements obtained was for temperature and + 0.01 unit for pH. In experiments with alkaline solutions, the vial was continuously flushed with nitrogen previously washed in a NaOH solution and water. This is of critical importance in order to prevent some pH drift due to the eventual presence of atmospheric CO2. In the pH-stat experiments, the pH was kept constant during the stepwise addition of Mg2+ and K +by the delivery of a precise volume of the titrant (NaOH 5 x 10-4N) controlled by the TT 100 titrator;. stock soluions of 0.1 M MgCI2 and 4 M KCI were used. The ratio (H+ (Rb) was calculated from the determination of the volume of the titrant after each addition of Mg2+ or K+ . Dilution effect was taken into account by using a. programmable calculator Tektronic 31. The accuracy
3426
of proton release measurements as a function of Mg 2+ or K +
Nucleic Acids Research was 10 % and depends of the ribosomal preparations. Continuous titration curves were recorded in the presence of different Mg2 and K concentrations. The initial pH (arbitrarily chosen as pH 8.5) was adjusted by addition of concentrated NaOH. Ribosome solutions were titrated from pH 8.5 to pH 6 by addition of 10o2 N HCI ; al the curves were reversibly titrated from pH 6 to pH 8.5 by addition of 10i2 N NaOH, and were calculated by substracting the blank curve from the experimental curve. The relative position of the titration curves can be obtained from the results of the pH-stat experiments.
RESULTS Protons released from ribosomal particles titrated by the pH-stat
method. Protons released from 70S ribosomes upon addition of Mg2 at selected concentrations of K , as well as upon addition of K at selected concentrations of Mg (arrow in Fig.1) were titrated by determining the amount of NaOH necessary to restore the initial pH of unbuffered solution. Fig. 1 shows the curves obtained at 25°C + 0.1 °, pH 7 + 0.01 and expressed as ( H+ ) / (Rb) versus (Mg2+). They were obtained with B type ribosomal particles. The curves are quite different from the association curves of the same particles (see insert in Fig. 1 showing association curves of B type ribosomes at the same Mg concentration range, i.e. 1.5 to 8 mM). As can be seen when the concentration of K+ is increased, the release of protons upon addition of Mg2 is proportionally decreased and reciprocally: (Hi) / (Rb) = 68 at 1 mM (K ) (curve 1); 36 at 100 mM (K ) (curve 3) and Mg concentrations between 1.5 and 8 mM; 50 at 1.5 mM (Mg ) (curve 2') ; and 10 at 8 mM (Mg ) (curve 5') and K+ concentration between 1 and 100 mM. much more efficient than K+ for determining proton release : it can be seen, comparing curves 1 and 1' that the same number of protons (36) is obtained at 1 .5 mM (Mg ) by increasing (K ) from
Mg2+
is
3427
Nucleic Acids Research [H
16-2
2
-(12
1.%.f
_a
%#
K[LMg--JmM
Figure 1 Protons released by ribosomes upon addition of Mg2+ or K measured by pH-stat method at 25°C, pH 7. Ribosomal solutions (from 40 to 50 A260 / ml) contained 7.3 mM ,8 -mercaptoethanol, 3 mM and 0.5 mM Tris. Each experiment depends on Mg2+ 1 mM and K+ concentration. Curves 1, 2, 3, 4 were obtained at fixed potassium concentration and stepwise increases of magnesium. Arrows 1', 2', 3', 4' were obtained at fixed Mg2+ concentration: a = 1 .5 mM ; b = 8 mM and stepwise additions of K+ to final concentrations of: 1 mM= 1' 50 mM =2'; 100 mM =3' ;and 500 mM =4'. The insert is a log log plot of the 70S association equilibrium as a function of magnesium concentration determined by relative scattering measurement at pH 7, 250C, 50 mM cacodylate buffer, 50 mM NH4 , ribosome concentration 4.3 : A260 / ml. "Rb" stands for ribosomes in molar concentrations, and 260P" for phosphate.
NH+
3428
AcJ
Nucleic Acids Research
r~~~~~~~~~~~2 1 to 50 mM, and at 1 mM (K ) by increasing (Mg )
only from 1.5 to 3.3 mM. Such a drastic difference is obtained with all the curves of Fig. 1. With isolated 30S and 50S particles and with their stoichiometric mixture, at the same temperature and pH as above, in the presence of 50 mM K (Fig. 2), the shape of the curves obtained is similar to that of the curves in Fig. 1.
Moreover, it can be seen that each subunit released approximately the same number of protons, and twice this amount for the stoichiometric mixtures. Comparison of Curve 4 in Fig. 2 and Curve 2 in Fig. 1 shows that two different ribosomal preparations do release a similar number of protons under the same conditions of medium. Fg. 3 shows the curves obtained with 70S ribosomes at 40C + 0.1 OC (dashed lines) compared to the curves obtained under similar conditions, i.e. between 1 and 8 mM Mg2+, at 250C.
As can be seen, protons released per particle [(H +) / (Rb)] are fewer when temperature is dropped from 250C to 40C. At pH 7.0, the number of protons decreased from about 45 to 30 whereas the difference is only of 5 protons at pH 6.0 between 1 and 8 mM Mg2.
Titration curves. The titration measurements of isolated subunits, of their mixture and of 70S ribosomes were carried out between pH 6 and 8.5 where ribosomes are stable, at 1 and 10 mM Mg2+ (Fig. 4 and 5) and varying the KCI concentration from 1 to 500 mM. In the pH range chosen, the charged groups of 16S and 23S RNA do not titrate and, according to previous results of Walter and Van Os (13), the change in pH only alters the charge of the ribosomal proteins. For direct comparison between the different curves obtained, the origin was arbitrarily chosen at pH 8.5, in the absence of possible determination of iso-electric points. All these titration curves are fully reversible between pH 6.0 and pH 8.5. They cannot show of course any resolution of pKl . It can be seen that they shift towards acidic values upon addition of K+ and of Mg2+, a behaviour indicating a decrease in the plk of 3429
Nucleic Acids Research [HXRb]
Fiqure 2 Protons released by ribosomal subunits upon addition of Mg2+ (pH-stat method)at pH 7, 251C in the presence of 7.3 mM , --mercaptoethanol ; 5 mM NH4 ; 2 mM Ac ; 0.5 mM Tris ; 50 mM K Final sibunit concentration was 10-6 M. Curve 1 = 30S alone ; curve 2 = 50S alone ; curve 3 = curve 1 + curve 2 ; curve 4 = stoichiometric mixture of 30S and 50S. .
0H11
10
rII
' tL 4 *C
12
2
20
I
I I I I t
30
!t 25-C
40
5 11
50
v
60
1 1
8 70 6.5
7
Figure 3: Protons released by ribosomes upon addition of Mg2 (p Hmethodjat 40C and 25°C as a function of pH. Ribosomal solutions (from 40 to 50 A260 / ml) contained 7.3 mM 0 -mercaptoethanol, 3 mM NH4+, 1 mM Ac- , 0.5 mM-Tris and 50 mM K+. The origin of (H+)/(Rb) = 0 was obtained from samples containing 1 mM Mg2 at each
stat
temperature. 3430
Nucleic Acids Research
[H/ 1
EH!/[PI
C
10012001 .5 10
30014001-
500F
KCI 500mM
1o-0
[Mg -1 mM
6001700k
1.5 10-1
8.5
7
5.5
pH
Figure 4: Titration curves of a mixture of 30S and 50 S particles in the presence of 1 mM Mg2+ ; temperature 25°C + 0.1. The titration mixture contained: 7.3 mM # -mercaptoethanol ; 3 mM NH4+; 1 mM Ac-; 0.5 mM Tris and KCI as indicated. The dilution of ribosome solution (60 A260 / ml) was about 20% after the experiment.
(Ht
[HI/
bl
_tP]
C
100F
.
QSiO
200 .0.5101
3004001
KCI 50rmM
500-
KCI It
[Mgj z10mM
6001
10-1
7001-
1.5 7
la,
pH
Figure 5: Titration curves of 70S ribosomes in the presence of 10 mM Mg The otFer conditions were the some as in Fig.4.
2+ .
3431
Nucleic Acids Research ionizing groups ; moreover, the net proton charge decreases markedly under these conditions. It has also been shown that at concentrations of 500 mM K , the curves obtained in the presence of 1 mM and 10 mM Mg2+ almost coincide, indicating that the net proton charge, Z, does not vary any further.
Titration curves were then performed at two different temperatures (Fig.6). We checked once again that at 25°C as well as at 40C, the 70S particles (10 mM Mg ) were fully reversible between pH 6.0 and 8.5. As can be seen (Fig. 6): 1) there are less titrable groups at 40C than at 250C, and 2) the net proton charge recorded at 40C is less sensitive to the addition of Mg than what is observed at 250C Which agrees with the results reported in Fig.3. Both experiments clearly show a somewhat un expected temperature effect on the potentiometric behaviour of ribosomal particles.
pH
Figure 6 : Titration curves of ribosomes in the presence of 50 mM K+; 7.3 mM 13-mercaptoethanol ; 3 mM NH +; 1 mM Ac- ; 0.5 mM - Tris and two concentrations of Mg2+: 1 and 10 mi. Ribosomes , 60 A260 / ml , dilution about 20%. Tempe rature 40C and 250C. 3432
Nucleic Acids Research Association equilibria as a function of temperature. The magnesium ion concentration-dependence of ribosome association equilibrium investigated by the above technique has been examined (Insert, Fig.1). Additional studies of such an equilibrium at selected temperatures were carried out to check any possible variation in (Mg ) 1/2, as a test for the kind of bond involved between the subunits. The results obtained between 370C and 120C are shown in Fig. 7. It can be clearly seen that (Mg2+) 1/2 decreases as a function of 1/T, from 4 mM at 37°C to2.5mM atl12C. DISCUSSION K+) or (Mg 2+ ) in Figures 1 and Rbvru versus (K From the plots of HH+/ Rb 2, it can be seen that these uni- and divalent cations chase protons from the ribosomal subunits. At fixed potassium concentration (50 mM (K+) ),
40 protons were released by 70S particles when (Mg2+ ) increased from 1.5 to 8 mM. Protons can also be released at fixed (Mg ) and increasing (K+), a result indicating that the number of H +released by subunits solely depends on the initial and final states of both uni- and divalent cations. From these different observations, we assume that proton release obtained by addition of Mg2+ or K is the consequence of a decrease in the electrostatic potential value of ribosomes favoring the association to E
j'5
[70S] 50%
4 'o
2
10
25a
40 1
32 3
34
3.5
TEMPERATURES in C
is+; (xio')
Evolution of (Mg2+) 1/2 of the asociation equilibrium of riboFigure 7 somes as a function of temperature. The ribosomes used for this experiment were of the A-type. 3433
Nucleic Acids Research 70S particles. If we accept that the association equilibrium is controlled by changes in the electrical charge of ribosomal subunits, we may consider the respective effects of uni- and divalent cations on such a charge and on the equilibrium. M92 is much more efficient than K in the release of H (Fig.1) which confirms that Mg 2+ is involved in a stronger interaction with ribosomes ; this interaction has been called "condensation" to point out that these cations concentrate in the immediate vicinity of the polyanion charged groups rather than at a few specific binding sites (24). The present results are in good agreement with the conclusions of Wishnia et al. (14) according to whom "the primary effect of Mg is to decrease the contribution of electrostatic repulsion to the free energy of activation". These authors postulate that interactions of divalent cations such as Mg with ribosomal substructures possibly modulate this effect, a supposition that can be translated in terms of proton release and of the polyelectrolyte theory. According to this theory,. the strongly negative and locally cylindrical electrostatic potential developped by the polyanionic backbone of RNA at low ionic strength determines an abnormal concentration in pro+ + + e I tons ( (H )in = (H )out exp / kT, where (H )out is the proton concentration in the aqueous medium, £ the charge unit and + the electrostatic potential) ; moreover, the pK of the ionizing groups (in fact weak electrolytes) is increased and pK4, = pKq,=o - 0.43 £/ kT, where pK k=0 is the pK value when 4t becomes zero. Thus protons released upon addition of cations could come both from the immediate vicinity of phosphate groups and from changes in pKs at the level of ionizing groups of nucleic bases and (or) amino-acid residues on protein surface. Calculations using data reported by Wishnia et al. (14) indicate that about 10 proton per particle would be released upon addition of K+, whereas 40 protons presence off550 mM rtn MK,wees4 tepeec 1 .5 to 7 mM Mg 2+ iin the are released under these conditions (Curve 2 of Fig.1). Since the protons recorded do not come from the immediate vicinity of phosphate groups, one could postulate that all or part of them 3434
Nucleic Acids Research originate from protonated bases such as cytosine, as a result of the decrease of the electrostatic potential I lowering their pK. However results reported with synthetic polynucleotides (15) and showing that protons were released by cytosine were obtained at pH 5.0. At pH 7.0, the number of protonated bases in the RNA fractions should be negligible even at low ionic strength. Local pH values (termed PH in and linked to pH out in the polyelectrolyte theory by the relationship pH in = pH out + 0.43 P-*/kT) should be lower than 5.5 since the pK of cytosine is 5.2 at low ionic strength. While it is known that cytosine content of the 50S particles is approximately twice that of the 30S, both ribosomal subunits release approximately the same number of protons upon addition of Mg (8 mM) (Fig. 2), further indicating that the contribution of protonated bases is ne-
gligible. Moreover Choi and Carr (21) have shown that the amount of Mg binding to each separate 30S and 50S subunit is essentially identical ; this suggests that the electrostatic potential of these subunits is practically the same for a given Mg2 concentration, and would imply that approximately half the phosphate of the RNA of 50S subunits is "neutralized" at low Mg2 ( 1 mM ). This was already suggested by theoretical calculations by Walters and Van Os (22) using a uniformly charged sphere as a model for the subunit. Figures 4 and 5 showing H + titrations within the range of pH 6.0 to pH 8.5 indicate drastic changes in the net proton charge upon addition an Mgg2+ . Changing the pH in of increasing concentrations of both KK+ and the same range, at low ionic strength, Walters and Van Os (13) found that only the charge of the ribosomal proteins was altered, and showed that the contribution of this charge to the total charge is no more than 10 per cent as compared to the contribution of the RNA phosphate groups. These authors assumed that there are no ionized groups on the ribosomal RNA, except the primary phosphate groups which bear no H + within this pH range. Therefore only the charge of ribosomal proteins is altered by pH changes. This conclusion can be accepted without restriction since the contribution of bases is negligible. 3435
Nucleic Acids Research It is thus evident that some proteins titrate "abnormally" at low ionic strength, which agrees with the foreseeable implications of the lyelectrolyte theory, and that these proteins undergo profound changes in their net proton charge Z when the electrostatic potential is modulated by changes in ionic strength. Such changes might have important quences when protein surfaces derive their polar character from the ionized groups more than from the un-ionized groups. In this case changes in ionization usually produce large changes in protein geometry (volume and surface area), which might be related to the well-known changes in volume of the ribosomal particles during association. po-
conse-
Two additional results deserve special attention and discussion.
These are: 1) The decrease of the number of protons released when the temperature is lowered from 25°C to 40C (Figures 3 and 6), and 2) The decrease of (Mg )1/2 under these conditions (Figure 7). It is granted that the dissociation constants of strong electrolytes such as neutral sals RNA phosphate groups, acidic and basic residues titrating at extreme pH values, are unaltered by temperature changes in the pH range explored (6.0 - 8.0). The observed "retention" of protons when temperature is dropped would result from changes in the dissociation constants of ionizing groups of ribosomal proteins with pK values located in the interval of the pH's explored. Under these conditions, lowering the temperature increases the pKs of the ,
groups:
B
+
H+-
BH+
and
A
+
H
+
-
AH.
The fact that (Mg )1/2 is then markedly lowered underlines once more the importance of the electrical state of ribosomal proteins in the association equilibrium of the sub-paoiticles and clearly indicates that such a state can be modified both through direct effect on the ionizing groups and through indirect effect, via changes in the electrostatic potential. and AH groups Increase in pK values with the concomitant increase in BH might influence a number of protein - protein, protein - RNA, RNA - RNA interactions, in addition to the foreseeable effects on protein geometry and ,
+
subsequent phase variations. 3436
Nucleic Acids Research These preliminary findings show the validity of the polyelectrolyte model applied to ribosomal sub-particles and open new conceptual and experimental perspectives now under examination in these laboratories. ACKNOWLEDGEMENTS: The authors are most grateful to Dr.A. Wishnia for fruitful discussions. This work was supported by the Institut Natienal de la Sant6 et de la Recherche M6dicale ( U 128 ) and the Centre National de la Recherche Scientifique(6quipe 262 and G.R.18), the D6l6gation G6n6rale 6 la Recherche Scientifique et Technique (Convention N°74.7.0356), the Fondation pour la Recherche M6dicale Frangaise ; the Ligue Nationale Frongaise contre le Cancer; N.A.T.O. Research grant N0894.
*Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France REFERENCES (1) Petermann,M.L. (1964) The physical and chemical properties of ribo somes (Elsevier, Amsterdam) pp 108 (2) Goldberg, A. (1966) J. Mol. Biol. 15, 663-673. (3) Spirin, A.S., Sabo,B. and Kovalenko,V.A. (1971) Febs Letters 15, 197-200 (4) Spirin,A.S. (1972) Febs Symposium 23, 197-228 (5) Watson,J.D. (1964) Bull. Soc. Chim. Biol. 46, 1399-1425 (6) Tamaoki, T. and Miyazawa, F. (1967) J. Mol. Biol. 23, 35-46 (7) Morgan,R.S., Greenspan, C. and Cunningham, B. (1963) Biochim. Biophys. Acta 68, 642-644 (8) Zak,R., Nair, K.G. and Rabinowitz, M. (1966) Nature 210, 169-172 (9) Miyazawa,F. and Tamaoki,T. (1967) J. Mol. Biol. 24, 485-489 (10) Marcot-Queiroz, J. and Monier,R. (1965) J. Mol. Biol. 14, 490-505 (11) Moore,P.B. (1966) J.Mol.Biol. 22, 145-163 (12) Petermann, M.L. and Pavlovec,A. (1967) Biochemistry 6, 2950-2958 (13) Walters, J.A.L.I. and Van Os, G.A.J. (1970) Biochim. Biophys. Acta 199, 453-463 (14) Wishnia, A., Boussert,A., Graffe,M., Dessen,Ph. and GrunbergManago,M. (1975) J.Mol.Biol. 93, 499-515 (15) Aronssohn, G. and Travers,F. (1976) Nucleic Acids Research 3, 1373-1385. (16) Godefroy-Colburn, Th., Wolfe,A.D., Dondon,J., GrunbergManago,M., Dessen,Ph. and Pantaloni,D. (1975) J.Mol.Biol. 94, 461-478 3437
Nucleic Acids Research (17) Debey,P., Hui Bon Hoa, G. Douzou,P., Godefroy-Colburn,Th., Graffe,M., and Grunberg-Manago,M. (1975) Biochemistry 14, 1553-1559 (18) Dondon,J., Godefroy-Colburn,Th., Graffe,M., and GrunbergManago, M., (1974) Febs Letters 45, 82-87 (19) Noll,H., Noll,M., Hapke,B. and Van Dielen,G., (1973) in 24th Colloquium der Gesellschaft fOr Biologische Chimie 26-28 april in Mosbach/Baden, Springer Verlag, Heilelberg, pp. 257-311 (20) Harvey, A.E., Komarmy,J.M. and Wyatt,G.M. (1953) Anal. Chem. 25, 498-500 (21) Yong Sung Choi and Carr,C.W. (1967) J.Mol.Biol. 25, 331-345 (22) Walters,J.A.L.I. and Van Os,G.A.J. (1971) Biopolymers 10, 11, 20
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