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Structural dynamics of bacterial ribosomes
J. Mol. Biol. (1976) 105, 97-109
Structural Dynamics of Bacterial Ribosomes IV?. Classification of Ribosomes by Subunit Interaction BERN
Department Northwestern
HAPKE$
AND HANS NOLL§
of Biochemistry and Molecular Biology LTniuersity, Evanston, Ill. 60201, U.S.A.
(Received 1 March 1974, and
in revised
form
10 Novem,ber 1975)
Previous studies in this series (M. No11 et al., 1973a,b; No11 & Noll, 1974) have established that in Es&e&h&z coli the ability of subunits to form vacant 70 S condition for activity in the transribosome couples at 10 mM-Mg2+ is a stringent lation of natural messenger (R 17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into t’wo classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mM-Mgzf and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mM-Mg2+ ill t,he gradient, both species sediment as a 705 peak. At 10 mM-Mg2+ and 60,000 r(tvs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples. Association is dependent on the state of each subunit. Removal of Mg2+ produces 30 S b-particles that are unable to associate with 50 S subunits unless rtaconverted to the 30 S a-form by thermal a&ration according to Zamir et al. ( 1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a + 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with KNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association strp.
Fresh S-30 extracts cont’ain only tight couples (approx. 80%) and subunits (Hpprox. 200/,). Our results suggest, that loose couples are a,rtefact,w derived from tight couples by a structural or conformational modification. Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA,,, (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization. are shown to consist of the b-form of each subunit. It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due t,o the lack of structural criteria required for standardizing the ribosome preparation used by different irlvestigators. This study provides simple methods and criteria to classify and sc,parate physically all ribosome and ribosome subunits that ha.ve been observed itlt,o well-defined classes of predictable act,ivity. t Paper III in this series is No11 & No11 (1974). 1 Present, address: Nuclear Div., 3M Center, St. Paul, &linn. 55101, U.S.A. S;To whom reprint requests should be addressed. 7
97
!)S
Ii.
HAPICE
r\,h;I)
H.
NOLI,
1. Introduction Experimental studies on the mechanism of protein synthesis are limited by the quality of the ribosome preparations in which only a fraction, I to SOS/,, are active. Recent work has shown that it is possible to prepare ribosomes all of which can be converted into initiation complexes with natural messenger (H. No11 et al., 1973 ; M. No11 et al., 1973a,b; No11 & Noll, 1972, 1974). A necessary condition for this activity is the ability of the subunits to associate in the absence of messenger RNA, transfer RNA and other factors; particles that cannot do so are invariably inactive in the translation of natural messengers, although they may be active with poly(U) (H. No11 et al., 1973). When vacant ribosomes prepared by common methods are analyzed on sucrose gradients at high resolution and Mg2+ concentrations above 5 mM, the patterns observed vary greatly with rotor speed, Mg2+ concentration and preparation of ribosomes. Thus, a number of variable peaks and shoulders between the 50 S and 70 S position appear as well as great shifts in the ratio of subunits to the faster sedimenting components. We found that all these observations can be explained by the occurrence in such preparations of two classes of ribosomes, tight and loose couples, whose 50 8 subunits differ greatly in their affinity for the 30 S partner in the physiological range It will be shown that much higher Mg2+ concentrations are of Mg2 + concentrations. required to keep the loose couples together and to protect them from dissociation by hydrostatic pressure in the ultracentrifuge (Herzog et al., 1971; Spirin, 1971; Noll, 1971; Van Diggelen et al., 1971a; Subramanian & Davis, 1971; M. No11 et al., 1973a) than in the case of tight couples. Moreover, evidence will be presented that loose couples originate from biologically intact tight couples by some form of denaturation involving the 50 S subunit. Published studies show dissociation curves (extent of dissociation versus Mg2+ concentration) that correspond to (1) tight couples, (2) loose couples or (3) a mixture of t’he two species. The curves for (1) and (2) are homogeneous and show that at 37°C tight couples dissociate in a narrow range between 1.5 and 2.5 mM-Mg2+ (Noll. 1972; H. No11 et al. 1973; No11 & Noll, 1976), while loose couples dissociate within 5 to 15 mM-Mg2+ (Spirin et al., 1971; Zitomer & Flaks, 1972). Curves corresponding t,o (3) are biphasic and span the range of both tight and loose couples (Van Duin et al.. 1970) with an inflection point somewhere in between (Spirin et aZ., 1971). Although tight couples were not recognized as a separate class, it had been noted during the earlier dissociation studies t’hat unwashed ribosomes that had never been required a higher Mg2+ concentration for exposed to low Mg2+ concentration reassociation than for dissociation, and that this hysteresis disappeared during subsequent cycles of association and dissociation (Spirin et al., 1971; Zitomer & Flaks, 1972). The present study implies that these authors observed the irreversible conversion of tight into loose couples. Bosch and co-workers, on the other hand, reported that couples formed from subunits fall into two classes characterized by different susceptibility to high hydrostatic pressure (Van Diggelen et al., 1971a,b,1973; Van Diggelen Q Bosch, 1973). According to their studies, active 30 S subunits regardless of origin associated with “native” 50 S subunits to form couples that, under conditions of high hydrostatic more slowly than similar couples obtained with “derived” pressure, sedimented 50 S subunits. The difference in stability was attributed to an intrinsic difference in the nature of the native 50 S particles present in the free state as opposed to 50 S
STRUCTURAL
DYNAMICS
OF BSCTERIAL
RIBOSOMES.
99
IT’
part’icles derived from couples by dissociation at low Mg2+ concentrations. The obvious question of how this view could be reconciled with the concept of a dynamic equilibrium between subunits and couples was not answered, nor was the possibility considered that one form was derived from the other by denaturation or other structural modifications. Neither was the relationship clarified between these two classes differing in their sensitivity to dissociation by pressure and the t,wo classes with different stabilities with respect t’o Mg2+. The following experiments were performed to answer these questions.
2. Materials and Methods Most of the methods employed in these experiment,s paper I of this series (M. No11 et al., 1973a).
have
been described
in detail
in
(a) Reagents Mg2 + is always added together with EDTA in a molar ratio of Mg2 + : EDTA of 20 : 1, as recommended by Staehelin & Maglott (1971). Thus, if the Mg2+ concentration is given as 10 mM, we mean the efSective concentration, the actual concentration being 10.5 mMMg2+ plus 0.5 mM-EDTA. TMND buffer (Staehelin & Maglott, 1971) is 160 mM-ammonium chloride, 10 mM-magnesium acetate, 50 mM-Tris*HCl (pH 7.5), 10 mM-2-mercaptoethanol. Buffer G is the same as TMND buffer, except that the ammonium chloride is 60 mM and Tris.HCl is 20 mM. Buffer H is the same as buffer G, rxcept that t,he ammonium chlori& is 100 mM. (b) Preparation.
of ribosomes
and mbunits
Ribosomes and ribosome subunits were prepared by the ammonium sulfate precipitation procedure of Kurland (1966). Pure tight vacant (aA) couples were prepared as follows. The S-30 extract from freshIS ground cells was centrifuged in a B-29 zonal rotor at> 6 mM-Mg2+ as described previously (M. No11 et al., 1973a). The fract,ions corresponding to the 70 S peak were pooled and the ribosomes centrifuged into a pellet (M. No11 et al. 1973a). The pellets were dissolved in 2 ml of 50 S subunits, 1000 A,,, units of the concentrated suspension in buffer G werfb diluted to a total volume of 20 ml with buffer F (same as buffer H except that the Mg2 ’ is 15 mM) and subjected to a second purification step in the B-29 zonal rotor. The prorcdure was the same as in the first purification, except that the Mg2 + concentration in the gradient was raised to 15 mM and centrifugation was for 18 h at 25.000 rers/min (Nell, 1972; No11 & No11 1976). 50 8 b-subunits were isolated as follows : 550 A,,, units of 30 S subunits in 10 ml bnf’el G were incubated at 37°C for 60 min and added to 550 A,,, units of 50 S subunit,s in 40 ml TMND buffer, and the mixture was kept at 0°C for 5 min. The solution was layered ovel a 1300-ml convex exponential sucrose gradient (c,t = 0.292 M, CR = 1.17 M, I’, = 500 ml) in buffer I (same as buffer H except that the Mg2 + concentration is 6 mM) and centrifuged in a B-29 IEC rotor for 18 h at 25,000 revs/min and 8°C. Fractions containing the 50 S subunits were combined and the ribosomes were centrifuged in the IEC A-170 rot,or for 8 h at 40,000 revs/min. Each pellet was dissolved in 0.5 ml buffer G and the solutions were st,ored in small portions at -60°C. The total yield of 50 S subunits was 360 A,,, units. Deactivation and activation of 30 S subunits was carried nut as described hy Znrnir et al. (1971). (c) Analytical The standard isokinetic sucrose in series of 6x 3.6 ml with the aid 1969). The isokinetic parameters for The parameters for ribosomal RNA t C,, sucrose concentration
method8
gradients (Noll, 1967) were prepared Simultaneously. of an automatic machine (McCarty et al., 1968; Noll, ribosomes have been published (M. No11 et al., 1973a). were as follows: C,t = 0.438 M, CR = 1.31 M, V, =
at top of gradient;
C,, reservoir
concentration;
V,, mixing
volumc~.
100
B.
HAPKE
ANI)
H.
NOLI,
5.3 ml. Tlre sucrose solutions w(‘r(’ msdo up iti 100 Irl~l-arlllllollillf~~ c*lrloride, 20 tnslTris .HCl (pH 7.5), 1 mM-magnesium actstat~e. Cent rifugat,ion was for :I 11 at 60,000 r(:vsl’ min and 12 to 15°C for ribosoma,l RNA. For ribosomes tile Mg2 + collcrntration irl t II{, gradients as well as the speed alld duration of crntrifllgat,ior> is givc,ll ill t/x, legends t,o tllcs Figures. The scanning of the gradients for bobh absorbancy and radioactivity was carried otlt with the automated MICO MR-747 radiograd system (Molecular instruments Co., P.O. Box 1652, Evanston, Ill. 60201, U.S.,4.). The system consists of a turbulence-free flowcell capable of detecting peaks corresponding to 20 ng RNA, and a programmable automatic fraction collector that collects fractions into scintillation vials with simultaneous addition of scintillation fluid (Noll, 1971). The optical scan of the gradients at 260 nm was recorded conventionally on a strip chart and, at the same time, in digitalized form on magnetic tape with the aid of a Hewlett-Packard model 9820A digital calculator, model 3480A digital voltmeter and model 9865A cassette memory. The radioactivity data obtained from the liquid scintillation counter were recorded on the tape cassettes separately by manual entry. For the final display, the dat’a. were combined by thcl comprlt,txr and recorded by the model 9060A plottcxr in the size drsirracl for rrprotlnc+io,r.
3. Results (a) Characterization
of tight and loose couples by Mg” +-dependence of subunit association
(> 10 miw) contain Crude extracts prepared rapidly at high Mg2 + concentrations only tight couples (80 to 95%) and free subunits. When such preparations are analyzed on sucrose gradients at low centrifugation speeds, all the couples are stable at 6 mM-Mg2 + and no significant increase in the proportion of couples is observed upon
70 I
50 I
30 I
Sedimentation value (S) 70 50 30 I I I
70 !50 30 I I
:
Effluent
(a)
volume (ml) (b)
FIG. 1. Different stabilities of tight and loose couples revealed by centrifugation at increasing 30 S subunits at a concentration of A,,, = 100 were activated by incubation Mg2+ concentrations. in buffer A (see Materials and Methods) at 37°C for 60 min. Samples of 5 ~1 were added to tubes . oontammg 0.6 A,,, unit of 60 S subunits in 0.12 ml buffer B and allowed to stand at O’C for 16 min. Buffer B had the same Mg2 + concentrations as the gradients: (a) 6 mr+r, (b) 10 mix, (c) 16 mx. Analysis on sucrose gradients under standard conditions (6 h at 30,000 revs/min).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
101
raising the Mg2+ concentration to 15 mM. However, if subunits prepared from the same extracts by centrifugation through sucrose gradients at 1 mM-Mg’+ are allowed and 37°C only a fraction (~30%) form to reassociate at high Mg2+ concentration couples at 6 mM (Fig. l(a)), while most of the remaining particles (~60O/~) reassociat’e between 10 and 15 mM (Fig. l(b) and (c)). Incubation at higher Mg2 + concentrations and/or heating to higher temperature failed to increase the proportion of couples stable at 6 mM-Mg 2+ (tight couples) (Stahli, 1975). The Mg2+ dependence of reassociation exhibits a biphasic curve of the general type published by Van Duin et al. (1970) and Spirin et al. (1971) with a plateau between 4 and 6 m&I and another steep rise above 10 mM-Mg’ + At 5 mM-Mg2 + t’hr association constants corresponding to these two classes of couples differ by four to five orders of magnitude (No11 L Noll, 1976). The difference in stability of reassociated subunits toward Mg2+ is paralleled hy a similar difference of susceptibility toward dissociation induced by hydrostatic pressure. For a given Mg2+ concentration the dissociation during sedimentation is enhanced by increasing the rotor speed and hence the hydrostatic pressure. Thus. it, has been shown for 10 miw-Mg 2+ that increasing the speed from 30,000 to 60.000 revs/min produces a shift in the s value of tight vacant couples from 70 S to about 62 S (M. No11 et al., 1973a). If reassociated subunits are analyzed at 60,000 revs/min (Fig. 2(b)) the loose couples present produce an additional 55 S peak, which at 30,000 revs/min is visible as a 60 S shoulder (Fig. 2(a)). That the loose couples. as Sedimentation
70 50 I I
value (S)
30 I
70 I
I
50 I
30 I
I.0
0.5
I A4 iii
Effluent
(al
2
3
volume
(ml)
(b)
4 (cl
FIG. 2. Reduced stability of loose couples to pressure-dependent, and Mg2+-dependent dissociation. Sedimentation patterns of association mixtures containing 30 S a, and a mixture of 50 S a and 50 S b-subunits as described in the legend to Fig. 1. Centrifugation for 5 h at 30,000 revs/mill (a) or for 1.1 h at 60,000 revs/min ((b) and (c)). The solid line in (c) shows the sedimentation profile of the association products with 50 S a and 60 S b, the broken line that of pure loose couples obtained by mixing 30 S a with 50 S b-subunits separated by collecting from a zonal rotor the 50 S particles that had failed to associate at 6 m&f-Mg2+. .411 gradients contained 10 mM-Mg2+.
B. HAPliE
102
AND
H.
NOLL
defined by their Mg2 + -dependent dissociation, arc responsible for the new 55 S peak was shown directly in t’he following experiment. Subunits capable of forming t’ight couples were removed from the mixture (Fig. 2(c)) by association at 6 m&r-Mg2+. The remaining subunits were allowed to recombine at 10 m&r-Mg2 + and were examined on a gradient (broken line in Fig. 2(c)). Only the 55 S peak is present. This proves that the Mg 2+ dependence of association and the susceptibility to pressure dissociation are equivalent characteristics of t,he two classes of ribosomes. Tt follows that pressure-induced dissociation on a high speed gradient provides a rapid assay for the composition ofribosome preparations with respect to tight and loose couples. So far: the results establish that loose couples originate from native tight couples by an irreversible alteration in structure that prevents association of the subunits at physiological Mg2 + concentrations (~4 mM). However, the reduced combining power can be compensated for by increasing the Mg2+ concentration in the solution to 15 mM. Further experiments were designed to determine which of the two subunits was responsible for the irreversible conversion of tight into loose couples and to elucidate the structural basis of the reduced combining power. (b) Reversible changes in the 30 S qlarticle Subunits, derived from vacant couples by exposure to 1 mM-Mg2 + , fail to combine when mixed together in the cold at a high (10 mM) Mg2+ concentration (Fig. 3(a)) but reassociate slowly upon incubation at 37°C. A similar observation was reported by Kikuchi & Monier (1970) who found that heating of the 30 S subunit alone restored association. Zamir et al. (1971) showed that exposure to low Mg2+ concentrations results in loss of translation activity that is restored by heating, and referred to unpublished results indicating that thermal reactivation also led t)o an increase in
Sedimentation 70 I
50 I
value (S) 70 I
30 1
50 I
30 1
!O-
.5 -
a -
,5-
o= 0
1:-
2
3
4 Effluent
(a)
valume (ml) (b)
FIG. 3. Association of 30 S and 60 S subunits before and after 30 S activation. 30 S particles were mixed with 60 S subunits at 0°C as described under Fig. 1. The 30 S subunits were either mixed immediately with 60 S (a) or after incubation for 60 min at 37°C (b). Analysis on sucrose gradients under standard conditions (10 mM-MgZ+, 30,000 revs/min for 6 h).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
I03
subunit interaction. We confirmed that couple formation was restored by thermal activation of isolated 30 S subunits derived from tight couples by exposure to 1 mMMg2+ (M. No11 et al., 19736). Indeed, when the 30 S subunits were thermally activated and then added to the 50 S particles in the cold, rapid reassociation was observed (Fig. 3(b)). A variable portion of the 30 S subunits fail to recover but it is often as little as 5%. We call the active 30 S particles 30 S a, the reversibly inactivated ones 30 S b and the irreversibly inactivated ones 30 S c. Similar treatment of 50 S subunits did not enhance their interaction with either 30 S a or 30 S b-particles. Moreover, free (“native”) 30 S subunit’s that had not been exposed to low Mg2+ concentrations associated readily with derived 50 S subunits and heating produced no furt,her increase (M. No11 et al., 19736). (c) Alteration
of the 50 ASparticle
The following experiments were carried out with subunits obtained from tight, couples by zonal centrifugation at 1 m&K-Mg 2 + . After resuspension of the particles activated and the 50 S particles at 10 mM-Mg 2+ , the 30 S subunits were thermallv kept in the cold. When activated 30 S particles (30 S a) were allowed to combine in the cold at 10 mM-Mg2 + with a large excess of 50 S subunits, only tight couples were formed. This was shown by the conversion of all 30 S part’icles into couples that resist disis carried sociation at 6 mM-Mg2+ in the gradient. However, if the same experiment out with 30 S b-particles, no association product was detected even at 15 m&r-Mg2 + Thus, there is no demonstrable heterogeneity of 30 S particles with regard to the affinit,y of the 50 S subunit: in the a-state they are equally active, while in the b-state they are all unable to associate. However, when an excess of 30 S a-subunits is incubated with the same preparation of 50 S subunits at 10 m&I-Mg2+, both tight and loose couples are formed. As increasing concentrations of 30 S a-subunits are added to a constant concentration of 50 S particles, tight couples are formed exclusively at, first (Fig. 4(a)) and loose couples later, after all the 50 S particles capable of forming tight couples have reacted (Fig. 4(b) to (d)). Thus. when all reactive 50 S particles are converted into couples by addition of an excess of 30 S a-subunits, two populations of particles corresponding to tight and loose couples are observed (Fig. 4(d)). It follows that 50 S particles prepared by dissociation at low Mg2+ concentrations are present in two states : one of which (50 S a) is characterized by a high and the other (50 S b) by a low affinity for 30 S a. Dissociation of tight couples at 1 mM-Mg2 + always produces a mixture of 50 S a and 50 S b> as well as a variable fraction of 50 S particles that have lost the ability to form couples with 30 S a even at high Mg2 + concentrations (50 S c). Apparently, during dissociation at low Mg2 + concentrations some of the 50 S part,icles undergo an irreversible transition to the b-form. For this reason it is impoasible to prepare pure 50 S a-particles by methods based on dissociation at low Mg2 + concentrations. On the other hand, pure 50 S b are easily obtained by removing 50 S a from a mixture by association with 30 S a at A mM-Mg2 + as described in section (a), above. The fact that loose couples are stable and form sharp peaks at 15 mM-Mg2+ and 30,000 revs/min in the gradient implies that 50 S b-subunits have a defined lower limit of binding affinity. This is in contrast to the population of couples produced by exposure to RNase. This treatment leads t,o particles that sediment over a broad range, reflecting binding affinities t’hat decrease continuously to zero.
13. HAL’KE
I0.l
AND
Sedimentation
H. value
lvl I
2
3 Effluent
(a)
(b)
(S)
70 50 30 I I I
70 50 30 I I I
.I
NOLI,
4
b!u I
volume
2
3
4
(ml) (c)
(d)
FIG. 4. Demonstration of two classes of 50 S subunits (50 S a and 60 S b) by titration with increasing concentrations of 30 S subunits. 50 S subunits (0.5 A,,, unit/assay) were incubated in 0.126 ml TMND buffer (see Materials and Methods) with 30 S subunits (not pre-activated) at 40°C ((a) to (d)) for 20 min. The amounts of 30 S subunits (in A,,, units) added were 0.1 (a), 0.2 (b), 0.3 (c) and I.0 (d). Centrifugation as described in the legend to Fig. 3.
If we denote the states of the large subunit by capital A,B,C and those of the small subunit by a,b,c, tight couples may be written as aA and loose couples as aB. Couples corresponding to bA or bB do not exist in the absence of mRNA and tRNA. (d) Conversion
of 50 S a into 50 S b-subunits
The conversion of 50 S a to the less effective 50 S b-form is always produced by dissociation at low Mg2+ concentrations and enhanced by washing with high salt (1 M-NH,Cl), prolonged storage at 0°C. or incubation at 37°C in standard buffers. Attachment to the 30 S partner seems to have a stabilizing influence, because 50 S subunits in tight couples are stable, whereas isolated 50 S subunits are converted to the b-form with a half-time of about 60 minutes by incubation in buffer at 10 mmMg2 + and 37°C. This was shown by t’he increase of loose couples in association tests with 30 S a-particles. (e) 60 S ribosomes formed from, “interaction-free” subunits. poly( U) and HO-tRNA,,, are bB couples Schreier & No11 (1970,1971) reported that subunits that had lost the ability to associate upon incubation at 30°C and 10 mM-Mg2+ contained a variable fraction that could form a complex sedimenting at 60,000 revs/min when both poly(U) and were present. This “primitive” initiation complex was converted to HO-tRNA,,, a 70 S complex upon further incubation with Phe-tRNA, elongation factor T and GTP. We now find that both subunits in these complexes are in the b-form (bB). Although incubation at 30°C was necessary for the formation of the complexes, the
STRUCTURAL
DYNAMICS
OF
BACTERIAL
RIHOSOMES.
I\
10.5
t,hermal energy was not sufficient for the 30 S b -+ 30 S a activation. The shift from 60 5 to 70 S was found to result from a stabilization against dissociation by pressure. For at 30,000 revs/min the sedimentation rate of both the complex containing and the more stable complex containing HO-tRNA,,, and Phe-tRNA HO-tRNA,,,, was 70 S. (f) Couples produced after treatment of 50 S subunits with RNase It is possible that the irreversible inactivation of the 50 S particle is produced by RNase. However, treatment with fibre-bound RNase failed to produce the changes characteristic of 50 S a + 50 S b conversion and the loss of both tight and loose couples was observed. In particular, 23 S RNA readily undergoes a break into A 17 S/15 S pair of fragments. There was, however, no correlation between the extent of this break and the proportion of tight and loose couples. Thus, we have seen tight couples (Fig. 5(a)) with nearly all the 23 S RNA broken (Fig. 5(b)) and loose couples (Fig. 5(c)) with nearly all the 23 S RNA intact (Fig. 5(d)). In contrast to the highly specific event responsible for the 50 S a --f 50 S b conversion, mild treatment with RNase gradually reduces the affinity of the 50 S subunits for their 30 S partners until the interaction disappears (Fig. 6(a) to (d)) and. parallel to it, the Phe-tRNA binding ability in response to poly(U) (Fig. 6(e) to (h)). Couples formed by association of 30 S a with RNase-treated 50 S subunits dissociate over a broad range in conditions (10 to 15 mM-Mg ‘+ , 30.000 revs/min) under which aB couples are still perfectly stable.
Sedimenation 23 17 15 I I,
value (S) 70 50 30 I 1 I
23 I7 I5 8 II
L AL I
(a)
2
(b)
3 Effluent
12301234 volume (ml) (cl
Cd)
E’Ic:. 5. Lack of correlation between 60 S subunit association and major break in 23 S rRNA. Pure tight couples washed with 1 M-ammonium sulfate were analyzed on a sucrose gradient, (1.2 h at 60,000 revs/min, 15 mM-Mg2+) before (a) and after (b) treatment with sodium dodecyl sulfat,e to release rRNA (No11 & Stutz, 1968). To 0.05 ml of each 50 S subunit 0.15 ml water was added and 0.05 ml 10% (w/v) sodium dodecyl sulfate containing 1% (w/v) EDTA. Analysis on isokinetic sucrose gradients after incubation at 37°C for 4 min. Gradient (b) was non-isokinetic (convex exponential 16% to 35.6% sucrose, 1 mM-i%ig’+, 3 h at 60,000 revs/min). Ribosome preparation containing mostly loose couples was analyzed on a sucrose gradient before (c) and after (d) treatment with sodium dodecyl sulfate. The subunit association mixture in (0) was centrifuged for 1.1 h at 60,000 revs/min and 10 mM-Mg 2+, t,he RNA product in (d) for 3.5 h at 17°C and 60,000 revs/min.
B.
HAl’KE
AND
Sedimention
H.
NOLI,
volue (S)
70 50 30
l-
b)
-T
Effluent
volume
(ml)
FIG. 6. Parallel loss of ability to form couples ((a) to (d)) and poly(U)-dependent phenylalanine binding) (e) to (h)) on treatment of 50 S subunits with fiber-bound RNase. 60 S subunits (1.1 A 260 units) were incubated in 0.25 ml TMND buffer (see Materials and Methods) for 10 min at 30°C at the following concentrations (pg/ml) of fiber-bound RNase (Enzite-RNase, GallardSchlesinger): (a) 0, (b) 200, (c) 500 and (d) 1000. The RNase was removed by centrifugation for 10 min at 20,000 revs/min in the IEC SB-406 rotor. To 0.1.ml portions of the supernatant containing 0.44 AsGo unit were added activated 30 S subunits in 6 ~1 buffer G for association analysis on sucrose gradients. Another 0.1.ml portion of each supernatant was added to 0.125 ml TMND buffer containing 10 pg poly(U), 100 pg tRNA-OH, 0.5 mM-GTP, 0.6 A,,, unit of 30 S a(2800 cts/min per pmol) and saturating amounts of subunits, 1.0 A,,, unit of [sHIphe-tRNA elongation factor T. After incubation for 20 min at 3O”C, the mixtures were analyzed on sucrose ) Absorbance at 260 nm; gradients (1.2 h at 60,000 revs/min and 10 mix-Mg”). ( (--O--O-3H radioactivity.
4. Discussion Vacant ribosome couples prepared by a variety of methods fall into two broad classes, tight and loose couples, that differ in stability with respect to pressuredependent and Mg2+ concentration-dependent dissociation. The existence of two populations corresponding to tight and loose couples was suggested by the biphasic dissociation curve obtained as a function of the Mg2+ concentration by Van Duin et al. (1970) and Spirin et al. (1971).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
107
The two classes of particles can be separated preparatively into tight couples, loose couples and subunits by two successive centrifugations. In the first step at 6 mM-Mg2 + only the tight couples resist dissociation while the loose couples appear as subunits. Recentrifugation of the pooled subunits at 15 mMMg2 + separates the loose couples as 70 S particles from contamination by inactive above 6 mM, the t*wo subunits that are unable to associate. At Mg2+ concentrations populations can be distinguished by the greater sensitivity of the loose couples to pressure-dependent dissociation. Each subunit exhibits a characteristic pattern with regard to subunit interaction. the active form of the 30 S particle (30 S a) tends Thus, at low Mg2+ concentrations, to undergo a transition to a conformational modification (30 S b) that cannot associat’e with active 50 S particles unless returned to the a-state by thermal activation according to the procedure of Zamir et al. (1971). The 50 S subunits, on the other hand, are responsible for the formation of loose couples (50 S b) by changing irreversibly to a state of much reduced affinity for their 30 S partners. Since fresh extracts that have are devoid of loose not been exposed to low Mg2 + or high NH + or K + concentrations couples, the latter must be derived from tight couples by some kind of structura)l alteration. Att’empts to convert 50 S a-subunits into the b-form by treatment’ with RNase were not successful because the resulting particles exhibited a much widei spect’rum of reduced affinities for active 30 S subunit,s than the typical 50 S b-particles formed by incubation at 37°C. Neither could the transition t,o t,he b-form be related bo breaks of 23 S rRNA into fragments that are detectable by sedimentation analysis. More recent studies rule out the removal of more than three nucleotides from the ends of 23 S or 5 S RNA or alterations in the 50 S proteins as the cause of the 50 S a to 50 S b conversion and suggest that the change is conformational (Stahli, 1975). We have implied that the differences in sensitivity to dissociation by pressure exhibited by tight and loose couples are a reflection of the affinity of the subunit’s for each other. This notion is based on the observation that dissociation in the gradient depends in opposite ways on pressure and Mg2+ concentration. We may explain this relationship qualit’atively as follows. Dissociation by pressure implies that the overall reaction involves a net decrease in t’he molar volume of the particles. We postulate that the resulting deformation of the subunits distorts the complrmen&y interfaces and thus reduces the interaction. At increased Mg2+ concentrations the particles are st’abilized to resist deformation. Since the phosphate groups of ribosomal RNA are the primary Mg 2+ binding sites, the rigidity of the particles appears to be largely determined by the three-dimensional structure of the RNA backbone. Removal of Mg2+ promotes repulsion of the negative phosphate charges and rearrangement of the secondary and tertiary RNA structure. Breaks in the 23 S RNA at, critical regions would have a similar effect and explain the much greater sensitivity to dissociation by pressure observed after mild treatment of ribosomes v&h RNase. Bosch and co-workers (Van Diggelen et al., 1971b; Van Diggelen $ Bosch. 1973) observed that the association products of 30 S subunits with native 50 S particles sedimented more slowly than the corresponding couples formed with derived 50 S subunits and concluded that the ability to form two association products was an intrinsic capacity of 50 S subunits. Our examination of native subunits in paper II of this series (M. No11 et al., 19733) showed that native 50 S in contrast to native 30 S subunits are severely damaged. Most of them had lost the ability to combine with 30 S a and the remaining subunits resembled 50 S b and RNase particles.
10X
K. Ht\PKE
;\SU
H.
NOLL
Furthermore, the data of Bosch do not support’ his interpretation that derived 50 S particles are a homogeneous population of 50 S a-particles. On the contra,ry, the presence of more than SO:/;, of subunits at 5 mM-Mg2+ and about 30”,, at 10 mM-Mg2 ’ (Van Diggelen et aZ.: 1973, Fig. 7) implies that their preparation of derived 50 S contained about one-third each of 50 S a, 50 S b and 50 S c-particles, in good agreement with our own findings. Moreover, in t’he light of our finding that all 50 S b and 50 S c particles are derived from the 50 S a-form, t’he classification of couples according t’o the origin of their 50 S subunit (native or derived) is misleading. Conversion of tight to loose couples profoundly affects their biochemical activities such as tRNA binding, translocation, response to initiation factors, Mg2+ optimum for initiation etc. (H. No11 et al., 1973). Although these will be described in detail in in preparation), it should be pointed a subsequent paper (Hapke et al.. manuscript out that many of the conflicting reports on the detailed mechanism of translation can be attributed to differences in the quality of t’hr ribosomes employed. The authors are indebted to Professor Ada Zamir for stimulating discussion. This work was supported by research grant no. P381F from the American Cancer Society and grant no. CA-11797 from the United States Public Health Service. One of us (H. N.) is a Lifetime Career Professor of fhe American Cancer Societ,g.
REFERENCES Herzog, H., Ghysen, A. & Bollen, A. (1971). PEBS Letters, 15, 291-294. Kikuchi, A. & Monier, R. (1970). FEBS Letters, 11, 157. Kurland, C. G. (1966). J. Mol. Biol. 18, 90p108. McCarty, K. S., Stafford, D. & Brown, 0. (1968). Anal. Biochem. 24, 31&329. Noll, H. (1967). Nature (London), 215, 360-363. Noll, H. (1969). In Techniques in Protein Biosynthesis (Sargent, J. $ Campbell, P. N., eds), vol. 2, pp. 101-171, Academic Press, London. Noll, H. (1971). BierteZjahresschr. Naturforsch. Gee. Zurich, 116, 377-402. Noll, H. & Stutz, E. (1968). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12B, pp. 129-155, Academic Press, New York and London. Noll, H., Noll, M., Hapke, B. & Van Dieijen, G. (1973). In Regulation of Transcription and Translation, Mosbach Colloquium ivo. 24 (Bautz, E., ed.), pp. 257-311, Springer Verlag, New York and Heidelberg. Noll, M. (1972). Thesis, Northwestern University. Noll, M. & Noll, H. (1972). Nature New BioZ. 238, 225-228. Noll, M. & Noll, H. (1974). J. Mol. BioZ. 90, 237-251. Noll, M. & Noll, H. (1976). J. Mol. BioZ. 105, 111.-130. Noll, M., Hapke, B., Schreier, M. & Noll, H. (1973a). J. Mol. BioZ. ‘75, 281-294. Noll, M., Hapke, B. & Noll, H. (1973b). J. Mol. BioZ. 80, 519-529. Schreier, M. H. & Noll, H. (1970). Nature (London), 227, 128-133. Schreier, M. H. & Noll, H. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 805.-809. Spirin, A. S. (1971). FEBS Letters, 14, 349-353. Spirin, A. S., Sabo, B. & Kovalenko, V. A. (1971). FEBS Letters, 15, 197-200. Staehelin, T. & Maglott, D. R. (1971). In Methods in Enzymology (Moldave, K. & Grossman, L., eds), vol. 20, pp. 449-456, Academic Press, New York. Stahli, C. (1975). Thesis, Northwestern University. Subramanian, A. R. & Davis, B. D. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 2453-2457. Van Diggelen, 0. P. & Bosch, L. (1973). Eur. J. Biochem. 39, 499-510. Van Diggelen, 0. P., Oostrom, H. & Bosch, L. (197la). FEBS Letters, 19, 115-120. Van Diggelen, 0. P., Heinsius, H. L., Kalousek, F. & Bosch, L. (1971b). .T. Mol. BioZ. 55, 277-281. Van Diggelen, 0. P., Oostrom, H. & Bosch, L. (1973). Eur. ,I. Biochem. 39, 511-523.
STRUCTURAL
DYNAMICS
Van Duin, J ., Van Dieijen, Biochem.
OF BACTERIAL
G.. va,n Knippenberg,
RIBOSOMES.
IV
1’. H. & Bosch, L. (1!)70). Eur.
17, 433-440.
Zamir. A., Miskin, R. & Elson, 1). (1971). J. Mol. Biol. 60, 347-364. Zitomrr, K. S. & Flaks, ,J. G. (1!372). J. Mol. Biol. 71. 263.~279.
109 .I.
Structural Dynamics of Bacterial Ribosomes IV?. Classification of Ribosomes by Subunit Interaction BERN
Department Northwestern
HAPKE$
AND HANS NOLL§
of Biochemistry and Molecular Biology LTniuersity, Evanston, Ill. 60201, U.S.A.
(Received 1 March 1974, and
in revised
form
10 Novem,ber 1975)
Previous studies in this series (M. No11 et al., 1973a,b; No11 & Noll, 1974) have established that in Es&e&h&z coli the ability of subunits to form vacant 70 S condition for activity in the transribosome couples at 10 mM-Mg2+ is a stringent lation of natural messenger (R 17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into t’wo classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mM-Mgzf and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mM-Mg2+ ill t,he gradient, both species sediment as a 705 peak. At 10 mM-Mg2+ and 60,000 r(tvs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples. Association is dependent on the state of each subunit. Removal of Mg2+ produces 30 S b-particles that are unable to associate with 50 S subunits unless rtaconverted to the 30 S a-form by thermal a&ration according to Zamir et al. ( 1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a + 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with KNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association strp.
Fresh S-30 extracts cont’ain only tight couples (approx. 80%) and subunits (Hpprox. 200/,). Our results suggest, that loose couples are a,rtefact,w derived from tight couples by a structural or conformational modification. Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA,,, (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization. are shown to consist of the b-form of each subunit. It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due t,o the lack of structural criteria required for standardizing the ribosome preparation used by different irlvestigators. This study provides simple methods and criteria to classify and sc,parate physically all ribosome and ribosome subunits that ha.ve been observed itlt,o well-defined classes of predictable act,ivity. t Paper III in this series is No11 & No11 (1974). 1 Present, address: Nuclear Div., 3M Center, St. Paul, &linn. 55101, U.S.A. S;To whom reprint requests should be addressed. 7
97
!)S
Ii.
HAPICE
r\,h;I)
H.
NOLI,
1. Introduction Experimental studies on the mechanism of protein synthesis are limited by the quality of the ribosome preparations in which only a fraction, I to SOS/,, are active. Recent work has shown that it is possible to prepare ribosomes all of which can be converted into initiation complexes with natural messenger (H. No11 et al., 1973 ; M. No11 et al., 1973a,b; No11 & Noll, 1972, 1974). A necessary condition for this activity is the ability of the subunits to associate in the absence of messenger RNA, transfer RNA and other factors; particles that cannot do so are invariably inactive in the translation of natural messengers, although they may be active with poly(U) (H. No11 et al., 1973). When vacant ribosomes prepared by common methods are analyzed on sucrose gradients at high resolution and Mg2+ concentrations above 5 mM, the patterns observed vary greatly with rotor speed, Mg2+ concentration and preparation of ribosomes. Thus, a number of variable peaks and shoulders between the 50 S and 70 S position appear as well as great shifts in the ratio of subunits to the faster sedimenting components. We found that all these observations can be explained by the occurrence in such preparations of two classes of ribosomes, tight and loose couples, whose 50 8 subunits differ greatly in their affinity for the 30 S partner in the physiological range It will be shown that much higher Mg2+ concentrations are of Mg2 + concentrations. required to keep the loose couples together and to protect them from dissociation by hydrostatic pressure in the ultracentrifuge (Herzog et al., 1971; Spirin, 1971; Noll, 1971; Van Diggelen et al., 1971a; Subramanian & Davis, 1971; M. No11 et al., 1973a) than in the case of tight couples. Moreover, evidence will be presented that loose couples originate from biologically intact tight couples by some form of denaturation involving the 50 S subunit. Published studies show dissociation curves (extent of dissociation versus Mg2+ concentration) that correspond to (1) tight couples, (2) loose couples or (3) a mixture of t’he two species. The curves for (1) and (2) are homogeneous and show that at 37°C tight couples dissociate in a narrow range between 1.5 and 2.5 mM-Mg2+ (Noll. 1972; H. No11 et al. 1973; No11 & Noll, 1976), while loose couples dissociate within 5 to 15 mM-Mg2+ (Spirin et al., 1971; Zitomer & Flaks, 1972). Curves corresponding t,o (3) are biphasic and span the range of both tight and loose couples (Van Duin et al.. 1970) with an inflection point somewhere in between (Spirin et aZ., 1971). Although tight couples were not recognized as a separate class, it had been noted during the earlier dissociation studies t’hat unwashed ribosomes that had never been required a higher Mg2+ concentration for exposed to low Mg2+ concentration reassociation than for dissociation, and that this hysteresis disappeared during subsequent cycles of association and dissociation (Spirin et al., 1971; Zitomer & Flaks, 1972). The present study implies that these authors observed the irreversible conversion of tight into loose couples. Bosch and co-workers, on the other hand, reported that couples formed from subunits fall into two classes characterized by different susceptibility to high hydrostatic pressure (Van Diggelen et al., 1971a,b,1973; Van Diggelen Q Bosch, 1973). According to their studies, active 30 S subunits regardless of origin associated with “native” 50 S subunits to form couples that, under conditions of high hydrostatic more slowly than similar couples obtained with “derived” pressure, sedimented 50 S subunits. The difference in stability was attributed to an intrinsic difference in the nature of the native 50 S particles present in the free state as opposed to 50 S
STRUCTURAL
DYNAMICS
OF BSCTERIAL
RIBOSOMES.
99
IT’
part’icles derived from couples by dissociation at low Mg2+ concentrations. The obvious question of how this view could be reconciled with the concept of a dynamic equilibrium between subunits and couples was not answered, nor was the possibility considered that one form was derived from the other by denaturation or other structural modifications. Neither was the relationship clarified between these two classes differing in their sensitivity to dissociation by pressure and the t,wo classes with different stabilities with respect t’o Mg2+. The following experiments were performed to answer these questions.
2. Materials and Methods Most of the methods employed in these experiment,s paper I of this series (M. No11 et al., 1973a).
have
been described
in detail
in
(a) Reagents Mg2 + is always added together with EDTA in a molar ratio of Mg2 + : EDTA of 20 : 1, as recommended by Staehelin & Maglott (1971). Thus, if the Mg2+ concentration is given as 10 mM, we mean the efSective concentration, the actual concentration being 10.5 mMMg2+ plus 0.5 mM-EDTA. TMND buffer (Staehelin & Maglott, 1971) is 160 mM-ammonium chloride, 10 mM-magnesium acetate, 50 mM-Tris*HCl (pH 7.5), 10 mM-2-mercaptoethanol. Buffer G is the same as TMND buffer, except that the ammonium chloride is 60 mM and Tris.HCl is 20 mM. Buffer H is the same as buffer G, rxcept that t,he ammonium chlori& is 100 mM. (b) Preparation.
of ribosomes
and mbunits
Ribosomes and ribosome subunits were prepared by the ammonium sulfate precipitation procedure of Kurland (1966). Pure tight vacant (aA) couples were prepared as follows. The S-30 extract from freshIS ground cells was centrifuged in a B-29 zonal rotor at> 6 mM-Mg2+ as described previously (M. No11 et al., 1973a). The fract,ions corresponding to the 70 S peak were pooled and the ribosomes centrifuged into a pellet (M. No11 et al. 1973a). The pellets were dissolved in 2 ml of 50 S subunits, 1000 A,,, units of the concentrated suspension in buffer G werfb diluted to a total volume of 20 ml with buffer F (same as buffer H except that the Mg2 ’ is 15 mM) and subjected to a second purification step in the B-29 zonal rotor. The prorcdure was the same as in the first purification, except that the Mg2 + concentration in the gradient was raised to 15 mM and centrifugation was for 18 h at 25.000 rers/min (Nell, 1972; No11 & No11 1976). 50 8 b-subunits were isolated as follows : 550 A,,, units of 30 S subunits in 10 ml bnf’el G were incubated at 37°C for 60 min and added to 550 A,,, units of 50 S subunit,s in 40 ml TMND buffer, and the mixture was kept at 0°C for 5 min. The solution was layered ovel a 1300-ml convex exponential sucrose gradient (c,t = 0.292 M, CR = 1.17 M, I’, = 500 ml) in buffer I (same as buffer H except that the Mg2 + concentration is 6 mM) and centrifuged in a B-29 IEC rotor for 18 h at 25,000 revs/min and 8°C. Fractions containing the 50 S subunits were combined and the ribosomes were centrifuged in the IEC A-170 rot,or for 8 h at 40,000 revs/min. Each pellet was dissolved in 0.5 ml buffer G and the solutions were st,ored in small portions at -60°C. The total yield of 50 S subunits was 360 A,,, units. Deactivation and activation of 30 S subunits was carried nut as described hy Znrnir et al. (1971). (c) Analytical The standard isokinetic sucrose in series of 6x 3.6 ml with the aid 1969). The isokinetic parameters for The parameters for ribosomal RNA t C,, sucrose concentration
method8
gradients (Noll, 1967) were prepared Simultaneously. of an automatic machine (McCarty et al., 1968; Noll, ribosomes have been published (M. No11 et al., 1973a). were as follows: C,t = 0.438 M, CR = 1.31 M, V, =
at top of gradient;
C,, reservoir
concentration;
V,, mixing
volumc~.
100
B.
HAPKE
ANI)
H.
NOLI,
5.3 ml. Tlre sucrose solutions w(‘r(’ msdo up iti 100 Irl~l-arlllllollillf~~ c*lrloride, 20 tnslTris .HCl (pH 7.5), 1 mM-magnesium actstat~e. Cent rifugat,ion was for :I 11 at 60,000 r(:vsl’ min and 12 to 15°C for ribosoma,l RNA. For ribosomes tile Mg2 + collcrntration irl t II{, gradients as well as the speed alld duration of crntrifllgat,ior> is givc,ll ill t/x, legends t,o tllcs Figures. The scanning of the gradients for bobh absorbancy and radioactivity was carried otlt with the automated MICO MR-747 radiograd system (Molecular instruments Co., P.O. Box 1652, Evanston, Ill. 60201, U.S.,4.). The system consists of a turbulence-free flowcell capable of detecting peaks corresponding to 20 ng RNA, and a programmable automatic fraction collector that collects fractions into scintillation vials with simultaneous addition of scintillation fluid (Noll, 1971). The optical scan of the gradients at 260 nm was recorded conventionally on a strip chart and, at the same time, in digitalized form on magnetic tape with the aid of a Hewlett-Packard model 9820A digital calculator, model 3480A digital voltmeter and model 9865A cassette memory. The radioactivity data obtained from the liquid scintillation counter were recorded on the tape cassettes separately by manual entry. For the final display, the dat’a. were combined by thcl comprlt,txr and recorded by the model 9060A plottcxr in the size drsirracl for rrprotlnc+io,r.
3. Results (a) Characterization
of tight and loose couples by Mg” +-dependence of subunit association
(> 10 miw) contain Crude extracts prepared rapidly at high Mg2 + concentrations only tight couples (80 to 95%) and free subunits. When such preparations are analyzed on sucrose gradients at low centrifugation speeds, all the couples are stable at 6 mM-Mg2 + and no significant increase in the proportion of couples is observed upon
70 I
50 I
30 I
Sedimentation value (S) 70 50 30 I I I
70 !50 30 I I
:
Effluent
(a)
volume (ml) (b)
FIG. 1. Different stabilities of tight and loose couples revealed by centrifugation at increasing 30 S subunits at a concentration of A,,, = 100 were activated by incubation Mg2+ concentrations. in buffer A (see Materials and Methods) at 37°C for 60 min. Samples of 5 ~1 were added to tubes . oontammg 0.6 A,,, unit of 60 S subunits in 0.12 ml buffer B and allowed to stand at O’C for 16 min. Buffer B had the same Mg2 + concentrations as the gradients: (a) 6 mr+r, (b) 10 mix, (c) 16 mx. Analysis on sucrose gradients under standard conditions (6 h at 30,000 revs/min).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
101
raising the Mg2+ concentration to 15 mM. However, if subunits prepared from the same extracts by centrifugation through sucrose gradients at 1 mM-Mg’+ are allowed and 37°C only a fraction (~30%) form to reassociate at high Mg2+ concentration couples at 6 mM (Fig. l(a)), while most of the remaining particles (~60O/~) reassociat’e between 10 and 15 mM (Fig. l(b) and (c)). Incubation at higher Mg2 + concentrations and/or heating to higher temperature failed to increase the proportion of couples stable at 6 mM-Mg 2+ (tight couples) (Stahli, 1975). The Mg2+ dependence of reassociation exhibits a biphasic curve of the general type published by Van Duin et al. (1970) and Spirin et al. (1971) with a plateau between 4 and 6 m&I and another steep rise above 10 mM-Mg’ + At 5 mM-Mg2 + t’hr association constants corresponding to these two classes of couples differ by four to five orders of magnitude (No11 L Noll, 1976). The difference in stability of reassociated subunits toward Mg2+ is paralleled hy a similar difference of susceptibility toward dissociation induced by hydrostatic pressure. For a given Mg2+ concentration the dissociation during sedimentation is enhanced by increasing the rotor speed and hence the hydrostatic pressure. Thus. it, has been shown for 10 miw-Mg 2+ that increasing the speed from 30,000 to 60.000 revs/min produces a shift in the s value of tight vacant couples from 70 S to about 62 S (M. No11 et al., 1973a). If reassociated subunits are analyzed at 60,000 revs/min (Fig. 2(b)) the loose couples present produce an additional 55 S peak, which at 30,000 revs/min is visible as a 60 S shoulder (Fig. 2(a)). That the loose couples. as Sedimentation
70 50 I I
value (S)
30 I
70 I
I
50 I
30 I
I.0
0.5
I A4 iii
Effluent
(al
2
3
volume
(ml)
(b)
4 (cl
FIG. 2. Reduced stability of loose couples to pressure-dependent, and Mg2+-dependent dissociation. Sedimentation patterns of association mixtures containing 30 S a, and a mixture of 50 S a and 50 S b-subunits as described in the legend to Fig. 1. Centrifugation for 5 h at 30,000 revs/mill (a) or for 1.1 h at 60,000 revs/min ((b) and (c)). The solid line in (c) shows the sedimentation profile of the association products with 50 S a and 60 S b, the broken line that of pure loose couples obtained by mixing 30 S a with 50 S b-subunits separated by collecting from a zonal rotor the 50 S particles that had failed to associate at 6 m&f-Mg2+. .411 gradients contained 10 mM-Mg2+.
B. HAPliE
102
AND
H.
NOLL
defined by their Mg2 + -dependent dissociation, arc responsible for the new 55 S peak was shown directly in t’he following experiment. Subunits capable of forming t’ight couples were removed from the mixture (Fig. 2(c)) by association at 6 m&r-Mg2+. The remaining subunits were allowed to recombine at 10 m&r-Mg2 + and were examined on a gradient (broken line in Fig. 2(c)). Only the 55 S peak is present. This proves that the Mg 2+ dependence of association and the susceptibility to pressure dissociation are equivalent characteristics of t,he two classes of ribosomes. Tt follows that pressure-induced dissociation on a high speed gradient provides a rapid assay for the composition ofribosome preparations with respect to tight and loose couples. So far: the results establish that loose couples originate from native tight couples by an irreversible alteration in structure that prevents association of the subunits at physiological Mg2 + concentrations (~4 mM). However, the reduced combining power can be compensated for by increasing the Mg2+ concentration in the solution to 15 mM. Further experiments were designed to determine which of the two subunits was responsible for the irreversible conversion of tight into loose couples and to elucidate the structural basis of the reduced combining power. (b) Reversible changes in the 30 S qlarticle Subunits, derived from vacant couples by exposure to 1 mM-Mg2 + , fail to combine when mixed together in the cold at a high (10 mM) Mg2+ concentration (Fig. 3(a)) but reassociate slowly upon incubation at 37°C. A similar observation was reported by Kikuchi & Monier (1970) who found that heating of the 30 S subunit alone restored association. Zamir et al. (1971) showed that exposure to low Mg2+ concentrations results in loss of translation activity that is restored by heating, and referred to unpublished results indicating that thermal reactivation also led t)o an increase in
Sedimentation 70 I
50 I
value (S) 70 I
30 1
50 I
30 1
!O-
.5 -
a -
,5-
o= 0
1:-
2
3
4 Effluent
(a)
valume (ml) (b)
FIG. 3. Association of 30 S and 60 S subunits before and after 30 S activation. 30 S particles were mixed with 60 S subunits at 0°C as described under Fig. 1. The 30 S subunits were either mixed immediately with 60 S (a) or after incubation for 60 min at 37°C (b). Analysis on sucrose gradients under standard conditions (10 mM-MgZ+, 30,000 revs/min for 6 h).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
I03
subunit interaction. We confirmed that couple formation was restored by thermal activation of isolated 30 S subunits derived from tight couples by exposure to 1 mMMg2+ (M. No11 et al., 19736). Indeed, when the 30 S subunits were thermally activated and then added to the 50 S particles in the cold, rapid reassociation was observed (Fig. 3(b)). A variable portion of the 30 S subunits fail to recover but it is often as little as 5%. We call the active 30 S particles 30 S a, the reversibly inactivated ones 30 S b and the irreversibly inactivated ones 30 S c. Similar treatment of 50 S subunits did not enhance their interaction with either 30 S a or 30 S b-particles. Moreover, free (“native”) 30 S subunit’s that had not been exposed to low Mg2+ concentrations associated readily with derived 50 S subunits and heating produced no furt,her increase (M. No11 et al., 19736). (c) Alteration
of the 50 ASparticle
The following experiments were carried out with subunits obtained from tight, couples by zonal centrifugation at 1 m&K-Mg 2 + . After resuspension of the particles activated and the 50 S particles at 10 mM-Mg 2+ , the 30 S subunits were thermallv kept in the cold. When activated 30 S particles (30 S a) were allowed to combine in the cold at 10 mM-Mg2 + with a large excess of 50 S subunits, only tight couples were formed. This was shown by the conversion of all 30 S part’icles into couples that resist disis carried sociation at 6 mM-Mg2+ in the gradient. However, if the same experiment out with 30 S b-particles, no association product was detected even at 15 m&r-Mg2 + Thus, there is no demonstrable heterogeneity of 30 S particles with regard to the affinit,y of the 50 S subunit: in the a-state they are equally active, while in the b-state they are all unable to associate. However, when an excess of 30 S a-subunits is incubated with the same preparation of 50 S subunits at 10 m&I-Mg2+, both tight and loose couples are formed. As increasing concentrations of 30 S a-subunits are added to a constant concentration of 50 S particles, tight couples are formed exclusively at, first (Fig. 4(a)) and loose couples later, after all the 50 S particles capable of forming tight couples have reacted (Fig. 4(b) to (d)). Thus. when all reactive 50 S particles are converted into couples by addition of an excess of 30 S a-subunits, two populations of particles corresponding to tight and loose couples are observed (Fig. 4(d)). It follows that 50 S particles prepared by dissociation at low Mg2+ concentrations are present in two states : one of which (50 S a) is characterized by a high and the other (50 S b) by a low affinity for 30 S a. Dissociation of tight couples at 1 mM-Mg2 + always produces a mixture of 50 S a and 50 S b> as well as a variable fraction of 50 S particles that have lost the ability to form couples with 30 S a even at high Mg2 + concentrations (50 S c). Apparently, during dissociation at low Mg2 + concentrations some of the 50 S part,icles undergo an irreversible transition to the b-form. For this reason it is impoasible to prepare pure 50 S a-particles by methods based on dissociation at low Mg2 + concentrations. On the other hand, pure 50 S b are easily obtained by removing 50 S a from a mixture by association with 30 S a at A mM-Mg2 + as described in section (a), above. The fact that loose couples are stable and form sharp peaks at 15 mM-Mg2+ and 30,000 revs/min in the gradient implies that 50 S b-subunits have a defined lower limit of binding affinity. This is in contrast to the population of couples produced by exposure to RNase. This treatment leads t,o particles that sediment over a broad range, reflecting binding affinities t’hat decrease continuously to zero.
13. HAL’KE
I0.l
AND
Sedimentation
H. value
lvl I
2
3 Effluent
(a)
(b)
(S)
70 50 30 I I I
70 50 30 I I I
.I
NOLI,
4
b!u I
volume
2
3
4
(ml) (c)
(d)
FIG. 4. Demonstration of two classes of 50 S subunits (50 S a and 60 S b) by titration with increasing concentrations of 30 S subunits. 50 S subunits (0.5 A,,, unit/assay) were incubated in 0.126 ml TMND buffer (see Materials and Methods) with 30 S subunits (not pre-activated) at 40°C ((a) to (d)) for 20 min. The amounts of 30 S subunits (in A,,, units) added were 0.1 (a), 0.2 (b), 0.3 (c) and I.0 (d). Centrifugation as described in the legend to Fig. 3.
If we denote the states of the large subunit by capital A,B,C and those of the small subunit by a,b,c, tight couples may be written as aA and loose couples as aB. Couples corresponding to bA or bB do not exist in the absence of mRNA and tRNA. (d) Conversion
of 50 S a into 50 S b-subunits
The conversion of 50 S a to the less effective 50 S b-form is always produced by dissociation at low Mg2+ concentrations and enhanced by washing with high salt (1 M-NH,Cl), prolonged storage at 0°C. or incubation at 37°C in standard buffers. Attachment to the 30 S partner seems to have a stabilizing influence, because 50 S subunits in tight couples are stable, whereas isolated 50 S subunits are converted to the b-form with a half-time of about 60 minutes by incubation in buffer at 10 mmMg2 + and 37°C. This was shown by t’he increase of loose couples in association tests with 30 S a-particles. (e) 60 S ribosomes formed from, “interaction-free” subunits. poly( U) and HO-tRNA,,, are bB couples Schreier & No11 (1970,1971) reported that subunits that had lost the ability to associate upon incubation at 30°C and 10 mM-Mg2+ contained a variable fraction that could form a complex sedimenting at 60,000 revs/min when both poly(U) and were present. This “primitive” initiation complex was converted to HO-tRNA,,, a 70 S complex upon further incubation with Phe-tRNA, elongation factor T and GTP. We now find that both subunits in these complexes are in the b-form (bB). Although incubation at 30°C was necessary for the formation of the complexes, the
STRUCTURAL
DYNAMICS
OF
BACTERIAL
RIHOSOMES.
I\
10.5
t,hermal energy was not sufficient for the 30 S b -+ 30 S a activation. The shift from 60 5 to 70 S was found to result from a stabilization against dissociation by pressure. For at 30,000 revs/min the sedimentation rate of both the complex containing and the more stable complex containing HO-tRNA,,, and Phe-tRNA HO-tRNA,,,, was 70 S. (f) Couples produced after treatment of 50 S subunits with RNase It is possible that the irreversible inactivation of the 50 S particle is produced by RNase. However, treatment with fibre-bound RNase failed to produce the changes characteristic of 50 S a + 50 S b conversion and the loss of both tight and loose couples was observed. In particular, 23 S RNA readily undergoes a break into A 17 S/15 S pair of fragments. There was, however, no correlation between the extent of this break and the proportion of tight and loose couples. Thus, we have seen tight couples (Fig. 5(a)) with nearly all the 23 S RNA broken (Fig. 5(b)) and loose couples (Fig. 5(c)) with nearly all the 23 S RNA intact (Fig. 5(d)). In contrast to the highly specific event responsible for the 50 S a --f 50 S b conversion, mild treatment with RNase gradually reduces the affinity of the 50 S subunits for their 30 S partners until the interaction disappears (Fig. 6(a) to (d)) and. parallel to it, the Phe-tRNA binding ability in response to poly(U) (Fig. 6(e) to (h)). Couples formed by association of 30 S a with RNase-treated 50 S subunits dissociate over a broad range in conditions (10 to 15 mM-Mg ‘+ , 30.000 revs/min) under which aB couples are still perfectly stable.
Sedimenation 23 17 15 I I,
value (S) 70 50 30 I 1 I
23 I7 I5 8 II
L AL I
(a)
2
(b)
3 Effluent
12301234 volume (ml) (cl
Cd)
E’Ic:. 5. Lack of correlation between 60 S subunit association and major break in 23 S rRNA. Pure tight couples washed with 1 M-ammonium sulfate were analyzed on a sucrose gradient, (1.2 h at 60,000 revs/min, 15 mM-Mg2+) before (a) and after (b) treatment with sodium dodecyl sulfat,e to release rRNA (No11 & Stutz, 1968). To 0.05 ml of each 50 S subunit 0.15 ml water was added and 0.05 ml 10% (w/v) sodium dodecyl sulfate containing 1% (w/v) EDTA. Analysis on isokinetic sucrose gradients after incubation at 37°C for 4 min. Gradient (b) was non-isokinetic (convex exponential 16% to 35.6% sucrose, 1 mM-i%ig’+, 3 h at 60,000 revs/min). Ribosome preparation containing mostly loose couples was analyzed on a sucrose gradient before (c) and after (d) treatment with sodium dodecyl sulfate. The subunit association mixture in (0) was centrifuged for 1.1 h at 60,000 revs/min and 10 mM-Mg 2+, t,he RNA product in (d) for 3.5 h at 17°C and 60,000 revs/min.
B.
HAl’KE
AND
Sedimention
H.
NOLI,
volue (S)
70 50 30
l-
b)
-T
Effluent
volume
(ml)
FIG. 6. Parallel loss of ability to form couples ((a) to (d)) and poly(U)-dependent phenylalanine binding) (e) to (h)) on treatment of 50 S subunits with fiber-bound RNase. 60 S subunits (1.1 A 260 units) were incubated in 0.25 ml TMND buffer (see Materials and Methods) for 10 min at 30°C at the following concentrations (pg/ml) of fiber-bound RNase (Enzite-RNase, GallardSchlesinger): (a) 0, (b) 200, (c) 500 and (d) 1000. The RNase was removed by centrifugation for 10 min at 20,000 revs/min in the IEC SB-406 rotor. To 0.1.ml portions of the supernatant containing 0.44 AsGo unit were added activated 30 S subunits in 6 ~1 buffer G for association analysis on sucrose gradients. Another 0.1.ml portion of each supernatant was added to 0.125 ml TMND buffer containing 10 pg poly(U), 100 pg tRNA-OH, 0.5 mM-GTP, 0.6 A,,, unit of 30 S a(2800 cts/min per pmol) and saturating amounts of subunits, 1.0 A,,, unit of [sHIphe-tRNA elongation factor T. After incubation for 20 min at 3O”C, the mixtures were analyzed on sucrose ) Absorbance at 260 nm; gradients (1.2 h at 60,000 revs/min and 10 mix-Mg”). ( (--O--O-3H radioactivity.
4. Discussion Vacant ribosome couples prepared by a variety of methods fall into two broad classes, tight and loose couples, that differ in stability with respect to pressuredependent and Mg2+ concentration-dependent dissociation. The existence of two populations corresponding to tight and loose couples was suggested by the biphasic dissociation curve obtained as a function of the Mg2+ concentration by Van Duin et al. (1970) and Spirin et al. (1971).
STRUCTURAL
DYNAMICS
OF BACTERIAL
RIBOSOMES.
IV
107
The two classes of particles can be separated preparatively into tight couples, loose couples and subunits by two successive centrifugations. In the first step at 6 mM-Mg2 + only the tight couples resist dissociation while the loose couples appear as subunits. Recentrifugation of the pooled subunits at 15 mMMg2 + separates the loose couples as 70 S particles from contamination by inactive above 6 mM, the t*wo subunits that are unable to associate. At Mg2+ concentrations populations can be distinguished by the greater sensitivity of the loose couples to pressure-dependent dissociation. Each subunit exhibits a characteristic pattern with regard to subunit interaction. the active form of the 30 S particle (30 S a) tends Thus, at low Mg2+ concentrations, to undergo a transition to a conformational modification (30 S b) that cannot associat’e with active 50 S particles unless returned to the a-state by thermal activation according to the procedure of Zamir et al. (1971). The 50 S subunits, on the other hand, are responsible for the formation of loose couples (50 S b) by changing irreversibly to a state of much reduced affinity for their 30 S partners. Since fresh extracts that have are devoid of loose not been exposed to low Mg2 + or high NH + or K + concentrations couples, the latter must be derived from tight couples by some kind of structura)l alteration. Att’empts to convert 50 S a-subunits into the b-form by treatment’ with RNase were not successful because the resulting particles exhibited a much widei spect’rum of reduced affinities for active 30 S subunit,s than the typical 50 S b-particles formed by incubation at 37°C. Neither could the transition t,o t,he b-form be related bo breaks of 23 S rRNA into fragments that are detectable by sedimentation analysis. More recent studies rule out the removal of more than three nucleotides from the ends of 23 S or 5 S RNA or alterations in the 50 S proteins as the cause of the 50 S a to 50 S b conversion and suggest that the change is conformational (Stahli, 1975). We have implied that the differences in sensitivity to dissociation by pressure exhibited by tight and loose couples are a reflection of the affinity of the subunit’s for each other. This notion is based on the observation that dissociation in the gradient depends in opposite ways on pressure and Mg2+ concentration. We may explain this relationship qualit’atively as follows. Dissociation by pressure implies that the overall reaction involves a net decrease in t’he molar volume of the particles. We postulate that the resulting deformation of the subunits distorts the complrmen&y interfaces and thus reduces the interaction. At increased Mg2+ concentrations the particles are st’abilized to resist deformation. Since the phosphate groups of ribosomal RNA are the primary Mg 2+ binding sites, the rigidity of the particles appears to be largely determined by the three-dimensional structure of the RNA backbone. Removal of Mg2+ promotes repulsion of the negative phosphate charges and rearrangement of the secondary and tertiary RNA structure. Breaks in the 23 S RNA at, critical regions would have a similar effect and explain the much greater sensitivity to dissociation by pressure observed after mild treatment of ribosomes v&h RNase. Bosch and co-workers (Van Diggelen et al., 1971b; Van Diggelen $ Bosch. 1973) observed that the association products of 30 S subunits with native 50 S particles sedimented more slowly than the corresponding couples formed with derived 50 S subunits and concluded that the ability to form two association products was an intrinsic capacity of 50 S subunits. Our examination of native subunits in paper II of this series (M. No11 et al., 19733) showed that native 50 S in contrast to native 30 S subunits are severely damaged. Most of them had lost the ability to combine with 30 S a and the remaining subunits resembled 50 S b and RNase particles.
10X
K. Ht\PKE
;\SU
H.
NOLL
Furthermore, the data of Bosch do not support’ his interpretation that derived 50 S particles are a homogeneous population of 50 S a-particles. On the contra,ry, the presence of more than SO:/;, of subunits at 5 mM-Mg2+ and about 30”,, at 10 mM-Mg2 ’ (Van Diggelen et aZ.: 1973, Fig. 7) implies that their preparation of derived 50 S contained about one-third each of 50 S a, 50 S b and 50 S c-particles, in good agreement with our own findings. Moreover, in t’he light of our finding that all 50 S b and 50 S c particles are derived from the 50 S a-form, t’he classification of couples according t’o the origin of their 50 S subunit (native or derived) is misleading. Conversion of tight to loose couples profoundly affects their biochemical activities such as tRNA binding, translocation, response to initiation factors, Mg2+ optimum for initiation etc. (H. No11 et al., 1973). Although these will be described in detail in in preparation), it should be pointed a subsequent paper (Hapke et al.. manuscript out that many of the conflicting reports on the detailed mechanism of translation can be attributed to differences in the quality of t’hr ribosomes employed. The authors are indebted to Professor Ada Zamir for stimulating discussion. This work was supported by research grant no. P381F from the American Cancer Society and grant no. CA-11797 from the United States Public Health Service. One of us (H. N.) is a Lifetime Career Professor of fhe American Cancer Societ,g.
REFERENCES Herzog, H., Ghysen, A. & Bollen, A. (1971). PEBS Letters, 15, 291-294. Kikuchi, A. & Monier, R. (1970). FEBS Letters, 11, 157. Kurland, C. G. (1966). J. Mol. Biol. 18, 90p108. McCarty, K. S., Stafford, D. & Brown, 0. (1968). Anal. Biochem. 24, 31&329. Noll, H. (1967). Nature (London), 215, 360-363. Noll, H. (1969). In Techniques in Protein Biosynthesis (Sargent, J. $ Campbell, P. N., eds), vol. 2, pp. 101-171, Academic Press, London. Noll, H. (1971). BierteZjahresschr. Naturforsch. Gee. Zurich, 116, 377-402. Noll, H. & Stutz, E. (1968). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12B, pp. 129-155, Academic Press, New York and London. Noll, H., Noll, M., Hapke, B. & Van Dieijen, G. (1973). In Regulation of Transcription and Translation, Mosbach Colloquium ivo. 24 (Bautz, E., ed.), pp. 257-311, Springer Verlag, New York and Heidelberg. Noll, M. (1972). Thesis, Northwestern University. Noll, M. & Noll, H. (1972). Nature New BioZ. 238, 225-228. Noll, M. & Noll, H. (1974). J. Mol. BioZ. 90, 237-251. Noll, M. & Noll, H. (1976). J. Mol. BioZ. 105, 111.-130. Noll, M., Hapke, B., Schreier, M. & Noll, H. (1973a). J. Mol. BioZ. ‘75, 281-294. Noll, M., Hapke, B. & Noll, H. (1973b). J. Mol. BioZ. 80, 519-529. Schreier, M. H. & Noll, H. (1970). Nature (London), 227, 128-133. Schreier, M. H. & Noll, H. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 805.-809. Spirin, A. S. (1971). FEBS Letters, 14, 349-353. Spirin, A. S., Sabo, B. & Kovalenko, V. A. (1971). FEBS Letters, 15, 197-200. Staehelin, T. & Maglott, D. R. (1971). In Methods in Enzymology (Moldave, K. & Grossman, L., eds), vol. 20, pp. 449-456, Academic Press, New York. Stahli, C. (1975). Thesis, Northwestern University. Subramanian, A. R. & Davis, B. D. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 2453-2457. Van Diggelen, 0. P. & Bosch, L. (1973). Eur. J. Biochem. 39, 499-510. Van Diggelen, 0. P., Oostrom, H. & Bosch, L. (197la). FEBS Letters, 19, 115-120. Van Diggelen, 0. P., Heinsius, H. L., Kalousek, F. & Bosch, L. (1971b). .T. Mol. BioZ. 55, 277-281. Van Diggelen, 0. P., Oostrom, H. & Bosch, L. (1973). Eur. ,I. Biochem. 39, 511-523.
STRUCTURAL
DYNAMICS
Van Duin, J ., Van Dieijen, Biochem.
OF BACTERIAL
G.. va,n Knippenberg,
RIBOSOMES.
IV
1’. H. & Bosch, L. (1!)70). Eur.
17, 433-440.
Zamir. A., Miskin, R. & Elson, 1). (1971). J. Mol. Biol. 60, 347-364. Zitomrr, K. S. & Flaks, ,J. G. (1!372). J. Mol. Biol. 71. 263.~279.
109 .I.