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Significant changes in 16 S RNA conformation accompanying assembly of the 30 S ribosome in vitro
J. Mol.
Biol.
(1977)
Significant
113, 623-634
Changes in 16 [S-RNA Conformation Accompanying Assembly of the 30 S Ribosome in Vitro HEINZ-KURT Laboratory
of
I Tn&uersify
(Received
H~CHKEPPEL~
Molecular
Biology
qf Wiscon8in,.
AND GARY R. CRAVEN and
Ma,dison,
21 October 1976, and in revised
Department Wise.
53706.
of Genetics U.S.A.
form 28 Murch
197i”)
0ur previous studies have shown that 16 S RNA can assume two different c:onformational forms as det,ected by agarose gel electrophoresis, and that these two forms vary in their ability to bind individual 30 S ribosomal proteins specifically. In this paper we show that the faster electrophoretic form can be converted to the slower electrophoretic form by the binding of either protein 54, 88, S7 or S15. The slower form can then be transformed into a fast form by heatactivating the reconstitution intermediate (RI] particle, which has been constructed under reconstitution conditions at O”C, to RI*. We demonstrate that t)he transformation of the 16 S RNA conformation by binding of protein S7 permits the subsequent binding of protein S9 following deproteination. We propose that many of the classical assembly-dependent relationships are due to induced cha,nges in the 16 8 RNA conformation.
1. Introduction Ribosomal RNA is capable of assuming measurably different conformations dependent on ionic, pH, temperature and solvent conditions (Cox & Hirst, 1976; Morris et al., 1974). The importance of 16 S RNA secondary and tertiary structure in the assembly of the 30 S ribosome in vitro has been indirectly implicated by several investigations. The large change in the sedimentation velocity of the ribonucleoprotein particle at various stages of assembly can be best attributed to alterations in the conformation of the 16 S RNA (Held & Nomura, 1973). In earlier studies Sypherd (1971) demonstrated that the addition of 30 S ribosomal proteins to denatured 16 S RNA under the prescribed conditions for reconstitution causes a renaturation of the RNA. Finally, many workers have suggested that the assembly of the 30 S ribosome in vivo involves a reorganization of the 16 S RNA molecule (e.g. Nashimoto & Nomura, 1970: Wireman & Sypherd, 1967). However, there has bcxen meager evidence forthcoming to support this suggestion. In this paper we report experiments which show that conformational alterations in 16 S RNA in fact do occur during the process of 30 S ribosome assembly in vitro. We have found that the electrophoretic mobility of 16 S RNA on composite agarosr polyacrylamide gels (Peacock & Dingman, 1968; Hochkeppel & Craven, 1976) changes significantly at different defined steps in the self-assembly reaction. Furthermore, we have demonstrated that one of the interdependent assembly relationships described t Present, address: Friedrich
Miescher-Institut,
P.O. Bras 273, CH-4002 Basel, Switzerland. 623
024
H.-K.
HOCHKEPPEL
AND
G.
R.
CRAVEN
by Mizushima & Nomura (I 970) can be at least partially explained dependent induced conformational change in the 16 S RNA.
2. Materials (a)
as due to a, prrbein-
and Methods Buffers
Buffer
I :
0.03 M-tricine, dithiothreitol
0.4 M-pOtaSSiUIn chloride, 0.02 M-magnesium (Nmritional Biochem. Corp.), pH 7.4.
acetate.
0.001
Buffer
II :
0.09 M-tricine, dithiothreitol,
1.2 M-potassium pH 7.4.
acet,ate,
0.00 1 M-
Buffer
III:
6.03
0.02
M-tricine, (b)
chloride,
M-magnesium
Preparation
0.06
M-magnesium
acetate,
pH
and
ribosomal
of riboscmes
M-
86. proteins
Ribosomeo were isolated from Escherichia co&i MREGOO and 30 S ribosomal subunits were purified as previously described by Craven & Gupta (1970). The purity of the 30 S subunits was routinely checked on 5% to 20% sucrose gradients and on 10% polyacrylamide gels (protein composition). The 30 S ribosomes were found to contain less than 1 y. contamination of 30 S subunits. Ribosomal proteins were extracted from the 30 S subunits with 67% acetic acid and fractionated by phosphocellulose (Mannex P-l) chromatography following the procedure described by Hardy et al. (1969). The nomenclature of the 30 S ribosomal proteins is taken from Wittmann et al. (1971). (c)
Isolation
of ribosomal
RNA
from
30 S ribosomes
The phenol and sodium dodecyl sulfate extraction procedure described by Traub et al. (1971) was used without modification to purify 16 S RNA from isolated 30 S subunits. The acetic acid/urea procedure published by Hochkeppel et al. (1976) was used as originally described. The RNA was precipitated from 30 S ribosomes by a mixture of acetic acid, urea and magnesium acetate (final concns were 75%, 1.0 M and 0.2, respectively). The RNA precipitate was suspended in buffer I and then extracted with acetic acid in the presence of magnesium acetate (final concns were 75% and 0.2 M, respectively). The precipitate was washed with a magnesium-tricine solution to remove excess acetic acid and urea. Finally, the precipitated RNA was dissolved in 0.03 M-tricine (pH 8.0). The quantity of protein which remained associated with the RNA was found to be less than 1% for both acetic acid/urea 16 S RNA and phenol 16 S RNA. This estimate was obtained by extracting ribosomes containing radioactive proteins (prepared by labeling in wiwo). Neither polyacrylamide gels nor composite agarose/polyacrylamide gels of acetic acid/urea 16 S RNA and phenol 16 S RNA showed any contamination of 23 S RNA. (d)
Radioactive
protein
30 S ribosomal proteins were made radioactive by the reductive alkylation procedure described by Rice & Means ( 1971) as modified by Held et al. (1974) using [3H]formaldehyde (320 mCi/mmol; New England Nuclear). The reductive alkylation does not effect the binding ability (Spicer & Craven, unpublished results). (e) Formation
and
isolation
of RNA-protein
complexes
With modifications previously described (Hochkeppel et al., 1976) the reconstitution conditions developed by Traub & Nomura (1969) were used to form specific RNAprotein complexes. The molar ratio of individual 30 S protein was calculated on the basis The concentration of protein was of individual molecular weight (Craven et al., 1969). determined by the ninhydrin method of Moore & Stein (1948). The molarity of RNA was calculated from the assumption that one absorbance unit of 260 nm corresponds to 7.5 x lo-l1 mol RNA/ml. The RNA and protein were suspended in buffer I and in buffer II, respectively, and the RNA suspensions were preheated at 42°C for 10 min. The 2 SUSpensions were mixed along with the appropriate amount of water to give a final concen-
RNA
CONFORMATION
DURING
RIBOSOME
ASSEMBLY
625
tration of salts identical to buffer I. The protein and RNA mixture wae incubated at 30°C for 10 min and then at 42°C for 30 min. The resultant RNA-protein complexes were purified by ultracentrifugation through a layer of 12% sucrose dissolved in buffer I. The binding of non-labeled 30 S proteins to 16 S RNA was tested by polyacrylamide gel electrophoresis. The amount of protein bound to the RNA was estimated by comparing the densitometer scan of the stained protein band with the scan of a control gel containing known amounts of protein. The amount of radioactive protein bound was determined by counting samples of the purified RNA-protein complex and calculating the spec. act. defined aa cts/min per 1 unit A,,, of 16 S RNA. The reconstitution intermediate (RI) particles were constructed by reconstituting 16 S RNA and total 30 S protein for 30 min, at 0°C in buffer I. The particles were purified as described above. The RI particles were then converted to RI* particles by heat-act,ivating the particles in buffer I for 10 min to 4O’C‘. (f)
Composite
agarose/polyacrylamide
gel electrophoresis
RNA
of ribosomal
The gel and buffer systems employed for electrophoresis of intact 16 S RNA are modifications of those described by Peacock & Dingman (1968). The electrode buffer system contains 0.06 M-tricine, 0.06 fir-boric acid, 0.06 M-KOH. The final pH is 8.0. The gel mixture is composed of 6 g sucrose, 1 g agarose (Sigma Chemical Co.) and 6 ml of an acrylamide solution (28.5 g recrystallized acrylamide, 1.5 g bisacrylamide, 48 g urea, dissolved in water to a final vol. of 100 ml), 10 ml of 10 x concentrated electrode buffer, made to a vol. of 100 ml with water. The resultant concentration of acrylamide and agarose are l*S’$/$ and I%, respectively. In all experiments 2 different preparations of RNA were analyzed side by side on the same gel. The gel was partitioned by inserting into t,he gel tube, which was sealed with melted parafin, a small piece of a plastic microscope cover slip prior to the addition of the gel solution. (g) Staining
and
densitometry
of agarose/polyacrylamide
gels
Composite agarose/polyacrylamide gels were stained for RNA in a 1% acetic acid solution containing 0.1% toluidine. After 0.5 h staining time the gels were destained in a 1% acetic acid solution. The stained RNA bands were then scanned in a microdensitometer (Gilford, model 2000) at a wavelength of 540 nm. The scans were used to determine the relative difference in mobility between the RNA bands. Each test channel was always found to contain a single compouent. There was in no case a second band overlapping the control.
3. Results We have recently shown that 16 S RNA derived from E. coli 30 S ribosomal subunits by extraction with phenol (16 S RNApj-) h as a faster mobility on composite agarose/polyacrylamide gels than 16 S RNA derived by acetic acid/urea extraction (16 S RNA8; Fig. 1; standard Fig. for Figs 2 to 4). The previous results indicated that 16 S RNA can exist, under identical conditions of temperature, pH and ionic strength in two readily distinguishable conformations as a consequence of the method of RNA preparation employed. It was also shown that 16 S RNA” was capable of binding independently six “new binders” (53, S5. S9, Sll, S12, S18), in addition to the “primary binders” (S4, 57, S8, 513, S15, 517, S20) bound by 16 S RNAP (Hochkeppel & Craven, 1976; Hochkeppel et al.. 1976). 16 S RNAp must bind the p-proteins t We propose the following terminology which will 16 8 RNAp, RNA deproteinized with phenol and deproteinized with acetic acid and urea; p-proteins, originally shown to bind 16 S RNA extracted by (Mizushima & Nomura, 1970). These proteins include the 30 S ribosomal proteins which bind 16 S RNA (Hochkeppel et al., 1976). These proteins are S3, SS,
be used throughout this paper: sodium dodecyl sulfate; 16 S RNAa, RNA the 30 S ribosomal proteins which were the phenol/sodium dodecyl sulfate method S4,57, 58, S13, S15, S17 and 520; a-proteins, prepared by t.he acetic acid/urea procedure S9, Sll, S12 and 518.
H.-l<.
HO(‘HKEL’i’E1,
ANI)
(:.
K.
(‘l<.\V15N
I Fast
FIG. 1. Densitometer scan of a composite agarose/polyacrylamide (slow peak) and 16 S RNAP (fast peak) at 540 nm. This Figure serves
split, gel with 16 S RNAa as a standard for Figs 2 to 4.
before being capable of binding the six a-proteins. This addition of individual p-proteins to 16 S RNAP led to a the RNA which would simultaneously create new binding bility we examined the effect of individual p-proteins on RNAP as measured by composite agarose/polyacrylamide
suggested to us that the conformational change in sites. To test this possithe conformation of 16 8 gel electrophoresis.
(a) The e#ect of protein
X4 on the conformation
of 16 S RNAp
In order to examine the inlluence of S4 on the conformation of 16 8 RNAP, a. specific complex between 16 S RNAP and protein S4 was formed in buffer I verified as described in Materials and Methods. The RNA was then deproteinized from the complex by the acetic acid/urea extraction procedure. This deproteinized “S4-RNA” was immediately analyzed by “split’‘-gel electrophoresis. Figure 2(a) shows that &l-RNA has the same low mobility as 16 S RNA”, and (c) slower than 16 8 RNAP. However, if the RNA is extracted from the same 16 S RNA”-S4 complex by the phenol method, the mobility is faster t,han that of 16 S RNA” (Fig. 2(b)) and identical
RNA
CONFORMATION
DURING
RIBOSOME
ASSEMBLY
627
(d)
i Fm. 2. Split-gel densitometer scans of RNA extracted from 16 S RNAP-S4 complex. The complex was prepared as described in Materials and Methods and deproteinized in (a) and (c) with acetic acid/urea and in (b) with phenol/sodium dodecyl sulfate. (a) Acetic acid/urea extracted 84-16 S RNA versus 16 S RNA” (both slow); (b) phenol extracted SP16 S RNA (fast) versus 16 S RNA* (slow); (c) SP16 S RNA (slow) www 16 S RNAP (fast); (d) control: 16 S RNAP re-extracted with acetic acid/urea in the absence of protein 54 v&vu8 16 S RNAp (both fast).
to our standard 16 S RNAp preparation (data not shown). If 16 S RNAp is re-extracted with acetic acid/urea in the absence of protein S4, there is no alteration in mobility (d). Thus any changes in RNA mobility found in RNA extracted from specific protein-RNA complexes are due to the interaction of the protein with the RNA only and not to the extraction procedure employed. This also demonstrates that the acetic acid/urea procedure reveals differences in the 16 S RNA conformations within specific RNA-protein complexes that the phenol extraction procedure obliterates. We conclude that when 16 S RNAp binds protein 54, the conformation of the RNA is altered so that it has a lower mobility during gel electrophoresis. (b) The effect of proteins
X7, 85, S15 and 520 on the 16 S RNAP
conformation
Figure 3 demonstrates the influence of the p-proteins 57, S8, S15 and S20 on the conformation of 16 S RNAP. On the left (a) are agarose/polyacrylamide split-gel patterns of RNA extracted with acetic acid/urea from the individual 16 S RNAPprotein complexes compared to 16 S RNAP. On the right (b) are gel patterns of the sameRNA preparations co-electrophoresedwith I6 S RNA&. As can be seen,S7-RNA and S&RNA have a mobility identical to that of 16 S RNA” (b) and lower than that of 16 S RNAP (a). SlB-RNA appears to have an intermediate mobility between 16 S RNAP and 16 S RNA”, whereas“SBO-RNA” hasa mobility unchanged from the original 16 S RNAP. Thus many, but not all, of the p-proteins can induce conformational alterations in 16 S RNA. Furthermore, the fact that $15RNA has an intermediate 42
628
H.-K.
HOCHKEI’I’EL
AND
(:.
It.
CRAVEK
J 3. Split-gel densitometer scans of RNA extracted with acetic acid/urea from 16 RNAP-87 16 S RNAP-S8, 16 S RNAP-S15 and 16 S RNAQZO complexes. The deproteinized RNA preparations were compared in (a) with 16 S RNAP and in (b) with 16 S RNA&. (a) 57-16 S RNA (slow) VWBUS 16 S RNAP (fast); M-16 S RNA (slow) versus 16 S RNAP (fast); 516-16 S RNA (slow) ~UCMLS 16 S RNAP (fast); S20-16 S RNA wewu8 16 S RNAP (both fast). (b) 57-16 S RNA ~tersu~ 16 S RNA* (both slow); SS-16 S RNA WYWUS 16 S RNA” (both slow); S15-16 S RNA (fast,) aersz .Y 16 S RNA” (slow); S20-16 S RNA (fast) oemus 16 S RNAR (slou’). FIG.
electrophoretic mobility suggests that these conformational alterations in RNA structure are different depending upon the protein bound to the RNA. There may also be more subtle differences between the conformers with low electrophoretic mobility, which are not detectable in our system. (c) The binding
of one protein to 16 S RNA induces a conformational which permits a second protein to bind
change in the RNA
This finding that the binding of proteins S4, S7 or S8 transforms the 16 S RNAP conformation into the 16 S RNA” conformation, which binds many new proteins, stimulated us to investigate the possibility that these proteins individually could change the RNA conformation in such a way that would permit a new protein to associate with the RNA. Other workers (e.g. Schaup & Kurland, 1972) have suggested that some of the co-operative assembly relationships for the 30 S ribosomal proteins could be best attributed to induced RNA conformational changes giving rise to new site-specific protein binding sites. We have not as yet made an exhaustive study but we report here several experiments which substantiate this hypothesis. Our approach in these experiments was to prepare first a complex between a single protein and 16 S RNAP. This complex was then deproteinized by the acetic acid/urea procedure.
RNA
CONFORMATION
Fra. 4. Densitometer re-extracted RI-RNA RNA”; (c) RI*-RN.4
DURING
RTBOSOME
ASSEMBLY
scans of composite agarose polyacrylamide split verau8 16 S RNA*; (b) acetic acid/urea re-extracted versus 16 S RNAP; (d) RI*-RNA wraus RI-RNA.
629
gels. (a) Acetic acid/urea RI*-RNA 2)er8’8us 16 S
The resultant preparation of 16 S RNA” was quantitatively tested for its ability t’o bind a second protein. Figure 6 shows the binding curve performed with 3H-labeled protein S9 and 16 S RNA extracted by acetic acid and urea from a complex involving protein S7. As can be seen. the S9 protein can bind the 16 S RNA directly, reaching a plateau value of 0.27 molecules of S9 per molecule of RNA. In contrast, protein X9 does not bind the I6 S RNAP used in the formation of the S7-Rn’A complex nor does S9 bind 16 S RNAp which has been re-extracted by acetic acid and urea in the absence of protein S7 (latter data not shown). Thus the binding of S7 to 16 S RNAp must induce a structural transition in the RNA which provides an RNA binding site for protein S9. The fact that the protein S9 binding reaches saturation at, less than one molecule per molecule of RNA could be due to incomplete binding of protein S7, which normally requires other proteins (S4, S8 and S20) to reach maximum binding. Indeed, we have conducted the same experiment with 16 S RNA re-ext’racted from a complex involving 16 S RNAP, protein S7 and in addition protein S4. The binding of protein S9 (Fig. 7) to the acetic acid/urea r-e-extracted “X4, S7-RNA” is enhanced, reaching a maximum of 0.40 copies per molecule of RNA. In contrast to that acetic acid/urea re-extracted S4-RNA from a 16 S RNAP-S4 complex did not bind protein S9. In an earlier paper (Hochkeppel et al., 1976) it was reported that close to 1 mole of protein S9 was bound per mole of acetic acid/urea 16 S RNA. The reason for the relatively 10~ binding of protein S9 to 54, S7-RNA could be that, even if 16 S RNAP has been transformed into the “slow” form, this slow form may not be completely identical with t,he slow form of 16 S RNA”.
FIG. 5. Densitometer scan 30 S subunits (fast peak).
of a composite
agarose/polyacrylamide
split
gel with
Fost
I
Slow I
16 S ltNAa
from
inactive
30 S subunits
(slow
peak),
and
frown
active
-RNA
CONFORMATION
DURING
RIBOSOME
631
ASSEMBLY
I 0.28
-
0.24
-
I:1
2:i
Mel S9/mol
3:1
4:l
6:l
5:l
16s RNA I” reoctlon
Fro. 6. Saturation binding curve for protein S9 and re-extracted S’I-RNA. The reconstitution was carried out in buffer I. The purified RNA-protein complexes were assayed directly for 3H cts/min bound. The calculations of copies protein bound/molecule RNA were based on the molecular weight data of Craven e2 al. (1969) and the concentration of protein was determined by the ninhydrin method of Moore & Skin (1948). It was assumed that one absorbance unit at 260 nm corresponds to 7.6 x lo-” mol RNA/ml. -o-o--. Re-extracted S7-RN4; -A-A--, phenol RN4 conkol.
Mol protemhol
F1o. 7. Saturation Fig. 6. --e-e---
(d) An RNA
binding Re-extracted
curve for protein S4, S’I-RNA;
conformational
alteration
16s RNA in reactlon
mixture
89 and re-extracted -o-(j -~, phenol
occurs during
S4, S’i-RNA. RNA cont,rol.
the RI to RI*
For
details
see
transition
Having established that the 16 S RNA conformation can vary after association with specific ribosomal proteins, we proceeded to determine whether or not other conformations of 16 S RNA can be distinguished at certain stages in ribosome assembly. Held & Nomura (1973) have shown that under certain ionic conditions the reconstitution intermediate (RI) particle has a sedimentation constant of 21 S whereas the heat-activated particle (RI*) is dramatically more compact having a
H.-Ii.
032
HO(IHl
i\Sl)
(:. Ii.
C:KAVES
sedimentation constant) of 26 S. As the pr&rin c*ontcnt of t,htt t u’o part,iclrs is presumablv identical, it, is reasonable to suggtxst, that’ t,hcl RNA has rrncic~go~w n structural transition. We have examined this Ilypothcbsis by using the acetic: acid/urea pro~~lurc: 60 extract RKA from t)he Rl and Rl* preparations. In each case the RI and the RI* preparations were constructed wit,h 16 R RXA* and 30 S ribosomal prot,eins. Figure 4(a,) shows the split gel containing I6 S RNA” and acetic acid/urea re-ext’racted “RI-RNA”, both having identical mobility. In contrast, Figure 4(b) represents acetic acid/urea re-extracted “RI*-RKA” co-electrophoresed with 16 S RNA”. In this case the “RI*-RNA has a faster mobility as verified by Figure! 4(c) which is “RI*-RNA” against 16 S RNAP. Thus the RNA ext’racted from Rl particles is of t’he slow or 16 S RNA” variet,y and RNA obtained from RI* part,icles has a fast mobility similar to that of 16 S RNA*. This is verified in Figure 4(d) which shows that RI-RNA and RI*-RNA have distinctly different electrophorotic mobilities. This is the first direct demonstration that a conformational transit,ion in the RNA moiety actually occurs during the conversion of RI to Rl*. However, it should be noted that the 16 8 RNA” and 16 S RNAP conformations cannot bt: distinguished by sedimentation velocity (Hochkeppel & Craven. 1976). Thus the RNA conformational changes we observe for the RI to RI* transit,ion cannot fully account for the dramatic change in sedimentation constant reported by Held & Komura (1973). RNA-REA and protein-protein int,eractions may also have some influence 011 t’lie tjransit’ion of the ribonucleoprotein complex. (e) “Active”
and “‘nactive”
30 S ribosomes conformations
contain
RNA
with different
Zamir et al. (1971) were the first to suggest that the 30 S ribosome is capable of a reversible conformational change which causes loss of capacity to bind transfer RNB at 0°C. They showed that this activity is reversibly lost when the ribosomes are depleted of either monovalent or divalent cations. Later, Ginsberg et al. (1973) demonstrated that these inactive 30 S ribosomes contain proteins with a different reactivity to N-ethyl maleimide than the proteins in the active particles, thus indicating that the two ribosomes have different conformations at least within the protein moiety. We have already sholvn that, 16 S RNA extracted wit’h acetic acid/ urea from such inactive 30 S ribosomal subunits yields RNA containing sites for the a-proteins (Hochkeppel & Craven, 1976). We therefore undertook to determine whether the RNA from such inactive particles is a different conformation. Figure 5 presents an experiment which directly demonstrates that inactive 30 S ribosomes contain RNA with an electrophoretic mobility distinctly slower than RNA extracted from active ribosomes. In this experiment separate portions of 30 S subunits (in buffer III) were diluted 1 to 10 into an activation buffer (0.02 M-Tris (pH 7-O), 0.02 M-Mg acetate, 0.2 M-NH,Cl and 2 mM-dithiothreitol and an inactivation buffer (0.02 M-Tris (pH 7.0), 0.5 miw-Mg acetate, 0.1 M-NH,Cl and 2 m&l-dithiothreitol). Both samples were heated to 40°C for 25 minutes and then extracted by the acetic acid/urea procedure. We routinely find this procedure yields the activation of ribosomes reported by Zamir et al. (1971). The RNA extracts were then co-electrophoresed on agarose polyacrylamide split gels. Figure 5 shows that the RNA from the inactive particle has a lower mobility in our system. We conclude that the RNA conformation within the 30 S ribosome can assume two distinctly different configurations.
RNA
CONFORMATION
DURING
RIHOSOME
633
ASSEMBLY
4. Discussion In all of the experiments described, we have shown changes in RNA conforma’tion of RNA re-extracted with acetic acid/urea from specific 16 S RNAP-protein complexes by comparing its electrophoretic mobility with t’hat observed for 16 S RNA” and 16 S RXAP. Thus we have shown that when proteins S4, S7, S8 and, to a lesser extent. S15 are individually bound to 16 S RNA”, the re-extracted RNA possesses a mobilit,y similar to that found for 16 S RNA”. Protein S20 has no effect on t’he RNA conformation as determined by this technique. Furthermore, this slow form, induced by t,he binding of these several proteins, can be reversed t’o a, form having roughly the fast elrctrophoretic mobility, by heating t’he RI particle, transforming it into the Rl* part’icle. All of these observations ana summa.rizcd in Figure 8. We emphasize t)hat the fast) form of RNA extra&d from t#hr RI* particle has not been shown to actually have the same conformation as the original 16 S RNAP, only that they have similar mobilities on this separabion system, The sa,me comment holds in comparing so-called S-I-RNA with S&RNA or S7-RNA. Indeed. we have some reason t’o believe that, although these RXA preparations have indist,inguishable electrophoretic mobilities. there almost certainly must be subtle differences in conformations. We propose that 16 8 RKA has the potential for a large numbrr of different conformations inherent in its complex st,ruct’ure and that many of thcbsc different forms arc assumed at different stages of assembly in vitro. S4-RNA complex 1
c
Other early protem
Fost conflgurahon
SIOW conflgurotlon
Slow conflguratlon
FInal protems
Fast conflguratlon
Fast conflguratlon
The RI particles we employed in these experimenbs were made by incubation of 16 S RNAP with total protein in the cold followed by pelleting through a layer of sucrose. The protein content of these particles is roughly that seen by Held & Nomura, (1973), and contains about 13 proteins. Clearly one of the next, steps in the exploitation of our techniques to analyze RNA conformabional changes is to determine which of these 13 proteins are required to transform the slow form of RNA into the fast form. Held & Nomura (1973) have found only five prot’eins which are strongly required t,o obtain an RI to RL* transformation. Another two are partially required and tive are only weakly required. We have t)ested two of these proteins and have, found that’ an RI particle made with all proteins present except protein S19 cannot undergo the change from slow to fast RNA induced by heating (Hochkeppel $
634
H.-K.
HOCHKEPPEL
ANI)
G.
It.
CRAVE:N
Craven, unpublished results). Further studies along this line should be very germane to a molecular understanding of the Rl to RI* transition. The most important aspect of the results presented here is t’hat these observed alterations of the RNA conformation seen at various stages in 30 S assembly in vitro could have actual functional significance with regard to the proposed assembly map of Mizushima & Nomura (1969). We have demonstrated that the binding of protein X7 to phenol RNA changes the RNA such that the deproteinated RNA is now capable of binding significant amounts of protein S9. Thus the assembly-dependence relat’ionship originally described by Mizushima & Nomura for these two proteins can now bc given a rational molecular interpretation. Protein S7 induces an alteration in a nearby section of the 16 S RNA which allows for S9 interaction. We propose that this type of molecular interpretation must pertain to many of the assembly-interdependent, relationships, especially those involving the proteins which we have shown can bind 16 S RNA” (Hochkeppel et al., 1976). We thank Dr Julian Gordon for critically reading the manuscript. We would like to express our appreciation to Cathy Bloomer for providing all the purified ribosomal proteins used in this investigation. This work was supported by the Graduate School and the College of Agriculture and Life Sciences, University of Wisconsin, Madison, and by research grant GM15422 from the National Institutes of Health. We also acknowledge use of the Biochemistry Department pilot plant, directed by Dr John Garver and supported by U.S Public Health Service grant FR-00214. REFERENCES Cox, R. A. & Hirst, W. (1976). Biochem. J. 160, 505-519. Craven, G. R. & Gupta, V. (1970). Froc. Nat. Acad. Sci., U.S.A. 67, 1329~-1336. Craven, G. R., Voynow, P., Hardy, S. J. S. & Kurland, C. G. (1969). Biochemistry, 8, 29062915. Ginsburg, J., Miskin, R. & Zamir, A. (1973). J. Mol. Biol. 79, 481-494. Hardy, S. Y. S., Kurland, C. G., Voynow, P. & Mora, G. (1969). Biochemistry, 8,2897--2905. Held, W. A. & Nomura, M. (1973). Biochemistry, 12, 3273-3281. Held, W. A., Ballon, B., Mizushima, S. & Nomura, M. (1974). J. Biol. Chem. 249,3103~3111. Hochkeppel, H.-K. & Craven, G. R. (1976). Nucl. Acids Res. 3, 188331902. Hochkeppel, H.-K., Spicer, E. & Craven, G. R. (1976). J. Mol. BioZ. 101, 155-170. Mizushima, S. & Nomura, M. (1970). Nature (London), 226, 1214-1218. Moore, S. & Stein, W. H. (1948). J. BioZ. Chem. 176, 367-388. Morris, D. R., Dahlberg, J. E. & Dahlberg, A. E. (1974). NucZ. Acids Res. 1, 1249-1258. Nashimoto, H. & Nomura, M. (1970). PTOC. Xat. Acad. Sci., U.X.A. 67, 1440. Peacock, A. C. & Dingman, C. W. (1968). Biochemistry, 7, 668-674. Rice, R. H. & Means, G. E. (1971). J. Biol. Chem. 246, 831-832. Schaup, H. W. & Kurland, C. G. (1972). Mol. Gen. Genet. 114, 350-357. Sypherd, P. S. (1971). J. Mol. BioZ. 56, 311-318. Traub, P. & Nomura, M. (1969). J. Mol. BioZ. 40, 391l413. Traub, P., Mizushima, S., Lowry, C. V. & Nomura, M. (1971). In Methods in Enzymology (Moldave, K. & Grossman, L., eds), vol. 20, part C, pp. 391-407, Academic Press, New York. Wireman, J. W. & Sypherd, P. S. (1975). Biochem. Biophys. Res. Commun. 66, 570-~577. Wittmann, H. G., Stoffler, G., Hindennach, I., Kurland, C. G., Randall-Zahelbauer, L., Birge, E. A., Nomura, M., Kaltschmidt, E., Mizushima, S., Traut, R. R. & Bickle, T. A. (1971). Mol. Gen. Genet. 111, 327-333. Zamir, R., Miskin, R. $ Elson, D. (197 1). ,7. Mol. BioZ. 60, 347-364.
Biol.
(1977)
Significant
113, 623-634
Changes in 16 [S-RNA Conformation Accompanying Assembly of the 30 S Ribosome in Vitro HEINZ-KURT Laboratory
of
I Tn&uersify
(Received
H~CHKEPPEL~
Molecular
Biology
qf Wiscon8in,.
AND GARY R. CRAVEN and
Ma,dison,
21 October 1976, and in revised
Department Wise.
53706.
of Genetics U.S.A.
form 28 Murch
197i”)
0ur previous studies have shown that 16 S RNA can assume two different c:onformational forms as det,ected by agarose gel electrophoresis, and that these two forms vary in their ability to bind individual 30 S ribosomal proteins specifically. In this paper we show that the faster electrophoretic form can be converted to the slower electrophoretic form by the binding of either protein 54, 88, S7 or S15. The slower form can then be transformed into a fast form by heatactivating the reconstitution intermediate (RI] particle, which has been constructed under reconstitution conditions at O”C, to RI*. We demonstrate that t)he transformation of the 16 S RNA conformation by binding of protein S7 permits the subsequent binding of protein S9 following deproteination. We propose that many of the classical assembly-dependent relationships are due to induced cha,nges in the 16 8 RNA conformation.
1. Introduction Ribosomal RNA is capable of assuming measurably different conformations dependent on ionic, pH, temperature and solvent conditions (Cox & Hirst, 1976; Morris et al., 1974). The importance of 16 S RNA secondary and tertiary structure in the assembly of the 30 S ribosome in vitro has been indirectly implicated by several investigations. The large change in the sedimentation velocity of the ribonucleoprotein particle at various stages of assembly can be best attributed to alterations in the conformation of the 16 S RNA (Held & Nomura, 1973). In earlier studies Sypherd (1971) demonstrated that the addition of 30 S ribosomal proteins to denatured 16 S RNA under the prescribed conditions for reconstitution causes a renaturation of the RNA. Finally, many workers have suggested that the assembly of the 30 S ribosome in vivo involves a reorganization of the 16 S RNA molecule (e.g. Nashimoto & Nomura, 1970: Wireman & Sypherd, 1967). However, there has bcxen meager evidence forthcoming to support this suggestion. In this paper we report experiments which show that conformational alterations in 16 S RNA in fact do occur during the process of 30 S ribosome assembly in vitro. We have found that the electrophoretic mobility of 16 S RNA on composite agarosr polyacrylamide gels (Peacock & Dingman, 1968; Hochkeppel & Craven, 1976) changes significantly at different defined steps in the self-assembly reaction. Furthermore, we have demonstrated that one of the interdependent assembly relationships described t Present, address: Friedrich
Miescher-Institut,
P.O. Bras 273, CH-4002 Basel, Switzerland. 623
024
H.-K.
HOCHKEPPEL
AND
G.
R.
CRAVEN
by Mizushima & Nomura (I 970) can be at least partially explained dependent induced conformational change in the 16 S RNA.
2. Materials (a)
as due to a, prrbein-
and Methods Buffers
Buffer
I :
0.03 M-tricine, dithiothreitol
0.4 M-pOtaSSiUIn chloride, 0.02 M-magnesium (Nmritional Biochem. Corp.), pH 7.4.
acetate.
0.001
Buffer
II :
0.09 M-tricine, dithiothreitol,
1.2 M-potassium pH 7.4.
acet,ate,
0.00 1 M-
Buffer
III:
6.03
0.02
M-tricine, (b)
chloride,
M-magnesium
Preparation
0.06
M-magnesium
acetate,
pH
and
ribosomal
of riboscmes
M-
86. proteins
Ribosomeo were isolated from Escherichia co&i MREGOO and 30 S ribosomal subunits were purified as previously described by Craven & Gupta (1970). The purity of the 30 S subunits was routinely checked on 5% to 20% sucrose gradients and on 10% polyacrylamide gels (protein composition). The 30 S ribosomes were found to contain less than 1 y. contamination of 30 S subunits. Ribosomal proteins were extracted from the 30 S subunits with 67% acetic acid and fractionated by phosphocellulose (Mannex P-l) chromatography following the procedure described by Hardy et al. (1969). The nomenclature of the 30 S ribosomal proteins is taken from Wittmann et al. (1971). (c)
Isolation
of ribosomal
RNA
from
30 S ribosomes
The phenol and sodium dodecyl sulfate extraction procedure described by Traub et al. (1971) was used without modification to purify 16 S RNA from isolated 30 S subunits. The acetic acid/urea procedure published by Hochkeppel et al. (1976) was used as originally described. The RNA was precipitated from 30 S ribosomes by a mixture of acetic acid, urea and magnesium acetate (final concns were 75%, 1.0 M and 0.2, respectively). The RNA precipitate was suspended in buffer I and then extracted with acetic acid in the presence of magnesium acetate (final concns were 75% and 0.2 M, respectively). The precipitate was washed with a magnesium-tricine solution to remove excess acetic acid and urea. Finally, the precipitated RNA was dissolved in 0.03 M-tricine (pH 8.0). The quantity of protein which remained associated with the RNA was found to be less than 1% for both acetic acid/urea 16 S RNA and phenol 16 S RNA. This estimate was obtained by extracting ribosomes containing radioactive proteins (prepared by labeling in wiwo). Neither polyacrylamide gels nor composite agarose/polyacrylamide gels of acetic acid/urea 16 S RNA and phenol 16 S RNA showed any contamination of 23 S RNA. (d)
Radioactive
protein
30 S ribosomal proteins were made radioactive by the reductive alkylation procedure described by Rice & Means ( 1971) as modified by Held et al. (1974) using [3H]formaldehyde (320 mCi/mmol; New England Nuclear). The reductive alkylation does not effect the binding ability (Spicer & Craven, unpublished results). (e) Formation
and
isolation
of RNA-protein
complexes
With modifications previously described (Hochkeppel et al., 1976) the reconstitution conditions developed by Traub & Nomura (1969) were used to form specific RNAprotein complexes. The molar ratio of individual 30 S protein was calculated on the basis The concentration of protein was of individual molecular weight (Craven et al., 1969). determined by the ninhydrin method of Moore & Stein (1948). The molarity of RNA was calculated from the assumption that one absorbance unit of 260 nm corresponds to 7.5 x lo-l1 mol RNA/ml. The RNA and protein were suspended in buffer I and in buffer II, respectively, and the RNA suspensions were preheated at 42°C for 10 min. The 2 SUSpensions were mixed along with the appropriate amount of water to give a final concen-
RNA
CONFORMATION
DURING
RIBOSOME
ASSEMBLY
625
tration of salts identical to buffer I. The protein and RNA mixture wae incubated at 30°C for 10 min and then at 42°C for 30 min. The resultant RNA-protein complexes were purified by ultracentrifugation through a layer of 12% sucrose dissolved in buffer I. The binding of non-labeled 30 S proteins to 16 S RNA was tested by polyacrylamide gel electrophoresis. The amount of protein bound to the RNA was estimated by comparing the densitometer scan of the stained protein band with the scan of a control gel containing known amounts of protein. The amount of radioactive protein bound was determined by counting samples of the purified RNA-protein complex and calculating the spec. act. defined aa cts/min per 1 unit A,,, of 16 S RNA. The reconstitution intermediate (RI) particles were constructed by reconstituting 16 S RNA and total 30 S protein for 30 min, at 0°C in buffer I. The particles were purified as described above. The RI particles were then converted to RI* particles by heat-act,ivating the particles in buffer I for 10 min to 4O’C‘. (f)
Composite
agarose/polyacrylamide
gel electrophoresis
RNA
of ribosomal
The gel and buffer systems employed for electrophoresis of intact 16 S RNA are modifications of those described by Peacock & Dingman (1968). The electrode buffer system contains 0.06 M-tricine, 0.06 fir-boric acid, 0.06 M-KOH. The final pH is 8.0. The gel mixture is composed of 6 g sucrose, 1 g agarose (Sigma Chemical Co.) and 6 ml of an acrylamide solution (28.5 g recrystallized acrylamide, 1.5 g bisacrylamide, 48 g urea, dissolved in water to a final vol. of 100 ml), 10 ml of 10 x concentrated electrode buffer, made to a vol. of 100 ml with water. The resultant concentration of acrylamide and agarose are l*S’$/$ and I%, respectively. In all experiments 2 different preparations of RNA were analyzed side by side on the same gel. The gel was partitioned by inserting into t,he gel tube, which was sealed with melted parafin, a small piece of a plastic microscope cover slip prior to the addition of the gel solution. (g) Staining
and
densitometry
of agarose/polyacrylamide
gels
Composite agarose/polyacrylamide gels were stained for RNA in a 1% acetic acid solution containing 0.1% toluidine. After 0.5 h staining time the gels were destained in a 1% acetic acid solution. The stained RNA bands were then scanned in a microdensitometer (Gilford, model 2000) at a wavelength of 540 nm. The scans were used to determine the relative difference in mobility between the RNA bands. Each test channel was always found to contain a single compouent. There was in no case a second band overlapping the control.
3. Results We have recently shown that 16 S RNA derived from E. coli 30 S ribosomal subunits by extraction with phenol (16 S RNApj-) h as a faster mobility on composite agarose/polyacrylamide gels than 16 S RNA derived by acetic acid/urea extraction (16 S RNA8; Fig. 1; standard Fig. for Figs 2 to 4). The previous results indicated that 16 S RNA can exist, under identical conditions of temperature, pH and ionic strength in two readily distinguishable conformations as a consequence of the method of RNA preparation employed. It was also shown that 16 S RNA” was capable of binding independently six “new binders” (53, S5. S9, Sll, S12, S18), in addition to the “primary binders” (S4, 57, S8, 513, S15, 517, S20) bound by 16 S RNAP (Hochkeppel & Craven, 1976; Hochkeppel et al.. 1976). 16 S RNAp must bind the p-proteins t We propose the following terminology which will 16 8 RNAp, RNA deproteinized with phenol and deproteinized with acetic acid and urea; p-proteins, originally shown to bind 16 S RNA extracted by (Mizushima & Nomura, 1970). These proteins include the 30 S ribosomal proteins which bind 16 S RNA (Hochkeppel et al., 1976). These proteins are S3, SS,
be used throughout this paper: sodium dodecyl sulfate; 16 S RNAa, RNA the 30 S ribosomal proteins which were the phenol/sodium dodecyl sulfate method S4,57, 58, S13, S15, S17 and 520; a-proteins, prepared by t.he acetic acid/urea procedure S9, Sll, S12 and 518.
H.-l<.
HO(‘HKEL’i’E1,
ANI)
(:.
K.
(‘l<.\V15N
I Fast
FIG. 1. Densitometer scan of a composite agarose/polyacrylamide (slow peak) and 16 S RNAP (fast peak) at 540 nm. This Figure serves
split, gel with 16 S RNAa as a standard for Figs 2 to 4.
before being capable of binding the six a-proteins. This addition of individual p-proteins to 16 S RNAP led to a the RNA which would simultaneously create new binding bility we examined the effect of individual p-proteins on RNAP as measured by composite agarose/polyacrylamide
suggested to us that the conformational change in sites. To test this possithe conformation of 16 8 gel electrophoresis.
(a) The e#ect of protein
X4 on the conformation
of 16 S RNAp
In order to examine the inlluence of S4 on the conformation of 16 8 RNAP, a. specific complex between 16 S RNAP and protein S4 was formed in buffer I verified as described in Materials and Methods. The RNA was then deproteinized from the complex by the acetic acid/urea extraction procedure. This deproteinized “S4-RNA” was immediately analyzed by “split’‘-gel electrophoresis. Figure 2(a) shows that &l-RNA has the same low mobility as 16 S RNA”, and (c) slower than 16 8 RNAP. However, if the RNA is extracted from the same 16 S RNA”-S4 complex by the phenol method, the mobility is faster t,han that of 16 S RNA” (Fig. 2(b)) and identical
RNA
CONFORMATION
DURING
RIBOSOME
ASSEMBLY
627
(d)
i Fm. 2. Split-gel densitometer scans of RNA extracted from 16 S RNAP-S4 complex. The complex was prepared as described in Materials and Methods and deproteinized in (a) and (c) with acetic acid/urea and in (b) with phenol/sodium dodecyl sulfate. (a) Acetic acid/urea extracted 84-16 S RNA versus 16 S RNA” (both slow); (b) phenol extracted SP16 S RNA (fast) versus 16 S RNA* (slow); (c) SP16 S RNA (slow) www 16 S RNAP (fast); (d) control: 16 S RNAP re-extracted with acetic acid/urea in the absence of protein 54 v&vu8 16 S RNAp (both fast).
to our standard 16 S RNAp preparation (data not shown). If 16 S RNAp is re-extracted with acetic acid/urea in the absence of protein S4, there is no alteration in mobility (d). Thus any changes in RNA mobility found in RNA extracted from specific protein-RNA complexes are due to the interaction of the protein with the RNA only and not to the extraction procedure employed. This also demonstrates that the acetic acid/urea procedure reveals differences in the 16 S RNA conformations within specific RNA-protein complexes that the phenol extraction procedure obliterates. We conclude that when 16 S RNAp binds protein 54, the conformation of the RNA is altered so that it has a lower mobility during gel electrophoresis. (b) The effect of proteins
X7, 85, S15 and 520 on the 16 S RNAP
conformation
Figure 3 demonstrates the influence of the p-proteins 57, S8, S15 and S20 on the conformation of 16 S RNAP. On the left (a) are agarose/polyacrylamide split-gel patterns of RNA extracted with acetic acid/urea from the individual 16 S RNAPprotein complexes compared to 16 S RNAP. On the right (b) are gel patterns of the sameRNA preparations co-electrophoresedwith I6 S RNA&. As can be seen,S7-RNA and S&RNA have a mobility identical to that of 16 S RNA” (b) and lower than that of 16 S RNAP (a). SlB-RNA appears to have an intermediate mobility between 16 S RNAP and 16 S RNA”, whereas“SBO-RNA” hasa mobility unchanged from the original 16 S RNAP. Thus many, but not all, of the p-proteins can induce conformational alterations in 16 S RNA. Furthermore, the fact that $15RNA has an intermediate 42
628
H.-K.
HOCHKEI’I’EL
AND
(:.
It.
CRAVEK
J 3. Split-gel densitometer scans of RNA extracted with acetic acid/urea from 16 RNAP-87 16 S RNAP-S8, 16 S RNAP-S15 and 16 S RNAQZO complexes. The deproteinized RNA preparations were compared in (a) with 16 S RNAP and in (b) with 16 S RNA&. (a) 57-16 S RNA (slow) VWBUS 16 S RNAP (fast); M-16 S RNA (slow) versus 16 S RNAP (fast); 516-16 S RNA (slow) ~UCMLS 16 S RNAP (fast); S20-16 S RNA wewu8 16 S RNAP (both fast). (b) 57-16 S RNA ~tersu~ 16 S RNA* (both slow); SS-16 S RNA WYWUS 16 S RNA” (both slow); S15-16 S RNA (fast,) aersz .Y 16 S RNA” (slow); S20-16 S RNA (fast) oemus 16 S RNAR (slou’). FIG.
electrophoretic mobility suggests that these conformational alterations in RNA structure are different depending upon the protein bound to the RNA. There may also be more subtle differences between the conformers with low electrophoretic mobility, which are not detectable in our system. (c) The binding
of one protein to 16 S RNA induces a conformational which permits a second protein to bind
change in the RNA
This finding that the binding of proteins S4, S7 or S8 transforms the 16 S RNAP conformation into the 16 S RNA” conformation, which binds many new proteins, stimulated us to investigate the possibility that these proteins individually could change the RNA conformation in such a way that would permit a new protein to associate with the RNA. Other workers (e.g. Schaup & Kurland, 1972) have suggested that some of the co-operative assembly relationships for the 30 S ribosomal proteins could be best attributed to induced RNA conformational changes giving rise to new site-specific protein binding sites. We have not as yet made an exhaustive study but we report here several experiments which substantiate this hypothesis. Our approach in these experiments was to prepare first a complex between a single protein and 16 S RNAP. This complex was then deproteinized by the acetic acid/urea procedure.
RNA
CONFORMATION
Fra. 4. Densitometer re-extracted RI-RNA RNA”; (c) RI*-RN.4
DURING
RTBOSOME
ASSEMBLY
scans of composite agarose polyacrylamide split verau8 16 S RNA*; (b) acetic acid/urea re-extracted versus 16 S RNAP; (d) RI*-RNA wraus RI-RNA.
629
gels. (a) Acetic acid/urea RI*-RNA 2)er8’8us 16 S
The resultant preparation of 16 S RNA” was quantitatively tested for its ability t’o bind a second protein. Figure 6 shows the binding curve performed with 3H-labeled protein S9 and 16 S RNA extracted by acetic acid and urea from a complex involving protein S7. As can be seen. the S9 protein can bind the 16 S RNA directly, reaching a plateau value of 0.27 molecules of S9 per molecule of RNA. In contrast, protein X9 does not bind the I6 S RNAP used in the formation of the S7-Rn’A complex nor does S9 bind 16 S RNAp which has been re-extracted by acetic acid and urea in the absence of protein S7 (latter data not shown). Thus the binding of S7 to 16 S RNAp must induce a structural transition in the RNA which provides an RNA binding site for protein S9. The fact that the protein S9 binding reaches saturation at, less than one molecule per molecule of RNA could be due to incomplete binding of protein S7, which normally requires other proteins (S4, S8 and S20) to reach maximum binding. Indeed, we have conducted the same experiment with 16 S RNA re-ext’racted from a complex involving 16 S RNAP, protein S7 and in addition protein S4. The binding of protein S9 (Fig. 7) to the acetic acid/urea r-e-extracted “X4, S7-RNA” is enhanced, reaching a maximum of 0.40 copies per molecule of RNA. In contrast to that acetic acid/urea re-extracted S4-RNA from a 16 S RNAP-S4 complex did not bind protein S9. In an earlier paper (Hochkeppel et al., 1976) it was reported that close to 1 mole of protein S9 was bound per mole of acetic acid/urea 16 S RNA. The reason for the relatively 10~ binding of protein S9 to 54, S7-RNA could be that, even if 16 S RNAP has been transformed into the “slow” form, this slow form may not be completely identical with t,he slow form of 16 S RNA”.
FIG. 5. Densitometer scan 30 S subunits (fast peak).
of a composite
agarose/polyacrylamide
split
gel with
Fost
I
Slow I
16 S ltNAa
from
inactive
30 S subunits
(slow
peak),
and
frown
active
-RNA
CONFORMATION
DURING
RIBOSOME
631
ASSEMBLY
I 0.28
-
0.24
-
I:1
2:i
Mel S9/mol
3:1
4:l
6:l
5:l
16s RNA I” reoctlon
Fro. 6. Saturation binding curve for protein S9 and re-extracted S’I-RNA. The reconstitution was carried out in buffer I. The purified RNA-protein complexes were assayed directly for 3H cts/min bound. The calculations of copies protein bound/molecule RNA were based on the molecular weight data of Craven e2 al. (1969) and the concentration of protein was determined by the ninhydrin method of Moore & Skin (1948). It was assumed that one absorbance unit at 260 nm corresponds to 7.6 x lo-” mol RNA/ml. -o-o--. Re-extracted S7-RN4; -A-A--, phenol RN4 conkol.
Mol protemhol
F1o. 7. Saturation Fig. 6. --e-e---
(d) An RNA
binding Re-extracted
curve for protein S4, S’I-RNA;
conformational
alteration
16s RNA in reactlon
mixture
89 and re-extracted -o-(j -~, phenol
occurs during
S4, S’i-RNA. RNA cont,rol.
the RI to RI*
For
details
see
transition
Having established that the 16 S RNA conformation can vary after association with specific ribosomal proteins, we proceeded to determine whether or not other conformations of 16 S RNA can be distinguished at certain stages in ribosome assembly. Held & Nomura (1973) have shown that under certain ionic conditions the reconstitution intermediate (RI) particle has a sedimentation constant of 21 S whereas the heat-activated particle (RI*) is dramatically more compact having a
H.-Ii.
032
HO(IHl
i\Sl)
(:. Ii.
C:KAVES
sedimentation constant) of 26 S. As the pr&rin c*ontcnt of t,htt t u’o part,iclrs is presumablv identical, it, is reasonable to suggtxst, that’ t,hcl RNA has rrncic~go~w n structural transition. We have examined this Ilypothcbsis by using the acetic: acid/urea pro~~lurc: 60 extract RKA from t)he Rl and Rl* preparations. In each case the RI and the RI* preparations were constructed wit,h 16 R RXA* and 30 S ribosomal prot,eins. Figure 4(a,) shows the split gel containing I6 S RNA” and acetic acid/urea re-ext’racted “RI-RNA”, both having identical mobility. In contrast, Figure 4(b) represents acetic acid/urea re-extracted “RI*-RKA” co-electrophoresed with 16 S RNA”. In this case the “RI*-RNA has a faster mobility as verified by Figure! 4(c) which is “RI*-RNA” against 16 S RNAP. Thus the RNA ext’racted from Rl particles is of t’he slow or 16 S RNA” variet,y and RNA obtained from RI* part,icles has a fast mobility similar to that of 16 S RNA*. This is verified in Figure 4(d) which shows that RI-RNA and RI*-RNA have distinctly different electrophorotic mobilities. This is the first direct demonstration that a conformational transit,ion in the RNA moiety actually occurs during the conversion of RI to Rl*. However, it should be noted that the 16 8 RNA” and 16 S RNAP conformations cannot bt: distinguished by sedimentation velocity (Hochkeppel & Craven. 1976). Thus the RNA conformational changes we observe for the RI to RI* transit,ion cannot fully account for the dramatic change in sedimentation constant reported by Held & Komura (1973). RNA-REA and protein-protein int,eractions may also have some influence 011 t’lie tjransit’ion of the ribonucleoprotein complex. (e) “Active”
and “‘nactive”
30 S ribosomes conformations
contain
RNA
with different
Zamir et al. (1971) were the first to suggest that the 30 S ribosome is capable of a reversible conformational change which causes loss of capacity to bind transfer RNB at 0°C. They showed that this activity is reversibly lost when the ribosomes are depleted of either monovalent or divalent cations. Later, Ginsberg et al. (1973) demonstrated that these inactive 30 S ribosomes contain proteins with a different reactivity to N-ethyl maleimide than the proteins in the active particles, thus indicating that the two ribosomes have different conformations at least within the protein moiety. We have already sholvn that, 16 S RNA extracted wit’h acetic acid/ urea from such inactive 30 S ribosomal subunits yields RNA containing sites for the a-proteins (Hochkeppel & Craven, 1976). We therefore undertook to determine whether the RNA from such inactive particles is a different conformation. Figure 5 presents an experiment which directly demonstrates that inactive 30 S ribosomes contain RNA with an electrophoretic mobility distinctly slower than RNA extracted from active ribosomes. In this experiment separate portions of 30 S subunits (in buffer III) were diluted 1 to 10 into an activation buffer (0.02 M-Tris (pH 7-O), 0.02 M-Mg acetate, 0.2 M-NH,Cl and 2 mM-dithiothreitol and an inactivation buffer (0.02 M-Tris (pH 7.0), 0.5 miw-Mg acetate, 0.1 M-NH,Cl and 2 m&l-dithiothreitol). Both samples were heated to 40°C for 25 minutes and then extracted by the acetic acid/urea procedure. We routinely find this procedure yields the activation of ribosomes reported by Zamir et al. (1971). The RNA extracts were then co-electrophoresed on agarose polyacrylamide split gels. Figure 5 shows that the RNA from the inactive particle has a lower mobility in our system. We conclude that the RNA conformation within the 30 S ribosome can assume two distinctly different configurations.
RNA
CONFORMATION
DURING
RIHOSOME
633
ASSEMBLY
4. Discussion In all of the experiments described, we have shown changes in RNA conforma’tion of RNA re-extracted with acetic acid/urea from specific 16 S RNAP-protein complexes by comparing its electrophoretic mobility with t’hat observed for 16 S RNA” and 16 S RXAP. Thus we have shown that when proteins S4, S7, S8 and, to a lesser extent. S15 are individually bound to 16 S RNA”, the re-extracted RNA possesses a mobilit,y similar to that found for 16 S RNA”. Protein S20 has no effect on t’he RNA conformation as determined by this technique. Furthermore, this slow form, induced by t,he binding of these several proteins, can be reversed t’o a, form having roughly the fast elrctrophoretic mobility, by heating t’he RI particle, transforming it into the Rl* part’icle. All of these observations ana summa.rizcd in Figure 8. We emphasize t)hat the fast) form of RNA extra&d from t#hr RI* particle has not been shown to actually have the same conformation as the original 16 S RNAP, only that they have similar mobilities on this separabion system, The sa,me comment holds in comparing so-called S-I-RNA with S&RNA or S7-RNA. Indeed. we have some reason t’o believe that, although these RXA preparations have indist,inguishable electrophoretic mobilities. there almost certainly must be subtle differences in conformations. We propose that 16 8 RKA has the potential for a large numbrr of different conformations inherent in its complex st,ruct’ure and that many of thcbsc different forms arc assumed at different stages of assembly in vitro. S4-RNA complex 1
c
Other early protem
Fost conflgurahon
SIOW conflgurotlon
Slow conflguratlon
FInal protems
Fast conflguratlon
Fast conflguratlon
The RI particles we employed in these experimenbs were made by incubation of 16 S RNAP with total protein in the cold followed by pelleting through a layer of sucrose. The protein content of these particles is roughly that seen by Held & Nomura, (1973), and contains about 13 proteins. Clearly one of the next, steps in the exploitation of our techniques to analyze RNA conformabional changes is to determine which of these 13 proteins are required to transform the slow form of RNA into the fast form. Held & Nomura (1973) have found only five prot’eins which are strongly required t,o obtain an RI to RL* transformation. Another two are partially required and tive are only weakly required. We have t)ested two of these proteins and have, found that’ an RI particle made with all proteins present except protein S19 cannot undergo the change from slow to fast RNA induced by heating (Hochkeppel $
634
H.-K.
HOCHKEPPEL
ANI)
G.
It.
CRAVE:N
Craven, unpublished results). Further studies along this line should be very germane to a molecular understanding of the Rl to RI* transition. The most important aspect of the results presented here is t’hat these observed alterations of the RNA conformation seen at various stages in 30 S assembly in vitro could have actual functional significance with regard to the proposed assembly map of Mizushima & Nomura (1969). We have demonstrated that the binding of protein X7 to phenol RNA changes the RNA such that the deproteinated RNA is now capable of binding significant amounts of protein S9. Thus the assembly-dependence relat’ionship originally described by Mizushima & Nomura for these two proteins can now bc given a rational molecular interpretation. Protein S7 induces an alteration in a nearby section of the 16 S RNA which allows for S9 interaction. We propose that this type of molecular interpretation must pertain to many of the assembly-interdependent, relationships, especially those involving the proteins which we have shown can bind 16 S RNA” (Hochkeppel et al., 1976). We thank Dr Julian Gordon for critically reading the manuscript. We would like to express our appreciation to Cathy Bloomer for providing all the purified ribosomal proteins used in this investigation. This work was supported by the Graduate School and the College of Agriculture and Life Sciences, University of Wisconsin, Madison, and by research grant GM15422 from the National Institutes of Health. We also acknowledge use of the Biochemistry Department pilot plant, directed by Dr John Garver and supported by U.S Public Health Service grant FR-00214. REFERENCES Cox, R. A. & Hirst, W. (1976). Biochem. J. 160, 505-519. Craven, G. R. & Gupta, V. (1970). Froc. Nat. Acad. Sci., U.S.A. 67, 1329~-1336. Craven, G. R., Voynow, P., Hardy, S. J. S. & Kurland, C. G. (1969). Biochemistry, 8, 29062915. Ginsburg, J., Miskin, R. & Zamir, A. (1973). J. Mol. Biol. 79, 481-494. Hardy, S. Y. S., Kurland, C. G., Voynow, P. & Mora, G. (1969). Biochemistry, 8,2897--2905. Held, W. A. & Nomura, M. (1973). Biochemistry, 12, 3273-3281. Held, W. A., Ballon, B., Mizushima, S. & Nomura, M. (1974). J. Biol. Chem. 249,3103~3111. Hochkeppel, H.-K. & Craven, G. R. (1976). Nucl. Acids Res. 3, 188331902. Hochkeppel, H.-K., Spicer, E. & Craven, G. R. (1976). J. Mol. BioZ. 101, 155-170. Mizushima, S. & Nomura, M. (1970). Nature (London), 226, 1214-1218. Moore, S. & Stein, W. H. (1948). J. BioZ. Chem. 176, 367-388. Morris, D. R., Dahlberg, J. E. & Dahlberg, A. E. (1974). NucZ. Acids Res. 1, 1249-1258. Nashimoto, H. & Nomura, M. (1970). PTOC. Xat. Acad. Sci., U.X.A. 67, 1440. Peacock, A. C. & Dingman, C. W. (1968). Biochemistry, 7, 668-674. Rice, R. H. & Means, G. E. (1971). J. Biol. Chem. 246, 831-832. Schaup, H. W. & Kurland, C. G. (1972). Mol. Gen. Genet. 114, 350-357. Sypherd, P. S. (1971). J. Mol. BioZ. 56, 311-318. Traub, P. & Nomura, M. (1969). J. Mol. BioZ. 40, 391l413. Traub, P., Mizushima, S., Lowry, C. V. & Nomura, M. (1971). In Methods in Enzymology (Moldave, K. & Grossman, L., eds), vol. 20, part C, pp. 391-407, Academic Press, New York. Wireman, J. W. & Sypherd, P. S. (1975). Biochem. Biophys. Res. Commun. 66, 570-~577. Wittmann, H. G., Stoffler, G., Hindennach, I., Kurland, C. G., Randall-Zahelbauer, L., Birge, E. A., Nomura, M., Kaltschmidt, E., Mizushima, S., Traut, R. R. & Bickle, T. A. (1971). Mol. Gen. Genet. 111, 327-333. Zamir, R., Miskin, R. $ Elson, D. (197 1). ,7. Mol. BioZ. 60, 347-364.