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Structural studies on Escherichia coli ribosomes
184
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96399
STRUCTURAL STUDIES ON E S C H E R I C H I A
COLI R I B O S O ' I I E S
III. D E N A T U R A T I O N AND SEDIMENTATION OF RIBOSOMAL SUBUNITS U N F O L D E D IN U R E A MARGARET E. ROBERTS AND I. O. W A L K E R
Biochemistry Department, University o/Ox[ord, Ox/ord (Great Britain) (Received July 3ist, I969) (Revised manuscript received October 29th, 1969)
SUMMARY
The addition of urea to 5o-S ribosomal subunits caused a lowering of the sedimentation coefficient to 32-38 S by a one-step co-operative process. This was interpreted as an unfolding of the native ribosome caused by the disruption of hydrophobic bonds between ribosomal proteins. In many cases slow-moving components were evident in tile ultracentrifuge which were probably dissociated protein. Treatment of 5o-S ribosomes with formaldehyde prevented the dissociation of the protein from RNA on subsequent reaction with urea. The ribosomes appeared to unfold to give slower-sedimenting species but the nature of the unfolding was different from that of untreated ribosomes. Thus the ribosomes were more sensitive to reaction with urea and tile unfolding was no longer highly co-operative. The addition of varying amounts of EDTA to formaldehyde-treated ribosomes produced a single unfolded component with a sedimentation coefficient of 34-38 S. Unlike untreated ribosomes no further unfolding occurred, either with time or with a large excess of EDTA. No loss of secondary structure in the RNA occurred when native or formaldehyde-treated ribosomes were reacted with urea or EDTA at room temperature. The melting temperatures decreased in urea and EDTA and the melting curves became less co-operative. The melting profiles of unfolded ribosomes in urea and EDTA were similar to RNA in the same solvent and showed that in tile unfolded form the protein had little effect on the thermal denaturation properties of the RNA. It is concluded that in the unfolded ribosomes, characterised by a sedimentation coefficient of 3o-35 S, the major bonds involved in the maintenance of ribosomal tertiary structure have been broken. These bonds involve interactions between the proteins of the ribosome.
INTRODUCTION
The tertiary structure of the ribosome and of the ribosomal subunits can be disrupted by removing Mg2+ which form an integral part of the native structure 1-'~. The great majority of these ions do not, however, appear to play a specific role in maintaining the tertiary structure of the ribosome since they may be completely replaced by Mn 2~ (ref. 4) with no loss in biological function 5 or by monovalent Biochim. Biophys. Acta, i99 (197 o) i84 I93
UNFOLDING OF RIBOSOMES IN UREA
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cations 2. On the basis of denaturation and sedimentation studies of ribosomal subunits which had been unfolded in E D T A it has been suggested that Mg 2+ functions as bound counter ion, thus decreasing the electrostatic free energy of the subunit and allowing other forces mediated by the protein to maintain the tertiary structure of the ribosome 1. Although the nature of these forces is not known it is reasonable to assume that they might at least in part be stabilised by hydrophobic interactions. We have therefore examined the sedimentation and thermal denaturation behaviour of 5o-S subunits in urea, since this reagent is thought to weaken hydrophobic interactions ~. Preliminary studies (S. H. MIALL AND I. O. WALKER,unpublished observations), which we have confirmed here, showed that urea unfolded the ribosome but it also caused some dissociation of the protein from the RNA. In order to avoid this the ribosomes were reacted with formaldehyde, which appeared to covalently bond the protein to tile RNA, and were then subjected to urea. The properties of the formaldehyde-treated ribosomes were examined in urea and EDTA and the results are interpreted in terms of the tertiary structure of the ribosome. METHODS
Preparation o/ribosomes, 5o-S subunits and RNA Ribosomes were prepared from Escherichia coli MRE 6oo, a ribonuclease-free strain which was obtained as a frozen paste from the Microbiological Research Establishment, Porton, by the method described previously 1. 5o-S subunits were prepared from a mixture of dissociated subunits by sucrose density gradient centrifugation. The ribosome suspension (2 ml, I5 mg/ml) was layered onto a 5-3o % linear sucrose gradient (28 ml) and centrifuged in a swinging bucket rotor for z2 h at 24.000 rev./min (6o ooo ×g.). The tubes containing the 5o-S subunits were pooled and dialysed against o.or M sodium phosphate-I mM magnesium acetate (pH 7.0) to remove the sucrose. The final concentration of 5o-S subunits was I - 2 mg/ml. RNA was prepared by a modification of the method of SPITNIK-ELsoN~. The precipitate of RNA was centrifuged, resuspended in 3 M LiC1-4 M urea and reprecipitated by centrifugation. The washed precipitate was resuspended in a small volume of o.oI M sodium phosphate buffer (pH 7.0) - I mM magnesium acetate and 2 vol. of ethanol were added. The precipitate of RNA was collected by centrifugation, dried in a fast stream of air until dry and powdery, and then dissolved in o.oi M sodium phosphate-i.o mM magnesium acetate (pH 7.0).
Analytical ultracentri/ugation Sedimentation coefficients were measured in a Spinco Model E analytical ultracentrifuge equipped with ultraviolet and schlieren optics. All measurements were made at zo °, and sedimentation coefficients (s20,~) were calculated from microdensitometer tracings of ultraviolet film, or from microcomparator measurements of schlieren photographs, with corrections made for the viscosity of the solvent where necessary. The quantities of sedimenting components were estimated from the areas under the schlieren peak by tracing enlarged projections of the photographs onto squared paper. Corrections were made for radial dilution. For most experiments the standard centrifuge cells were used, but for some, in which the solvent gave a very Biochim. Biophys. Acta, 199 (197 o) 184-I93
186
M. E. R O B E R T S , I. O. W A L K E R
curved baseline, a double sector cell, or a synthetic boundary cell was used to give a sharper boundary and to resolve minor slow-moving components.
Melting profiles Melting profiles were measured by heating ribosome solutions in stoppered cells in an electrically heated cell compartment attached to a Unicam SP5oo spectrophotometer using the procedure and precautions described previously I. The hyperchromicity is the percentage increase in absorbance at a given temperature compared with its value at room temperature. The melting temperature of the solution is defined as the temperature at which half of the hyperchromicity at 9°0 (where most of the curves have reached maximum hyperchromicity) has occurred. The maximum gradient of the curve was also measured.
Treatment with reagents A concentrated solution of formaldehyde was neutralised to pH 7.0 with NaOH and added directly to the ribosome solution at o ° in appropriate amounts to give a final concentration of 4 %- The usual reaction time was from 8-15 h although shorter times yielded essentially the same results. Urea and Na z EDTA were added directly to the solutions in appropriate amounts. Where urea and EDTA were added to the formaldehyde-treated ribosomes, this was either in the continued presence of formaldehyde, or after the formaldehyde had been diluted or dialysed out. There was no apparent difference in the results. In all cases the buffer was o.o~ M sodium phosphate I mM magnesium acetate (pH 7.o) and all reagents, except EDTA, were made up in this buffer.
RESULTS
Un/olding native 5o-S subunits in urea The treatment of native 5o-S subunits with varying concentrations of urea for 2- 5 h resulted in the conversion of some of the 5o-S particles to components moving with decreased sedimentation coefficients. Although the proportions of the various components in any particular urea concentration varied considerably with different preparations of ribosomes the sedimentation coefficients remained relatively constant. In I M urea there was either no change, or occasionally a small amount of a component sedimenting at 32 S-38 S appeared (Fig. Ia). In 2 M urea the proportion of the slower-
7i~:,1:4
I:i~4. i. T h e s e d i m e n t a t i o n p a t t e r n s a t 20 o f 5 o - S r i b o s o m a l s u b u n i t s a f t e r v a r i o u s t r e a t m e n t s . a. N a t i v e 5 o - S s u b u n i t s in I M u r e a . b. N a t i v e 5 o - S s u b u n i t s in 2 M u r e a ( l o w e r p i c t u r e ) a n d 4 M u r e a ( u p p e r p i c t u r e ) , c. F o r m a l d e h y d e - t r e a t e d 5 o - S s u b u n i t s in o.75 M u r e a a f t e r 6..5 h. d. F o r m a l ¢ l e h v d e - t r e a t e d 5 o - S s u b u n i t s in 3 M u r e a a f t e r 2. 5 h.
Biochim.
Biophys.
,4eta, 199 (197o} 1 8 4 - I 9 3
UNFOLDING OF RIBOSOMES IN UREA
18 7
moving component, which again sedimented at 32-38 S, increased, in some cases becoming the major component (Fig. Ib). However, in m a n y cases there was evidence of a heterogeneous slow-moving component at the meniscus. With increasing urea concentration the proportion of material at the meniscus tended to increase, and in 3 or 4 .'V[urea there was sometimes no rapidly-moving component visible (Fig. Ib). The nature of tile slow-moving components has not been studied; they were presumed to be dissociation products of the ribosome although degradation of the RNA cannot be ruled out. The process whereby the 5o-S subunit is converted to a slower moving particle appeared to be co-operative; in all cases where both components were present simultaneously, they separated completely, and no particles with intermediate sedimentation coefficients were seen.
The e/]ect o[ [ormaldehyde on 5o-S subunits and ribosomal R N A In an a t t e m p t to prevent dissociation of the protein from the nucleic acid ribosomes were first reacted with formaldehyde prior to the addition of urea. SPIRIN et al. 8 have shown that formaldehyde prevents the dissociation of protein from ribosomes in high concentrations of salt. The mode of action of the formaldehyde is unknown, but presumably covalent cross-links are formed between the proteins or the proteins and the RNA. Treatment with 4 % formaldehyde produced no change in the sedimentation coefficients of either 5o-S subunits or I6-S and 23-S RNA. When formaldehydetreated ribosomes were reacted with 3 M LiC1-4 M urea for 24 h at o ° no precipitate of RNA was formed. This procedure caused complete dissociation of the protein with the precipitation of the free RNA when applied to native 5o-S ribosomes 7. Concentrated LiCI will completely strip ribosomal proteins from RNA, provided that the ribosomes are first unfolded by removing Mg 2~ with EDTA 9. By analogy, it might be argued that in the experiment described above 4 M urea is the unfolding agent, and that the 3 M LiCI then dissociates the proteins from the RNA. As described later, 4 M urea extensively unfolded formaldehyde-treated ribosomes. This suggested that the action of the formaldehyde in this case was to covalently link at least some of the protein onto the RNA. The effect of formaldehyde treatment on the ribosome was found to persist even when the formaldehyde was extensively diluted out or removed by dialysis against buffer and it is concluded that the reaction is not appreciably reversible. U n/olding /ormaldehyde-treated 5o-S subunits E D T A treatment. When formaldehyde-treated subunits were reacted with EDTA at a concentration sufficient to remove all Mg~+ present in solution the 5o-S subunit appeared to be completely converted to a slower-moving component. The sedimenting boundary was very sharp and had a sedimentation coefficient in the range 34-38 S. This may be compared with the 35-S component found as the first stage in the EDTA unfolding of native 5o-S subunits t. The sedimentation coefficient remained remarkably constant with different concentrations of EDTA, and over reaction times of several hours (Table 1). There was no evidence of any slow-moving material at the meniscus. By contrast, native 5o-S ribosomes in 3 mM EDTA produced two components after 3o min, in approximately equal amounts, with sedimentation coefficients of 21 and 16 S (c/. ref. I). Biochim. Biophys. Acta, 199 (197 o) 184-193
188
M.E. ROBERTS, I. O. WALKER
TABI.E I SEDIMENTATION
COEFFICIENTS
OF UNFOLDED
IOUS T I M E S A F T E R T H E A D D I T I O N OF
FORMALDEHYDE-TREATED
I'2D7",4 conch. ( m M )
Time Onin)
s,o,,~
I. 5 1.5
3° 14{} qoo Iio
38 36 34 35
2.0
7-5
50-S
R I B O S O M E S AT VAR-
EDTA
Urea treatment. When formaldehyde-treated ribosomes were reacted with urea o n l y one c o m p o n e n t was o b s e r v e d a n d t h e s e d i m e n t a t i o n coefficient d e c r e a s e d p r o g r e s s i v e l y w i t h i n c r e a s i n g u r e a c o n c e n t r a t i o n (Fig. IC). T h e f o r m a l d e h y d e a p p a r e n t l y s e n s i t i s e d t h e r i b o s o m e s to u r e a as s o m e d e c r e a s e of t h e s e d i m e n t a t i o n coe f f i c i e n t o c c u r r e d in 0.25 M u r e a w h e r e a s l i t t l e effect was o b s e r v e d w i t h n a t i v e ribos o m e s in u r e a c o n c e n t r a t i o n s less t h a n I M. T h e s e d i m e n t a t i o n coefficients w e r e v a r i a b l e a n d d e p e n d e d b o t h on u r e a c o n c e n t r a t i o n a n d on t i m e . H o w e v e r , at least u p to 2.0 M urea, a n d for r e a c t i o n t i m e s of less t h a n IO h, a single c o m p o n e n t was o b s e r v e d w i t h a s e d i m e n t a t i o n c o e f f i c i e n t u s u a l l y in t h e r a n g e 2 5 - 5 0 S (Fig. 2). In h i g h e r u r e a c o n c e n t r a t i o n s , o r for l o n g e r r e a c t i o n times, s e d i m e n t a t i o n c o e f f i c i e n t s in t h e r a n g e 16-25 S or e v e n below, w e r e s o m e t i m e s o b s e r v e d , b u t g e n e r a l l y t h e v a l u e s ,
|
|
|
5G
40
3C
v~
2C
Urea concn,(M)
Fig. 2. The sediinentation coefficients at 20" of formaldehyde-treated 5o-S subunits, as a function of urea concentration after I-IO h treatment. Different symbols represent different ribosome preparations. Q-C), after 5-6 h treatment. Biochim. Biophys. Acta, 199 (I97 o) I84 ~93
UNFOLDING OF RIBOSOMES IN UREA
189
remained constant at about 25-28 S between 2 and 4.0 M urea (Figs. Id and 2). Within these limits the results tended to be rather variable between different 5o-S subunit preparations, and depended also on the age of the preparation. The variability is illustrated in Fig. 2. However, for any given experiment and ribosome preparation the sedimentation coefficient, was found to decrease fairly linearly with urea concentration, at least between o.25 and i.o M urea. Quantitative measurements made on the assumption that the molecular weight remained constant, indicated that the spreading of the boundary was greater than could be accounted for by diffusion alone, and it is concluded that the sedimenting component was heterogeneous. No slowly-sedimenting component was visible at the meniscus in any of the schlieren photographs and synthetic boundary cells showed no evidence of a slow-moving component. In a further attempt to show that no dissociation of protein had occurred the RNA:protein ratios were measured in the following way. The components were isolated from possible dissociation products on a density gradient of sucrose (5-3o %), made up in each case to the appropriate urea concentration. Protein was measured by Folin's reaction, using native 5o-S subunits as a standard, and RNA was estimated from the absorbance at 26o m/t. As shown in Table 1I, there was no evidence of any progressive loss of protein, although the results showed a fairly wide scatter around the value of 37 °'o protein found for native ribosomesL It is likely therefore that the components observed in urea solution result from the unfolding or swelling of the 5o-S subunits without loss of protein. "FABLE iI THE PERCENTAGE X V E I G t I T O F P R O T E I N IN F O R M A L D E I I Y D E - T R E A T E D IN UREA AND ISOLATED FROM DISSOCIATION PRODUCTS BY SUCROSE
Urea conch. (3I)
Protein in ribosome component (oo)
o.oo 0-25 0.50 0.75
32 34 38 35
1.0
41
50-.~ RIBOSOMES UNFOLDED GRADIENT CENTRIFUGATION
Reversibility Attempts were made to reverse the effect of the urea by dialysis against buffer. This treatment was applied to components which had been isolated on a sucrose density gradient as described previously, and on unfolded ribosomes dialysed directly against buffer after urea treatment for 2-4 h. In no case was any increase in sedimentation coefficient observed after dialysis. Thus no reversibility could be demonstrated.
Melting profiles No change in absorbance at 260 raft was obserw~d when native or formaldehydetreated ribosomes were reacted with EDTA or urea, and it is concluded that, at room temperature, the secondary structure of the RNA is unchanged by the addition of these reagents. When 5o-S subunits were melted in increasing concentrations of urea the melting temperatures progressively decreased. The gradient of the curves also decreased Biochim. Biophys. Acta, x99 (197 o)
I84-I93
I90
M . E . ROBERTS, I. O. WALKER
until in 3.0 M urea the gradient was about the same as that of RNA (Fig. 3). The decrease in the melting temperature may be explained by the destabilising effect of urea on the secondary structure of the RNA since a similar effect was observed for RNA alone, where the melting temperature in 2 M urea decreased by 8 °, from 62 to 54 ° (Fig. 5). The decrea.se in the gradient of the curve most likely results from the effect of urea in destroying the tertiary structure of the ribosome, thus causing the melting behaviour to become less co-operative and more like that of RNA. This is borne out by the observation that the melting curve of the s u b u n i t s in 2 M urea, at which concentration they have m a i n l y unfolded to form a 35-S c o m p o n e n t , was very similar to that of R N A in the same solvent except that the melting temperature of the s u b u n i t s was greater than that of the R N A b y 2 '~.
1+ I /
80 100
o 6(~r
E o 80
/ ///// /
/
/11 f /
+ooL/
~o 60 ~)
~.O M-.../
/////
"-- 20
E c
O
Z
~OM
20 °
40 ° Temp.
60 °
40 °
80 o
6(3°
80"
Fig. 3. The m e l t i n g profiles of n a t i v e 5o-S s u b u n i t s a n d R N A in urea: 5o-S s u b u n i t s in () M ( 0 - 0 } , I M (KI .'~1), 2 M ( ~ ~ ) a n d 3 M n r e a ( O - O ) ; I¢.NA in 2 M ure a ( A - A ) . Fig. 4. T h e m e l t i n g profiles of f o r m a l d e h y d e - t r e a t e d 5o-S s u b u n i t s in v a r i o u s c o n c e n t r a t i o n s of urea.
,
,
,
,
100
"~ 8 0 6
E 6o tv
Jc do ,~ 20 -6
E
o :7
O
20=
40"
EO °
80"
Temp. Fig.
5. "]'he m e l t i n g p r o f i l e s o f n a t i v e a n d f o r m a ] c l e h y d e - t r e a t e d
5o-S s u b u n i t s , in [ o m M ~ o d i u m
(pH 7.o)--1 mM m a g n e s i u m a c e t a t e and in 2 mM E D T A . N a t i v e 5o-S s u b u n i t s in buffer ( O - O ) a n d in 2 mM E D T A (A---&). F o r m a l d e h y d e - t r e a t e d 5o-S s u b u n i t s in buffer ( O O ) a n d in 2 mM E I ) T A ( A - & ) . The m e l t i n g profiles of I
el-D). Biochim. Biophys. Acta, 199 (i97 o) i84 .I93
UNFOLDING OF RIBOSOMES IN UREA
191
When 5o-S subunits were treated with formaldehyde, their melting behaviour was altered significantly. The gradient of the curve remained the same, but the melting temperature increased by 3-5 ° (Fig. 4). This must be due to the stabilisation of bonds normally broken during thermal denaturation. The addition of urea to formaldehyde-treated ribosomes resulted in the progressive lowering of the melting temperature, again presumably due to the destabilising effect of urea on the secondary structure of the rRNA. However, from 0.25 to i.o M urea tile gradient of tile curves, and the overall shape, remained the same, showing that the denaturation process was still highly co-operative, although judging by ttle decrease in the sedimentation coefficients, some change in the tertiary structure of the subunits had occurred under these conditions. In 2.0 and 3.0 M urea the melting temperature further decreased, and the gradients of the curves also decreased so that the shapes of the curves at these higher urea concentrations were essentially the same as those of native 5o-S subunits and RNA under the same conditions. In 2 mM EDTA, the melting behaviour was found to be the same for formaldehyde-treated and untreated ribosomes. Both curves were similar to RNA ill 2 mM EDTA (Fig. 5). This indicated that, in the 35-S component which is formed under these conditions, the protein has no effect on the melting of the RNA secondary structure. DISCUSSION
The sedimentation coefficients of native 5o-S ribosomal subunits in urea solution suggest that a co-operative conversion to a component of 32-38 S is occurring. EDTA has a similar effect on 5o-S subunits, causing a co-operative unfolding, via a 35-S particle, to 2I-S and I6-S particles. The melting profiles of the ribosomes in 2 M urea show that the tertiary structure has been largely destroyed. Again this behaviour m a y be compared to that of the 35-S unfolded particle in EDTA 1. These similarities strongly suggest that the particle formed in urea and the particle formed in the first stage of E D T A unfolding are the same. The hydrodynamic volumes of the 5o-S subunits and 23-S RNA, calculated by the method described previously 1, shows that a 35-S particle would occupy about the same volume as free 23-S rRNA in its most compact state (28 S), whereas 5o-S particles are much smaller. Clearly RNA is held in its more compact form in the native subunit by bonds involving the ribosomal proteins, and these bonds are broken in the 5o-S to 35-S transition. If ttle 35-S component observed in urea-treated subunits is indeed an unfolded component, analogous to tile first step in EDTA unfolding, then this would suggest that the bonds holding the 5o-S subunit in its highly compact conformation are mediated, at least in part, by hydrophobic interactions. These are likely to be protein.protein bonds since protein-RNA bonds are predo,ninantly electrostatic, judging by their lability to solvents of high ionic strength. However, the existence of a hydrophobic contribution from the latter cannot be excluded on the results presented here. The secondary structure of the RNA in 5o-S subunits is unchanged in urea at room temperature, but the lowering of the melting temperature indicates that the secondary structure has become destabilised. This destabilisation is independent of the tertiary structure of the ribosome, as it occurs in free RNA treated with urea. The melting profiles of 5o-S subunits in urea also show a decrease in gradient, and Biochim. Biophys. Acta, 199 (I97 o) I84-I93
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M.E.
R O B E R T S , I. O. W A I . K E R
become similar to RNA in 2 M urea, which shows that the protein in tile 35-S component which is present at this urea concentration has little effect on the denaturation of the RNA. This result is supported by tile melting behaviour of subunits in EDTA. Formaldehyde-treated ribosomes in 2 mM EDTA, in which they form a 35-S component, melt identically to untreated 5o-S subunits, and to RNA, in the same. solvent. This implies that tile bonds broken in the formation of the 35-S component are the ones responsible for the highly co-operative denaturation of 5o-S subunits. It has been argued elsewhere that these bonds are also responsible for maintaining the compact tertiary structure of the ribosome ~. However, it cannot be unequivocally shown that the 35-S comt)onent found in urea is the result of unfolding the wholc 5o-S subunit, and not tim result of somc dissociation of proteins. In many cases in 2 M urea, and higher concentrations, there is evidence of a slow-moving and heterogeneous component at the meniscus, which could bc dissociated protein. In an attempt to investigate the effect of urea on the ribosome tertiary structure without the colnplication of protein dissociation, prior trcatmcltt with formaldehyde was adopted. Reaction with formaldehyde, under the conditions described, does not appear to affect the secondary structure of the RNA, or the gross tertiary structure of the ribosome. There is evidence that formaldehyde covalcntlv links at least a proportion of the proteins onto the ribosome. Spirm has shown that a protein fraction, known as the "split proteins", which normally dissociates in a high-ionic-strength solvent, remains attached after formaldehyde treatment s. The work described hcrc showed that, when formaldehyde-treated ribosomes were unfolded in 4 M urea and reacted with 3 M LiC1, no precipitate of free RNA was formed, unlike native 5o-S subunits under the same conditions. This suggests that the action of the formaldehyde is to attach at least some of the protein onto tile RNA, presumably by cross-linking, although crosslinking between proteins may also bc important. Unfmtunately the mode of action of formaldehyde is unknown in this case. FELl)MANl° has shown that RNA can be cross-linked by methylene bridges between purine groups, and FRAENKEL-(~ONRAT AND Or.COTTl~ reported the formation of stable methylene bridges between amino groups and guanidyl groups, phenol, imadazole and indolc rings treated with formaldehyde. The latter could explain both protein RNA and t)rotein-protein cross-linking. In urea the sedimentation coefficient of formaldehyde-treated 5o-S subunits is decreased. This is due, apt)arently, to the unfolding or swelling of the whole subunit since no evidence of dissociation was found. These results lend further weight to the suggestion that the (:onapact tertiary structure of the 5o-.':, ribosome is maintained by hydrophobic bonds. Attempts to reverse the unfolding by dialysis against buffer were not successful. However, the process of unfolding is, in many respects, different from that shown by native 5o-S subunits in EDTA and urea. First, the formaldehyde appears to have sensitised the subunits to urea: appreciable unfolding occurs at concentrations below r M, whereas native 5o-S subunits show little or no unfolding in i M urea. In addition the stepwise co-operative nature of the unfolding appears to have changed, such that after formaldehyde treatment only a single sedimenting peak is seen in urea, which is considerably broader than the boundary of the native subunit. Thus unfolding, in this ease, appears to be a non-co-operative, continuous process. Similarly the EDTA unfolding of formaldehyde-treated subunits results in a single sedimenting Biochim. Biophys. Acta, 199 (r97o) t84-r93
UNFOLDING OF RIBOSOMES IN UREA
193
component. Furthermore, over a wide range of EDTA concentrations, and after different reaction times, the sedimentation coefficient of the unfolded component remained constant at 35 S. This could be the same as the 35-S component found as the first stage in the EDTA unfolding of native subunits. Tile melting profiles of formaldehyde-treated subunits in urea show a progressive lowering of the melting temperature, due to the destabilising effect of urea on the RNA secondary structure. However, a striking feature of the curves is that their gradient and overall shape remain very similar to that of the native 5o-S subunits over the range o.25-1.o M urea. in 2.o and 3.o M urea the curves flatten out and become very similar to tile curves of native 5o-S subunits in the same urea concentrations. Clearly at the lower urea concentrations at least some of the protein bonds causing the melting of the native subunits to be highly co-operative remain intact, and are only broken at the higher urea concentrations. The finding that the 35-S component found at the first stage in EDTA or urea unfolding has roughly the same hydrodynamic volume as free RNA suggests that it m a y be significant as the stage at which important protein bonds, holding the 5o-S subunit in its compact tertiary conformation, have been broken. In native subunits further unfolding can take place, as shown by the 2I-S and I6-S components found in EDTA ~, but after formaldehyde treatment, this further unfolding is abolished, probably as a result of cross-linking. It is also suggestive that unfolding below 35 S is not usually found in urea. Therefore the components with sedimentation coefficients less than 35 S probably result from the disruption of electrostatic or hydrogen bonds. In all cases the melting profiles of the 35-S components are similar to RNA under the same conditions, showing that the protein in these components is exerting no effect on the denaturation of the RNA. This provides further evidence that the 5o-S to 35-S transition involves the breaking of the major bonds maintaining ribosomal tertiary structure.
ACKNOXVLEDGEMENTS
The authors wish to thank the director and staff of the Microbiological Research Establishment, Porton, for supplying the frozen paste of E. coli MRE 600. One of them (M.E.R.) wishes to thank the Medical Research Council for financial support.
REFERENCES I 2 3 4 5 6 7 8 9 IO ii
S. H. MIALL AND I. O. WALKER, Biochim. Biophys. Acta, 174 (1969) 551. L. [-'. GAVRILOVA, D. A. IVANOV AND A. S. SPIRIN, J . Mol. Biol., 16 (1966) 473. R. I;. GESTELAND, J. Mol. Biol., I8 (1966) 356. B. SHEARI), S. H. MIALL, A. 1{. PEACOCKE, 1. O. WALKI-:R AND 1{. E. RICHARDS, J. Mol. Biol., 28 (1967) 389. A. TISSII~RES, n . SCHLESSINGER AND F. GROS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 145o. P. I.. WHITNEY AND C. TANFORD, J. Biol. Chem., 237 (1962) I735. P- SPlTNIK-ELSON, Biochem. Biophys. Res. Commun., 18 (i965) :557. A. S. SPIRIN, N. V. I'IEI.1TSINA AND M. I. LERMAN, .]. Mol. Biol., 14 (1965) 6 t l . P. SPITNIK-ELSON AND A. ATSMON, F E B S Letters, 2 (1968) 13. M. YA. FELDMAN, Biochim. Biophys. Acta, 149 (1967) 2o. H . FRAENKEL-CONRAT AND H. S. OLCOTT, J. Biol. Chem., I74 (I948) 827.
Biochim. Biophys. Acta, 199 (t97 o) 184-I93
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96399
STRUCTURAL STUDIES ON E S C H E R I C H I A
COLI R I B O S O ' I I E S
III. D E N A T U R A T I O N AND SEDIMENTATION OF RIBOSOMAL SUBUNITS U N F O L D E D IN U R E A MARGARET E. ROBERTS AND I. O. W A L K E R
Biochemistry Department, University o/Ox[ord, Ox/ord (Great Britain) (Received July 3ist, I969) (Revised manuscript received October 29th, 1969)
SUMMARY
The addition of urea to 5o-S ribosomal subunits caused a lowering of the sedimentation coefficient to 32-38 S by a one-step co-operative process. This was interpreted as an unfolding of the native ribosome caused by the disruption of hydrophobic bonds between ribosomal proteins. In many cases slow-moving components were evident in tile ultracentrifuge which were probably dissociated protein. Treatment of 5o-S ribosomes with formaldehyde prevented the dissociation of the protein from RNA on subsequent reaction with urea. The ribosomes appeared to unfold to give slower-sedimenting species but the nature of the unfolding was different from that of untreated ribosomes. Thus the ribosomes were more sensitive to reaction with urea and tile unfolding was no longer highly co-operative. The addition of varying amounts of EDTA to formaldehyde-treated ribosomes produced a single unfolded component with a sedimentation coefficient of 34-38 S. Unlike untreated ribosomes no further unfolding occurred, either with time or with a large excess of EDTA. No loss of secondary structure in the RNA occurred when native or formaldehyde-treated ribosomes were reacted with urea or EDTA at room temperature. The melting temperatures decreased in urea and EDTA and the melting curves became less co-operative. The melting profiles of unfolded ribosomes in urea and EDTA were similar to RNA in the same solvent and showed that in tile unfolded form the protein had little effect on the thermal denaturation properties of the RNA. It is concluded that in the unfolded ribosomes, characterised by a sedimentation coefficient of 3o-35 S, the major bonds involved in the maintenance of ribosomal tertiary structure have been broken. These bonds involve interactions between the proteins of the ribosome.
INTRODUCTION
The tertiary structure of the ribosome and of the ribosomal subunits can be disrupted by removing Mg2+ which form an integral part of the native structure 1-'~. The great majority of these ions do not, however, appear to play a specific role in maintaining the tertiary structure of the ribosome since they may be completely replaced by Mn 2~ (ref. 4) with no loss in biological function 5 or by monovalent Biochim. Biophys. Acta, i99 (197 o) i84 I93
UNFOLDING OF RIBOSOMES IN UREA
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cations 2. On the basis of denaturation and sedimentation studies of ribosomal subunits which had been unfolded in E D T A it has been suggested that Mg 2+ functions as bound counter ion, thus decreasing the electrostatic free energy of the subunit and allowing other forces mediated by the protein to maintain the tertiary structure of the ribosome 1. Although the nature of these forces is not known it is reasonable to assume that they might at least in part be stabilised by hydrophobic interactions. We have therefore examined the sedimentation and thermal denaturation behaviour of 5o-S subunits in urea, since this reagent is thought to weaken hydrophobic interactions ~. Preliminary studies (S. H. MIALL AND I. O. WALKER,unpublished observations), which we have confirmed here, showed that urea unfolded the ribosome but it also caused some dissociation of the protein from the RNA. In order to avoid this the ribosomes were reacted with formaldehyde, which appeared to covalently bond the protein to tile RNA, and were then subjected to urea. The properties of the formaldehyde-treated ribosomes were examined in urea and EDTA and the results are interpreted in terms of the tertiary structure of the ribosome. METHODS
Preparation o/ribosomes, 5o-S subunits and RNA Ribosomes were prepared from Escherichia coli MRE 6oo, a ribonuclease-free strain which was obtained as a frozen paste from the Microbiological Research Establishment, Porton, by the method described previously 1. 5o-S subunits were prepared from a mixture of dissociated subunits by sucrose density gradient centrifugation. The ribosome suspension (2 ml, I5 mg/ml) was layered onto a 5-3o % linear sucrose gradient (28 ml) and centrifuged in a swinging bucket rotor for z2 h at 24.000 rev./min (6o ooo ×g.). The tubes containing the 5o-S subunits were pooled and dialysed against o.or M sodium phosphate-I mM magnesium acetate (pH 7.0) to remove the sucrose. The final concentration of 5o-S subunits was I - 2 mg/ml. RNA was prepared by a modification of the method of SPITNIK-ELsoN~. The precipitate of RNA was centrifuged, resuspended in 3 M LiC1-4 M urea and reprecipitated by centrifugation. The washed precipitate was resuspended in a small volume of o.oI M sodium phosphate buffer (pH 7.0) - I mM magnesium acetate and 2 vol. of ethanol were added. The precipitate of RNA was collected by centrifugation, dried in a fast stream of air until dry and powdery, and then dissolved in o.oi M sodium phosphate-i.o mM magnesium acetate (pH 7.0).
Analytical ultracentri/ugation Sedimentation coefficients were measured in a Spinco Model E analytical ultracentrifuge equipped with ultraviolet and schlieren optics. All measurements were made at zo °, and sedimentation coefficients (s20,~) were calculated from microdensitometer tracings of ultraviolet film, or from microcomparator measurements of schlieren photographs, with corrections made for the viscosity of the solvent where necessary. The quantities of sedimenting components were estimated from the areas under the schlieren peak by tracing enlarged projections of the photographs onto squared paper. Corrections were made for radial dilution. For most experiments the standard centrifuge cells were used, but for some, in which the solvent gave a very Biochim. Biophys. Acta, 199 (197 o) 184-I93
186
M. E. R O B E R T S , I. O. W A L K E R
curved baseline, a double sector cell, or a synthetic boundary cell was used to give a sharper boundary and to resolve minor slow-moving components.
Melting profiles Melting profiles were measured by heating ribosome solutions in stoppered cells in an electrically heated cell compartment attached to a Unicam SP5oo spectrophotometer using the procedure and precautions described previously I. The hyperchromicity is the percentage increase in absorbance at a given temperature compared with its value at room temperature. The melting temperature of the solution is defined as the temperature at which half of the hyperchromicity at 9°0 (where most of the curves have reached maximum hyperchromicity) has occurred. The maximum gradient of the curve was also measured.
Treatment with reagents A concentrated solution of formaldehyde was neutralised to pH 7.0 with NaOH and added directly to the ribosome solution at o ° in appropriate amounts to give a final concentration of 4 %- The usual reaction time was from 8-15 h although shorter times yielded essentially the same results. Urea and Na z EDTA were added directly to the solutions in appropriate amounts. Where urea and EDTA were added to the formaldehyde-treated ribosomes, this was either in the continued presence of formaldehyde, or after the formaldehyde had been diluted or dialysed out. There was no apparent difference in the results. In all cases the buffer was o.o~ M sodium phosphate I mM magnesium acetate (pH 7.o) and all reagents, except EDTA, were made up in this buffer.
RESULTS
Un/olding native 5o-S subunits in urea The treatment of native 5o-S subunits with varying concentrations of urea for 2- 5 h resulted in the conversion of some of the 5o-S particles to components moving with decreased sedimentation coefficients. Although the proportions of the various components in any particular urea concentration varied considerably with different preparations of ribosomes the sedimentation coefficients remained relatively constant. In I M urea there was either no change, or occasionally a small amount of a component sedimenting at 32 S-38 S appeared (Fig. Ia). In 2 M urea the proportion of the slower-
7i~:,1:4
I:i~4. i. T h e s e d i m e n t a t i o n p a t t e r n s a t 20 o f 5 o - S r i b o s o m a l s u b u n i t s a f t e r v a r i o u s t r e a t m e n t s . a. N a t i v e 5 o - S s u b u n i t s in I M u r e a . b. N a t i v e 5 o - S s u b u n i t s in 2 M u r e a ( l o w e r p i c t u r e ) a n d 4 M u r e a ( u p p e r p i c t u r e ) , c. F o r m a l d e h y d e - t r e a t e d 5 o - S s u b u n i t s in o.75 M u r e a a f t e r 6..5 h. d. F o r m a l ¢ l e h v d e - t r e a t e d 5 o - S s u b u n i t s in 3 M u r e a a f t e r 2. 5 h.
Biochim.
Biophys.
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UNFOLDING OF RIBOSOMES IN UREA
18 7
moving component, which again sedimented at 32-38 S, increased, in some cases becoming the major component (Fig. Ib). However, in m a n y cases there was evidence of a heterogeneous slow-moving component at the meniscus. With increasing urea concentration the proportion of material at the meniscus tended to increase, and in 3 or 4 .'V[urea there was sometimes no rapidly-moving component visible (Fig. Ib). The nature of tile slow-moving components has not been studied; they were presumed to be dissociation products of the ribosome although degradation of the RNA cannot be ruled out. The process whereby the 5o-S subunit is converted to a slower moving particle appeared to be co-operative; in all cases where both components were present simultaneously, they separated completely, and no particles with intermediate sedimentation coefficients were seen.
The e/]ect o[ [ormaldehyde on 5o-S subunits and ribosomal R N A In an a t t e m p t to prevent dissociation of the protein from the nucleic acid ribosomes were first reacted with formaldehyde prior to the addition of urea. SPIRIN et al. 8 have shown that formaldehyde prevents the dissociation of protein from ribosomes in high concentrations of salt. The mode of action of the formaldehyde is unknown, but presumably covalent cross-links are formed between the proteins or the proteins and the RNA. Treatment with 4 % formaldehyde produced no change in the sedimentation coefficients of either 5o-S subunits or I6-S and 23-S RNA. When formaldehydetreated ribosomes were reacted with 3 M LiC1-4 M urea for 24 h at o ° no precipitate of RNA was formed. This procedure caused complete dissociation of the protein with the precipitation of the free RNA when applied to native 5o-S ribosomes 7. Concentrated LiCI will completely strip ribosomal proteins from RNA, provided that the ribosomes are first unfolded by removing Mg 2~ with EDTA 9. By analogy, it might be argued that in the experiment described above 4 M urea is the unfolding agent, and that the 3 M LiCI then dissociates the proteins from the RNA. As described later, 4 M urea extensively unfolded formaldehyde-treated ribosomes. This suggested that the action of the formaldehyde in this case was to covalently link at least some of the protein onto the RNA. The effect of formaldehyde treatment on the ribosome was found to persist even when the formaldehyde was extensively diluted out or removed by dialysis against buffer and it is concluded that the reaction is not appreciably reversible. U n/olding /ormaldehyde-treated 5o-S subunits E D T A treatment. When formaldehyde-treated subunits were reacted with EDTA at a concentration sufficient to remove all Mg~+ present in solution the 5o-S subunit appeared to be completely converted to a slower-moving component. The sedimenting boundary was very sharp and had a sedimentation coefficient in the range 34-38 S. This may be compared with the 35-S component found as the first stage in the EDTA unfolding of native 5o-S subunits t. The sedimentation coefficient remained remarkably constant with different concentrations of EDTA, and over reaction times of several hours (Table 1). There was no evidence of any slow-moving material at the meniscus. By contrast, native 5o-S ribosomes in 3 mM EDTA produced two components after 3o min, in approximately equal amounts, with sedimentation coefficients of 21 and 16 S (c/. ref. I). Biochim. Biophys. Acta, 199 (197 o) 184-193
188
M.E. ROBERTS, I. O. WALKER
TABI.E I SEDIMENTATION
COEFFICIENTS
OF UNFOLDED
IOUS T I M E S A F T E R T H E A D D I T I O N OF
FORMALDEHYDE-TREATED
I'2D7",4 conch. ( m M )
Time Onin)
s,o,,~
I. 5 1.5
3° 14{} qoo Iio
38 36 34 35
2.0
7-5
50-S
R I B O S O M E S AT VAR-
EDTA
Urea treatment. When formaldehyde-treated ribosomes were reacted with urea o n l y one c o m p o n e n t was o b s e r v e d a n d t h e s e d i m e n t a t i o n coefficient d e c r e a s e d p r o g r e s s i v e l y w i t h i n c r e a s i n g u r e a c o n c e n t r a t i o n (Fig. IC). T h e f o r m a l d e h y d e a p p a r e n t l y s e n s i t i s e d t h e r i b o s o m e s to u r e a as s o m e d e c r e a s e of t h e s e d i m e n t a t i o n coe f f i c i e n t o c c u r r e d in 0.25 M u r e a w h e r e a s l i t t l e effect was o b s e r v e d w i t h n a t i v e ribos o m e s in u r e a c o n c e n t r a t i o n s less t h a n I M. T h e s e d i m e n t a t i o n coefficients w e r e v a r i a b l e a n d d e p e n d e d b o t h on u r e a c o n c e n t r a t i o n a n d on t i m e . H o w e v e r , at least u p to 2.0 M urea, a n d for r e a c t i o n t i m e s of less t h a n IO h, a single c o m p o n e n t was o b s e r v e d w i t h a s e d i m e n t a t i o n c o e f f i c i e n t u s u a l l y in t h e r a n g e 2 5 - 5 0 S (Fig. 2). In h i g h e r u r e a c o n c e n t r a t i o n s , o r for l o n g e r r e a c t i o n times, s e d i m e n t a t i o n c o e f f i c i e n t s in t h e r a n g e 16-25 S or e v e n below, w e r e s o m e t i m e s o b s e r v e d , b u t g e n e r a l l y t h e v a l u e s ,
|
|
|
5G
40
3C
v~
2C
Urea concn,(M)
Fig. 2. The sediinentation coefficients at 20" of formaldehyde-treated 5o-S subunits, as a function of urea concentration after I-IO h treatment. Different symbols represent different ribosome preparations. Q-C), after 5-6 h treatment. Biochim. Biophys. Acta, 199 (I97 o) I84 ~93
UNFOLDING OF RIBOSOMES IN UREA
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remained constant at about 25-28 S between 2 and 4.0 M urea (Figs. Id and 2). Within these limits the results tended to be rather variable between different 5o-S subunit preparations, and depended also on the age of the preparation. The variability is illustrated in Fig. 2. However, for any given experiment and ribosome preparation the sedimentation coefficient, was found to decrease fairly linearly with urea concentration, at least between o.25 and i.o M urea. Quantitative measurements made on the assumption that the molecular weight remained constant, indicated that the spreading of the boundary was greater than could be accounted for by diffusion alone, and it is concluded that the sedimenting component was heterogeneous. No slowly-sedimenting component was visible at the meniscus in any of the schlieren photographs and synthetic boundary cells showed no evidence of a slow-moving component. In a further attempt to show that no dissociation of protein had occurred the RNA:protein ratios were measured in the following way. The components were isolated from possible dissociation products on a density gradient of sucrose (5-3o %), made up in each case to the appropriate urea concentration. Protein was measured by Folin's reaction, using native 5o-S subunits as a standard, and RNA was estimated from the absorbance at 26o m/t. As shown in Table 1I, there was no evidence of any progressive loss of protein, although the results showed a fairly wide scatter around the value of 37 °'o protein found for native ribosomesL It is likely therefore that the components observed in urea solution result from the unfolding or swelling of the 5o-S subunits without loss of protein. "FABLE iI THE PERCENTAGE X V E I G t I T O F P R O T E I N IN F O R M A L D E I I Y D E - T R E A T E D IN UREA AND ISOLATED FROM DISSOCIATION PRODUCTS BY SUCROSE
Urea conch. (3I)
Protein in ribosome component (oo)
o.oo 0-25 0.50 0.75
32 34 38 35
1.0
41
50-.~ RIBOSOMES UNFOLDED GRADIENT CENTRIFUGATION
Reversibility Attempts were made to reverse the effect of the urea by dialysis against buffer. This treatment was applied to components which had been isolated on a sucrose density gradient as described previously, and on unfolded ribosomes dialysed directly against buffer after urea treatment for 2-4 h. In no case was any increase in sedimentation coefficient observed after dialysis. Thus no reversibility could be demonstrated.
Melting profiles No change in absorbance at 260 raft was obserw~d when native or formaldehydetreated ribosomes were reacted with EDTA or urea, and it is concluded that, at room temperature, the secondary structure of the RNA is unchanged by the addition of these reagents. When 5o-S subunits were melted in increasing concentrations of urea the melting temperatures progressively decreased. The gradient of the curves also decreased Biochim. Biophys. Acta, x99 (197 o)
I84-I93
I90
M . E . ROBERTS, I. O. WALKER
until in 3.0 M urea the gradient was about the same as that of RNA (Fig. 3). The decrease in the melting temperature may be explained by the destabilising effect of urea on the secondary structure of the RNA since a similar effect was observed for RNA alone, where the melting temperature in 2 M urea decreased by 8 °, from 62 to 54 ° (Fig. 5). The decrea.se in the gradient of the curve most likely results from the effect of urea in destroying the tertiary structure of the ribosome, thus causing the melting behaviour to become less co-operative and more like that of RNA. This is borne out by the observation that the melting curve of the s u b u n i t s in 2 M urea, at which concentration they have m a i n l y unfolded to form a 35-S c o m p o n e n t , was very similar to that of R N A in the same solvent except that the melting temperature of the s u b u n i t s was greater than that of the R N A b y 2 '~.
1+ I /
80 100
o 6(~r
E o 80
/ ///// /
/
/11 f /
+ooL/
~o 60 ~)
~.O M-.../
/////
"-- 20
E c
O
Z
~OM
20 °
40 ° Temp.
60 °
40 °
80 o
6(3°
80"
Fig. 3. The m e l t i n g profiles of n a t i v e 5o-S s u b u n i t s a n d R N A in urea: 5o-S s u b u n i t s in () M ( 0 - 0 } , I M (KI .'~1), 2 M ( ~ ~ ) a n d 3 M n r e a ( O - O ) ; I¢.NA in 2 M ure a ( A - A ) . Fig. 4. T h e m e l t i n g profiles of f o r m a l d e h y d e - t r e a t e d 5o-S s u b u n i t s in v a r i o u s c o n c e n t r a t i o n s of urea.
,
,
,
,
100
"~ 8 0 6
E 6o tv
Jc do ,~ 20 -6
E
o :7
O
20=
40"
EO °
80"
Temp. Fig.
5. "]'he m e l t i n g p r o f i l e s o f n a t i v e a n d f o r m a ] c l e h y d e - t r e a t e d
5o-S s u b u n i t s , in [ o m M ~ o d i u m
(pH 7.o)--1 mM m a g n e s i u m a c e t a t e and in 2 mM E D T A . N a t i v e 5o-S s u b u n i t s in buffer ( O - O ) a n d in 2 mM E D T A (A---&). F o r m a l d e h y d e - t r e a t e d 5o-S s u b u n i t s in buffer ( O O ) a n d in 2 mM E I ) T A ( A - & ) . The m e l t i n g profiles of I
el-D). Biochim. Biophys. Acta, 199 (i97 o) i84 .I93
UNFOLDING OF RIBOSOMES IN UREA
191
When 5o-S subunits were treated with formaldehyde, their melting behaviour was altered significantly. The gradient of the curve remained the same, but the melting temperature increased by 3-5 ° (Fig. 4). This must be due to the stabilisation of bonds normally broken during thermal denaturation. The addition of urea to formaldehyde-treated ribosomes resulted in the progressive lowering of the melting temperature, again presumably due to the destabilising effect of urea on the secondary structure of the rRNA. However, from 0.25 to i.o M urea tile gradient of tile curves, and the overall shape, remained the same, showing that the denaturation process was still highly co-operative, although judging by ttle decrease in the sedimentation coefficients, some change in the tertiary structure of the subunits had occurred under these conditions. In 2.0 and 3.0 M urea the melting temperature further decreased, and the gradients of the curves also decreased so that the shapes of the curves at these higher urea concentrations were essentially the same as those of native 5o-S subunits and RNA under the same conditions. In 2 mM EDTA, the melting behaviour was found to be the same for formaldehyde-treated and untreated ribosomes. Both curves were similar to RNA ill 2 mM EDTA (Fig. 5). This indicated that, in the 35-S component which is formed under these conditions, the protein has no effect on the melting of the RNA secondary structure. DISCUSSION
The sedimentation coefficients of native 5o-S ribosomal subunits in urea solution suggest that a co-operative conversion to a component of 32-38 S is occurring. EDTA has a similar effect on 5o-S subunits, causing a co-operative unfolding, via a 35-S particle, to 2I-S and I6-S particles. The melting profiles of the ribosomes in 2 M urea show that the tertiary structure has been largely destroyed. Again this behaviour m a y be compared to that of the 35-S unfolded particle in EDTA 1. These similarities strongly suggest that the particle formed in urea and the particle formed in the first stage of E D T A unfolding are the same. The hydrodynamic volumes of the 5o-S subunits and 23-S RNA, calculated by the method described previously 1, shows that a 35-S particle would occupy about the same volume as free 23-S rRNA in its most compact state (28 S), whereas 5o-S particles are much smaller. Clearly RNA is held in its more compact form in the native subunit by bonds involving the ribosomal proteins, and these bonds are broken in the 5o-S to 35-S transition. If ttle 35-S component observed in urea-treated subunits is indeed an unfolded component, analogous to tile first step in EDTA unfolding, then this would suggest that the bonds holding the 5o-S subunit in its highly compact conformation are mediated, at least in part, by hydrophobic interactions. These are likely to be protein.protein bonds since protein-RNA bonds are predo,ninantly electrostatic, judging by their lability to solvents of high ionic strength. However, the existence of a hydrophobic contribution from the latter cannot be excluded on the results presented here. The secondary structure of the RNA in 5o-S subunits is unchanged in urea at room temperature, but the lowering of the melting temperature indicates that the secondary structure has become destabilised. This destabilisation is independent of the tertiary structure of the ribosome, as it occurs in free RNA treated with urea. The melting profiles of 5o-S subunits in urea also show a decrease in gradient, and Biochim. Biophys. Acta, 199 (I97 o) I84-I93
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M.E.
R O B E R T S , I. O. W A I . K E R
become similar to RNA in 2 M urea, which shows that the protein in tile 35-S component which is present at this urea concentration has little effect on the denaturation of the RNA. This result is supported by tile melting behaviour of subunits in EDTA. Formaldehyde-treated ribosomes in 2 mM EDTA, in which they form a 35-S component, melt identically to untreated 5o-S subunits, and to RNA, in the same. solvent. This implies that tile bonds broken in the formation of the 35-S component are the ones responsible for the highly co-operative denaturation of 5o-S subunits. It has been argued elsewhere that these bonds are also responsible for maintaining the compact tertiary structure of the ribosome ~. However, it cannot be unequivocally shown that the 35-S comt)onent found in urea is the result of unfolding the wholc 5o-S subunit, and not tim result of somc dissociation of proteins. In many cases in 2 M urea, and higher concentrations, there is evidence of a slow-moving and heterogeneous component at the meniscus, which could bc dissociated protein. In an attempt to investigate the effect of urea on the ribosome tertiary structure without the colnplication of protein dissociation, prior trcatmcltt with formaldehyde was adopted. Reaction with formaldehyde, under the conditions described, does not appear to affect the secondary structure of the RNA, or the gross tertiary structure of the ribosome. There is evidence that formaldehyde covalcntlv links at least a proportion of the proteins onto the ribosome. Spirm has shown that a protein fraction, known as the "split proteins", which normally dissociates in a high-ionic-strength solvent, remains attached after formaldehyde treatment s. The work described hcrc showed that, when formaldehyde-treated ribosomes were unfolded in 4 M urea and reacted with 3 M LiC1, no precipitate of free RNA was formed, unlike native 5o-S subunits under the same conditions. This suggests that the action of the formaldehyde is to attach at least some of the protein onto tile RNA, presumably by cross-linking, although crosslinking between proteins may also bc important. Unfmtunately the mode of action of formaldehyde is unknown in this case. FELl)MANl° has shown that RNA can be cross-linked by methylene bridges between purine groups, and FRAENKEL-(~ONRAT AND Or.COTTl~ reported the formation of stable methylene bridges between amino groups and guanidyl groups, phenol, imadazole and indolc rings treated with formaldehyde. The latter could explain both protein RNA and t)rotein-protein cross-linking. In urea the sedimentation coefficient of formaldehyde-treated 5o-S subunits is decreased. This is due, apt)arently, to the unfolding or swelling of the whole subunit since no evidence of dissociation was found. These results lend further weight to the suggestion that the (:onapact tertiary structure of the 5o-.':, ribosome is maintained by hydrophobic bonds. Attempts to reverse the unfolding by dialysis against buffer were not successful. However, the process of unfolding is, in many respects, different from that shown by native 5o-S subunits in EDTA and urea. First, the formaldehyde appears to have sensitised the subunits to urea: appreciable unfolding occurs at concentrations below r M, whereas native 5o-S subunits show little or no unfolding in i M urea. In addition the stepwise co-operative nature of the unfolding appears to have changed, such that after formaldehyde treatment only a single sedimenting peak is seen in urea, which is considerably broader than the boundary of the native subunit. Thus unfolding, in this ease, appears to be a non-co-operative, continuous process. Similarly the EDTA unfolding of formaldehyde-treated subunits results in a single sedimenting Biochim. Biophys. Acta, 199 (r97o) t84-r93
UNFOLDING OF RIBOSOMES IN UREA
193
component. Furthermore, over a wide range of EDTA concentrations, and after different reaction times, the sedimentation coefficient of the unfolded component remained constant at 35 S. This could be the same as the 35-S component found as the first stage in the EDTA unfolding of native subunits. Tile melting profiles of formaldehyde-treated subunits in urea show a progressive lowering of the melting temperature, due to the destabilising effect of urea on the RNA secondary structure. However, a striking feature of the curves is that their gradient and overall shape remain very similar to that of the native 5o-S subunits over the range o.25-1.o M urea. in 2.o and 3.o M urea the curves flatten out and become very similar to tile curves of native 5o-S subunits in the same urea concentrations. Clearly at the lower urea concentrations at least some of the protein bonds causing the melting of the native subunits to be highly co-operative remain intact, and are only broken at the higher urea concentrations. The finding that the 35-S component found at the first stage in EDTA or urea unfolding has roughly the same hydrodynamic volume as free RNA suggests that it m a y be significant as the stage at which important protein bonds, holding the 5o-S subunit in its compact tertiary conformation, have been broken. In native subunits further unfolding can take place, as shown by the 2I-S and I6-S components found in EDTA ~, but after formaldehyde treatment, this further unfolding is abolished, probably as a result of cross-linking. It is also suggestive that unfolding below 35 S is not usually found in urea. Therefore the components with sedimentation coefficients less than 35 S probably result from the disruption of electrostatic or hydrogen bonds. In all cases the melting profiles of the 35-S components are similar to RNA under the same conditions, showing that the protein in these components is exerting no effect on the denaturation of the RNA. This provides further evidence that the 5o-S to 35-S transition involves the breaking of the major bonds maintaining ribosomal tertiary structure.
ACKNOXVLEDGEMENTS
The authors wish to thank the director and staff of the Microbiological Research Establishment, Porton, for supplying the frozen paste of E. coli MRE 600. One of them (M.E.R.) wishes to thank the Medical Research Council for financial support.
REFERENCES I 2 3 4 5 6 7 8 9 IO ii
S. H. MIALL AND I. O. WALKER, Biochim. Biophys. Acta, 174 (1969) 551. L. [-'. GAVRILOVA, D. A. IVANOV AND A. S. SPIRIN, J . Mol. Biol., 16 (1966) 473. R. I;. GESTELAND, J. Mol. Biol., I8 (1966) 356. B. SHEARI), S. H. MIALL, A. 1{. PEACOCKE, 1. O. WALKI-:R AND 1{. E. RICHARDS, J. Mol. Biol., 28 (1967) 389. A. TISSII~RES, n . SCHLESSINGER AND F. GROS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 145o. P. I.. WHITNEY AND C. TANFORD, J. Biol. Chem., 237 (1962) I735. P- SPlTNIK-ELSON, Biochem. Biophys. Res. Commun., 18 (i965) :557. A. S. SPIRIN, N. V. I'IEI.1TSINA AND M. I. LERMAN, .]. Mol. Biol., 14 (1965) 6 t l . P. SPITNIK-ELSON AND A. ATSMON, F E B S Letters, 2 (1968) 13. M. YA. FELDMAN, Biochim. Biophys. Acta, 149 (1967) 2o. H . FRAENKEL-CONRAT AND H. S. OLCOTT, J. Biol. Chem., I74 (I948) 827.
Biochim. Biophys. Acta, 199 (t97 o) 184-I93