J.

Mol.

Biol.

(1978)

121, l-15

RNA-Protein 1II.t Conformation

Interactions in the Ribosome

and Stability of Ribosomal Protein Binding Sites in the 16 S RNA

AKIRA MUTO$ AND ROBERT A. ZIMMERMAXS~ D.4partment de Biologic MoECculairP Universite’ de CenPve 30, Quai Ernest Ansermet 1211 Genbve 4, Switzerland (Received 29 June 1976, and an revised form, 20 Decem,ber 1977) Following dialysis against distilled water, the 16 S ribosomal RNA of Escherichia coli is unable to interact with 30 S subunit protein S4 at 0°C. The dialysed RNA recovered this capacity, however, when heated at 40°C in the presence of 0.02~Furthermore, its sensitivity to riboMgCl, prior t’o addition of the protein. nuclease markedly declined and its sedimentation rate increased as a consequence of this treatment. Although no concomitant changes in secondary structure were detected by absorbance and fluorescence techniques, the rearrangement of a small number of base-pairs was not excluded. Kinetic measurements revealed that binding site reactivation satisfies the first-order rate law and that the process is highly temperature-dependent, exhibiting an Arrhenius activatioii energy of 40,800 cal/mol. Together, these data suggest that dialysed RNA undergoes a unimolecular conformational transition upon pre-incubation in Mg2 + containing buffers and that this transition leads to renaturation of the binding site for protein S4. Similar results were obtained for several other proteins of the 30 S subunit. In particular, S7, S16/S17 and 520 all failed to interact efficiently with dialysed 16 S RNA at 0°C. These proteins bound normally to the RNA, however, after it had been incubated at 40°C in the presence of Mg2 + ions. Ry contrast, prior dialysis of the 16 S RNA did not affect its ability to associate with S8 and 515 at 0°C. These two proteins interacted equally well with dialysed and preincubated 16 S RNA, indicating that their binding sites are not susceptible t,o tlic, reversible alterations in conformation which influence the attachment of the other RNA-binding proteins to the nucleic acid molecule. The effects of dialysis ancl pre-incubation on the interaction of 16 S RNA with an unfractionated mixture of 30 S subunit proteins were also investigated. The dialysed RNA bound onI> S6, SS, S15 and 518 at 0°C whereas, after heating at high Mg2+ concentrations, the RNA associated with S4, 57, S9, 513, S16/S17, Sl9 and S20 as well. These results leave little doubt that the protein-binding capacities of the 16 S RNA are intimately related to its three-dimensional configuration. although individual binding sites appear to differ significantly in their stability to small changes irk structure. i Paper II in this series is Zimmermann et al. (1974). $ Present address: Research Institute for Nuclear Medicine and Biology, Hiroshima 1Jniversit~. Kasumi-cho, Hiroshima, Japan. 0 Present address: Department of Biochemistry, Universit,y of Massachusetts, Amhrl,-;t, Mass. 01003, U.S.A. OO”L L!x3e/7x/12i

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%IhLRlEKM~\SS

1. Introduction A growing body of evidence indicates t,hat specific intc~rw&ons 11;~tw~,~nri 1)1);()tni~l proteins and ribosomal RNA depend not only upon thca base s~yur~ncc~and sc~~mtl;~~~~ struct,ure of the nucleic acid chain but upurl its three-dimenxiorlai org:anisat,ion as \\Y~II (Bollen et al.. 1970; Sypherd, 1971 ; Schult,e ef al.. 1974; Zimmerm:Lnn et (II.. 1975: Hochkeppel & Craven, 1977a). The binding of individual proteins to the 16 S RNA of Escherichia coli has generally been studied at 40°C in neutral buffers containing ~).3(, to 0.35 ~-Kc1 and 0.02 M-Mgcl,, the same temperature and ionic cnvironment~ that promote optimal reconstitution of the 30 S subunit (Traub & Nomura. 1969). It, is no\!’ firmly established that under the above conditions, proteins S4. S7, S8. 815, 817 and S20 combine with specific sites in the ribosomal RNA (Mizushima & Nomura. 1970; Schaup et al., 1970,1971; Garrett et al.. 1971; Muto et al., 1974; Held et al.. 1974) and several other protein-RNA associations have been detected using 30 S subunit components prepared in novel ways (Hochkeppel et al., 1976: Hochkeppel & Craven, 1977b; Littlechild et al., 1977). A detailed analysis of factors influencing the binding of S4 and S8 to the 16 S RNA has shown that interactions involving these prot,eins arc’ relatively insensitive to variations in pH and K + ion concentration, but) strongI) dependent upon incubation temperature and the presence of magnesium ions (S&&e below 0.01 I13.complex $ Garrett. 1972; Schulte et al.. 1974). Bt Mg2+ concentrations formation rapidly falls off and the RNA apparently assumes a less compact structure since its sedimentation rate sharply declines (Cammack et al.. 1970: Schultz et (~1.. 1974). Further correlations between the conformation and protein-binding activity of the nucleic acid molecule have been inferred from the properties of 16 S RNA prepared by acetic acid/urea extraction (Hochkeppel et al., 1976; Hockheppel & Craven, 1977u). The present investigation was prompt’ed by the discovery that 16 S RNA fresh]! extracted with phenol was often unable to associate with protein 84 at 0°C (Zimmerhowever. when heated at mann et al., 1972a). The RNA recovered this property. 40°C in the reconstitution buffer. In addition, the incubation transformed the RNA from a form that was highly ribonuclease-sensitive to one that n-as relatively resistant to enzymic degradation. We subsequently noticed that, any 16 S RNA preparat,ion could be converted to the inactive, ribonuclease-sensitive form by dialysis against distilled water. Here again, the capacity of the RNA to associat,c with S4 at 0°C. as well as its resistance to ribonuclease digestion, was restored by heating in buffer COIItaining high concentrations of MgCI,. The binding site for protein S4 could thus be “denatured” and “renatured” at will, and we speculated t’hat the transition was mediated by a reversible conformational rearrangement within t,he 16 S RNA molecule. The experiments reported here were designed to characterize the structure and binding capacities of dialysed 16 S RNA both before and after heating in the presence buffer was found to produce a of magnesium ions. Incubation in Mg2+ -containing small but reproducible alteration in the overall conformation of the RNA. Moreover. this treatment was shown to overcome the inability of dialysed 16 S RNA to efficient15 interact with a number of 30 S subunit proteins at 0°C. Differences in the stability of various protein binding sites are consistent with our current understanding of their size, complexity and structure (Zimmermann et al., 1975; Ungewickell et al.. 1975a). Additional characteristics of the RNA segments that interact with S4. S16/S17 and S20 are described in the accompanying paper (Mackie & Zimmermann. 1978).

(‘ONFORMATION

AND

STABILITY

OIi

16 S rRSA

3

2. Materials and Methods (a) Ribosomal

RNA

LJnlabelled and ‘V-labelled 16 S RNA were prepared from the 70 S ribosomes of E. coli strain MRE600 according to Muto et al. (1974). Purified 16 S RNA in Tris buffer (0.05 MTris.HCl, pH 7.6) was precipitated with ethanol and stored at -20°C. Prior t,o earh experiment, t,lle RNA was collected by centrifugation, dissolved in distilled water at a concentration of 1 mg/ml and dialysed extensively against distilled water at 4°C. Dialysed 16 S RNA, which will be referred to hereafter as D-16 S RNA, was used directly or after incubation in the presence of 0.02 M-Mg2+ as indicated in tttr text. Under standard conditions, incubation was carried out for 20 min at 40°C in either Tris/Mg buffer (0.05 or Tris/Mg/K buffer (0.05 bI-Tris.HCl (pH 7.6). I\l-Tris.HCl (pH 7.6), 0.02 M-M&X,) 0.02 tir-M&l,, 0.35 ~-Kcl, 0.006 M-8-mercaptoet.tlarlol). (b) Xedirnentation RNA samples were sedimented through 12-ml 3oi;, to 15% linear sucrose gradients ilr Tris/Mg/K buffer in either a Spinco SW41 or an IEC SB283 rotor at 2°C. After centrj fugation, the gradients were fractionated into 20 tubes and the radioactivity in each on was controlled by the use of a jacketed quartz cuvette through which water was circulated from a thermostated bath. Measurements were made at intervals of 5°C and after eactl increase in temperature, 5 min were allowed for the sample to reach thermal eyuilibriurn. (d) Pluorescence

measurements

Fifty pg D-16 S RNA were incubated in 1 ml of the appropriate buffer and mixed wittl ethidium bromide (Calbiochem) at 0°C to a final concentration of 2.5~ 1O-5 31. Changes in fluorescence emission were recorded with a Perkin-Elmer model MPF-2A fluorescenccspectrophotometer as the temperature of the RNA solution was increased from 5” to 50‘(! in increments of 5°C. Excitation and emission wavelengths were 480 and 590 nm. respectively. The temperature of the sample was maintained as described in section (c). above. and 5 min were permitted for thermal stabilization between eacll reading. (e) Ribosomal

proteins

lndividual 3H-labelled proteins from the 30 S subunit of E. coli MRE600 were purified by the method of Muto et al. (1974). Unfractionated 30 S subunit proteins were prepared according to Zimmermann et al. (1974). (f) RXA--protein

complexes

Interactions between ribosomal RNA and individual ribosomal proteins were st,udied by a modification of the procedure previously reported (Muto et al., 1974). From 20 to 25 pg D- 16 S RNA were incubated in Tris or Tris/Mg buffer as indicated for each experiment and chilled on ice. The RNA was mixed with approximately 2 molar equivalents of purifi wibhin this interval, however. and the profile wa,s nearly superposable upon on(’ obtained for a sample that had been incubated at 40°C in Tris/Mg buffer prior to th(a absorbance measurements (data not shown). Alt’hough these findings would exclude any large alterations in secondary structure below 5O”C, a shift, in a small number of hydrogen bonds is not ruled out. We estimate that a net rearrangement of fewer than trn base-pairs would not have been detected by t*his method. Quantitative chanprs in the fluorescence emission of RNA-et’hidium bromide complexes should also reflect va,riations in the amount of secondary structnre wit’hin the RNA molecule (Roll~w

6

rractlon

number (b)

FIG. 2. Sedimentation of D-16 8 RNA following incubation in the presence of 0.02 ax-Mg’+. D-16 S RNA were incubated in 100 ~1 Tris/Mg buffer for 20 min at (a) 0°C or 5 6% 14C-labelled (b) 40°C, chilled on ice, and mixed with 50 pg unlabelled D-16 S RNA. The samples were fractionated by sucrose gradient centrifugation for 16 h at 30,000 revs/min and analysed as described in Materials and Methods. 14C-labelled RNA (+oPPO--); absorbance at 260 nm (-m---m--). Insets show the rat,io of unlabelled:labelled RNA in the peak fractions (+-•-), calling attention to t,he difference in sedimentation rate (a) before and (b) after heating in the presence of 0.02 M-Mg2 +.

et al., 1970). Here again, the behavior of unheated D-16 S RNA did not differ from that of D-16 S RNA which had been heated to 40°C in Tris/Mg/K buffer and chilled prior to analysis (data not shown). These results confirm the conclusion that conditions under which the D-16 S RNA acquires a more compact’ structure and a heightened resistance to RNAase do not bring about an appreciable alteration in base-pairing. (b) Interaction

of D-16 S RNA

with individual

30 S subunit yroteim

We have extended our studies on the interaction of D-16 S RNA with S4 (Zimmermann et al., 1972a) to its association with each of six RNA-binding proteins of the 30 S subunit. For these assays, a solution of D-16 S RNA in Tris/Mg buffer was divided into two portions; one of them was held at 0°C for 20 minutes while the other was incubated at 40°C for an equal period of time and chilled on ice. Both samples were then tested for their ability to interact with 54, S7, S8, S15, S16/Sl7t and 520 at 0°C. In Table 1, the amount of each protein bound under t#hese conditions (lines I and 2) is expressed as a percentage of that bound when protein-RNA complex formation was allowed to proceed at 40°C (line 4). The association of unheated D-16 S RNA with 54, X16/X17 and S20 was less than 20% as efficient, and with S7 less than 50% as ef?icient, at 0°C as in the control. Following incubation at 40°C in the presence of 0.02 M-Mgcl,, the RNA recovered more than SO%, of its ability to bind each of the of D-16 S RNA four proteins. However, in the absence of Mg 2+ ions, pre-incubation t Since purified here were carried

S16 and A17 were unavailable during these studied, out with a mixt,ure of 816 and 817 that will be referred

the qxriment.s to throughout

reported as S16/817.

(‘ONFORMATION

AND

STABILITY

OF

16 S IEN.\

TABLE 1 Effects of temperature and Mg2 + ioa concer~tr~t~o)~ on the &eraction II-16 X RNA with individual 30 S r;ubunif protein,s Incubation I’ri(,r t rvat merit I,f RXX +31p2+. OH’ 1 MpZ + , 40°C’ -~Mg’ + , 411”( * + yg* + 40”(‘

conditions? Formation RNA-protein complexes

of

Amount s-k

protein ,s7

bound sx

-+. Mg2 + ) 0°C’ +hfg2+) 0°C’ +I\Ig2+, 0°C’ -- ivg* + , 4noc’

(i 82 10 100

43 85 54 100

108 8” x4 1 no

?f

as perccntagc of control: 815 S16/817 SW

1”” 99 107 100

li 18 :!I I on

19 93 “(1 lotI

or Tris/Mg ( t-M& + ) t Twenty-five pg D-16 8 RNA were first incubated in Tris ( - Mg”+) buffor for 20 min at) the indicated temperature and chilled to 0°C‘. The RNA samples were then mixed with about 2 molar equivalent of each protein in 100 ~1 Tris/hfg/K buffer and maint,ainrtl at &her 0” or 4o’C for an additional 30 min. Xftc,r fractionation of the mixture;l by sucrose gradient centrifugation. the amount of radioactive protein sedimcnting with the 16 S peak was measured. 1 The amount of protein bound to each RNA samplr at WY is expressed as a T)rrcbentagv ot that bound at, 40°C. s(0)

400

(b)

i

300

200

200 2 E \ E u u

f

!z 0

100

0

ir: L

400

(d)

I :+ T

(e)

r 100 -< +! 2 0 ; 400

e a T

300

L

87

300

200

t I!

: I

: I

L

200

100

‘00

0 0 Fraction

number

FIG. 3. React.ivat,ion of binding site for protein 54 following incubation of II-16 S RNA iu t.he presence of O-02 M.Mga + . 20 pg ‘W-labelled D-16 S RNA were incubated in Tris/Mg buffer for 20 min at (a) and (f) 40X’, (b) O”C, (c) lO”C, (d) 20°C and (e) 30°C and chilled on ice. RNA samplex were added to 1.4 pg 3H-labelled 54 in 100 ~1 Tris/Mg/K buffer and incubation was continued for 30 min at (a) 40°C or (b) to (f) 0°C. Mixtureswere fractionated bysucrose gradientcrntrifugation for 16 h at 27,000 revs/min and the radioactivit’y present in each tube was measured. ‘*C-1abe11otl RN.4 (--o-i I---); 3H-labelled 54 (-a-e--).

8

T\.

hllJ’l’0

.1X1)

Ii.

.A. %IhI>lli:IChl.\~S

at 40°C did not materially alt#cr its binding act#ivity (‘l’abk I. lint, 3). In ninrlct~ci contrast to the first group of proteins. S8 md Sl5 bound cquall~~ wc~ll to all of t,hck RNA samples. The binding &es for thcsc proteins are apparentjl,y uneft’ec*tc~tl 11) tjraGtion in RNA conformation described abovt~. MAT‘+-dependent These results clearly demonstratc~ t,hat ncbither heat,& nor a high M$ + (*oncet~tration is in itself sufficient to restore the ability of D-16 8 RNA t’o interact with S4. S7, SlS/S17 and S20. Rather, the RNA must he incubated in the presence of’ M$ + ions before an observable increase in binding activity occurs. ‘l’ht~ conditions rquirt~tl for binding site renaturat’ion thus coincide with t’hosc t,hat produce a oonforlnatiolial shift in the D-16 S RNA. Furthermore, the da,ta of Table 1 show that RNA-protein complex formation does not depend on heating the react’anta together at 40°C sinctta all of the proteins that interact independently with the 16 S RNA at 4OYI also associate efficiently with pre-incubated D-16 S RNA at 0°C. on the binding of S4 to The effects of temperature and Mg 2 + ion oonccntration D-16 S RKA are illustrated in Figure 3. The sedimentation profile of a control sample. in which RNA and protein were incubabed together at 40°C. is depicted in Figure 3(a). Figure 3(b) and (c) show that D-16 S RNA does not form a complex with S4 after incubation at 0” or 10°C in the presence of Mg 2 + ions. A small quantity of prot,t:in appears in the 16 S peak when the Rh’B is first heated at 20°C and t’his value increases substantially when pre-incubation is carried out at 30” or 40°C (Fig. 3(d), (e) and (f)). t’o form a dimer sediIt is noteworthy that a fraction of the II-16 S RNA tippears menting at about 25 S (see Fig. 3(b)). A progressive shift of 14C radioactivity from the 25 S peak to the 16 S peak occurs as t,he incubation temperature is increased. After heating at 4O”C, almost all of the material from the dimer peak is rccovrred

0 20

IO

20

Fraction number (b) FIG. 4. Formation of ribonuoleoprotein particles. (a) RI(D)-part,iole. 25 pg ‘V-labelled D-16 8 RNA were incubated with 14 pg unfraotionated 3H-labelled 30 S proteins (500 ots/min per pg) 25 pg 14C-labelled D-16 8 RNA were in 100 1.11Tris/Mg/K buffer for 60 min at 0°C. (b) RI-particle. incubated in 100 ~1 Tris/Mg/K buffer for 30 min at 4O”C, chilled on ice and further incubated with 14 pg 3H-labelled 30 S proteins for 60 min at 0°C. Sucrose gradient oentrifugation of the 2 mixture3 was carried out for 16 h at 25,000 revs/min. Radioactivity was measured in the usual way. 14Clabelled RNA (--O-O--); 3H-labelled protein (-•--•-).

(‘ONFORMATION

AND

STABILITY

OF

16

K rRSA

!I

in the 16 S peak. A parallel analysis showed that the binding site for 515 is fully active under conditions in which no 54 interacts with the D-16 S RNA. In addition. Sl5 associates with the 25 S dimer fraction as well as with t’he monomer (data not shown).

(c) Irderaction

of D-16 S RNA

with unfraction.atrd

30 S xubmit

potpins

The addition of an unfractionated mixture of 30 S subunit proteins to D-l 6 S RNA at 0°C results in the formation of an RNA-protein complex that sediment’s at approximately 18 S (Fig. 4(a)). A second component. sedimenting at about 28 S, is presumed to arise from the association of ribosomal prot,rins with the D-l 6 S RX.4

S3 54 Sl8SlIS9 99 S21 520 Sl4,Sl5 Sl2$13

516/517

400

100 5: p”

0

Sl6/S17

S3 54 SB,Sll,S9

Sl9

S21

520 600

Si4.Sl5 Sl2,Sl3

5 E \ 2 2 5 I s? a T fi

500

too 0 0

50

100

200

I50 Frochon

250

300

number

VIC. 5. Chromatographic analysis of 30 S subunit proteins present in RI(D)-particles and RIparticles. (a) RI(D)-pa&&. 75 rg D-16 S RNA were mixed directly with 40 pg 3H-labelled 30 S proteins (8000 cts/min per pg) in 300 ~1 Tris/Mg/K buffer at 0°C. (b) RI-particle. 75 pg D-16 S RNA were heated at 40°C for 30 ruin in Tris/Mg/K buffer, chilled on ice and t,hrn mixed with 3H-labelled 30 S proteins as in (a). In both cases, reaction mixtures were incubated for 60 min at, O”(’ and centrifuged through 12.ml 3% to 15:; sucrose gradients for 16 h at 25,000 revs/min. Ribonucleoprotein particles were concentrated by sedimentation. Proteins associated wit,h thck particles were extracted and chromatographed on phosphocellulose at pH 6.5 as described in Materials and Methods. Elution profile of 3H-labollcd protrins ( -- - --); elution profile of unlabelled carrier proteins (- - - - -).

10

;I . 11 177’0

.A S I)

Ii.

.\

% I 31 II 14:Ji RI A S S

dimer described in the previous s&ion. The 18 S ribonucleoprot,t,itl \~a;i d~*signatt~tl the RI(D)-particle by analogy with t’hc RI-particlet. a complex formed by the int,t+ action of roughly a dozen 30 S subunit proteins with fully active 16 S RX,1 at 0 (’ (Traub & Nomura, 1969; Nashimoto rt nl.. 1971 : Zimmermnnn rf al.. 1974). When thch D-16 S RNA is heated to 40°C in Tris/Mg/K b u ff er 1)rior to mixing with 30 S subunit proteins at O”C, a 20 S complex similar or identical to the RI-partick is protlu~tl (Fig. 4(b)). Proteins associated with the RI(D)-particle were identified by column chromatography and gel electrophoresis. Chromatography on phosphocellulose revealed that, the complex contained S6, S8 and at least one protein from each of t,he mixtures 89 + Sll + S18 and S14 -+ S15 (Fig. 5(a)). P ractionation of a parallel sa,mplc by polyacrylamide gel electrophoresis established that S15 and S18 lvere present in the RI(D)-particle. whereas S9, X11 and S14 were absent (dat)a not shown). The majo) protein constituents of the RI(D)-particle are t’herefore SB. S8. Rl5 and S18. An analysis of proteins bound to the 28 S dimer peak gave similar results. The results presented here are in good agreement with those> obtained for thcx binding of individual proteins to D-16 S RNA at 0°C (Table 1). Thus, 88 and S15, which directly interact’ with D-16 S R’NA. arc &o found in thcl R[(D) complex. Small amounts of Sl6/S17 and S2O were detected in the RI(D)-partick as well. consistent with their weak attachment to D-16 S RNA in the absence of other proteins. and S4 and S7 were completely missing. Although the occurrence of S7 was anticipated on the basis of its limited binding to D-16 S RR’A in t#hc individual assays. it,s failure to remain associated with the complex in the present CUSPmay br related to the instability of the ST-16 S RNA int’eraction during the numerous preparative steps involved. For comparison, the protein composition of the RI-part& formed with prc(Fig. 5(b)). In conformity with rarliel incubated D-16 S RNA was a,lso determined findings (Traub & Nomura, 1969; Nashimoto et al., 1971: Zimmermann et al.. 1974). this complex was found to contain X4, X7, S9. S13. S16/S17, S19 and S20 in addition to S6, S8, S15 and SlS. (d) Kir~,etics of bindiny

site rennturatiou

Renaturation of the binding site for protein S4 in the D-16 S RNA is strongly temperature-dependent (see Fig. 3). The kinetics of this process were investigated in greater detail in order to estimate the magnitude of the thermodynamic parameters involved. Accordingly, D-16 S RN$ was incubated at several temperatures from 0” to 40°C in Tris/Mg/K buffer for various periods of time, chilled and mixed with a slight molar excess of S4 at 0°C. The amount of 54 bound to each RNA sample was determined by sucrose gradient analysis and the results are shown in Figure 6(a). No increase in S4 binding activity was detected when the RNA was pre-incubated at temperatures below 20°C for 40 minutes, the longest time interval utilized. 7!h( capacity of the RNA to bind S4 rose steadily with incubation at temperatures between 25” and 40°C. After 30 minutes at t’he latter temperature, the RNA exhibited a binding activity equivalent to about 909