Structural studies of the ribosomes of Rhodopseudomonas spheroides

J. iKoZ. Biol. (1966) 22, 63436

Structural Studies of the Ribosomes of Rhodopseudomonasspheroides DAVID I. l?mmwt,

BERNARD

POU

AND ERNEST D. GRAY

Department8 of Pediatrics and Biochemistry Collqe of Medical Xciences, University of Minnesota Minneapolis, Minnesota, U.S.A. (Received 21 April 1966, and in revised form 23 August 1966) Riboeomes isolated from Rfwdopsewlonaonae qvheroidea have a sediment&ion coeffioient at infinite dilution of 60s. Values of 46s and 29s were obtained for the ribosomal subunits. The two ribosomal subunits of this organism have different proportions of RNA and protein. The 46s has 62% RNA, 38% protein, and the 29s has 61% RNA, and 49% protein. It was found that the 29 s subunit is structurally labile to both T1 and pancreatic RNase whereas the 46s particle is relatively stable. 60s ribosomes exhibit diverse responses to RNase depending on both the mode of isolation and the presence of potassium chloride. Evidence is presented to show that different states of intraribosomal binding are responsible for the apparent stability of some and lability of other 66s ribosomes to RNase action. Polyuridylio acid binds both to the 29s subunit and to the 66s ribosome and protects these particles from the a&ion of T1 RNase. It is suggested that polyuridylic acid binds to 66s and 29s particles in a similar manner. Since the binding of.polyuridylic acid protects against nuclease action, it is proposed that the structurally important RNA attacked by the nuclease is either associated with or part of the messenger binding site. 1. Introduction It has been shown that the 50 s and 30 s ribosomal subunits possessdiffering functional capabilities. Takanami & Okamoto (1963) haye demonstrated that the messenger RNA is associated with the 30s particle and Gilbert (1963) showed that both the newly formed polypeptide as well as the sRNA are associated with the 50s subunit. Compartmentalized function suggests that there could exist observable structural differences between these subunits. Studies of the properties and interaction of the ribosomal particles isolated from Rhodopaeudomonas a~heroides were undertaken with the object of elucidating some of these differences. The primary approach in the present investigation involved the use of ribonuclease to alter ribosomal structure. The effect of RNase on the structural integrity of ribosomes and subunits varies from organism to organism. Ribosomes isolated from Eacherichia wli maintain their structural integrity after RNase treatment whether in the 70s form or dissociated to the 50s and 30s subunits (Santer, 1963). Hess & Horn (1964) found that although the 70s ribosomes isolated from Streptococcus pyogenee were structurally resistant to pancreatic RN&se, the dissociated subunits were both sensitive to enzymic treatment. Our studies reveal that the smaller ribot Present Washiqton,

address: Department D.C., U.&A.

of Molecular

Biology, 68

Walter

Reed Institute

of Research,

D. I. FRIEDMAN,

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somal particle of R. spheroida is very sensitive to the actionof RNase while the larger subunit is relatively resistant. Structural lability of the smaller ribosomal subunit has been reported by others in several experimental systems (T’so & Vinograd, 1961; Tashiro & Siekevitz, 1965; Lamfrom & Glowacki, 1962).This differential sensitivity to RNase has been exploited in studying subunit interaction and structure. 2. Solution

Materials and Methods (a) Soluticns

A: 0.01 i%f-MgCls, 0.06 m-KC], 0.006 u-Tris-HCl buffer,pH 76. Solution B: 0.0001 M-MgCl,, O-01 M-Tris-HCI buffer, pH 76. Solution C: 0.01 m-MgClz, 0.01 M-Tris-HCl buffer, pH 7.6. Solution D: 0.6 a~-KCl, 0.04 M-Tris-HCl buffer, pH 7.6.

(b) Organisms Strain 241 -of R. spheroides, a facultative photoheterotroph, was originally obtained from R. K. Clayton. The organisms when grown anaerobically were cultivated at 26’C in Roux bottles and illuminated with 60-w incandescent light bulbs. Aerobically cultivated organisms were grown at 26% in the dark and aerated by shaking. The media used in the growth of both physiological types of R. spheroides was that described by Cohen-Bazire, Sistrom t Stanier (1957). E. co.% strain K12 used in these experiments was obtained from Dr Brooks Church. The organisms were grown at 37% in Difoo brain-heart infusion and were aerated by shaking. (0) Isokdon of ribosomee Three different methods of cellular disruption were used in these experiments: sonication, alumina, grinding and osmotic lysis. In these isolation procedures all centrifugation was carried out at 0%; cells were harvested in the logarithmic phase of growth. Cells were washed twice with the appropriate buffered salt solution, and then treated for 6 min in an ultrasonic disintegrator (Medical and Scientiilc Equipment Co.). The cell extract was centrifuged twice at 12,000 g for 20 mm, the supernatant decanted, and stored at 4°C. Al~w&a grinding Cells were washed with the appropriate buffered salt solution, and the sedimented cells frozen. The frozen cells were disrupted by grinding with twice their weight of ahunina and then extracted with 3 ml. of buffered salt solution containing 60 pg of DNase I. The solution was centrifuged as before and the supernatant was decanted and stored.

os??%otic lyei Cells were washed twice with deionized water, suspended in 6 ml. of solution D at 4”C, then 25 mg of lysozyme and O-6 ml. of 0.5% EDTA, pH 8.0, were added (Sadler). This fmal mixture was incubated for 30 min at 0°C. The solution was then centrifuged at 12,000 g and the sediment was resuspended in 2 ml. of deionized water. The treated cells were then lysed either by the addition of 0.2 ml. of 10% deoxycholate or by the addition of 100 vol. of solution B. These lysed samples were centrifuged, decanted, and the supernatants were stored. All cell extracts were stored at PC! and were freshly prepared every 3 days. Ribosomal subunits were obtained by dialyzing the above isolated material against 3 changes of buffered Mgs + (lo-* M) solutions. Reassociation of subunits was accomplished by another overnight dialysis against 3 changes of buffered lOba r,r-Mga+ solutions. All dialysis was carried out at 4’C. For isolation of subunits for RNA and protein analysis the following procedure was employed. An exponentially growing anaerobic cell suspension was harvested and washed

RIBOSOMES

OF R. BPHEROIDES

66

3 times with O-01 M-Tris (pH 7.6), O-0001 BGM~CI,. The cells were frozen and ground with alumina at 0°C in a chilled mortar. The paste was extracted with the same buffer and the debris and unbroken cells removed by centrifugation at 10,000 g for 16 min. The supernate (20 to 60 ml.) was immediately introduced onto a 10 to 20% sucrose gradient containing the same buffer. other salts if indicated) Ribosomes isolated in lOma i+r-Mge+ (buffered and containing will be referred to as native ribosomes, since 811 evidence indicates that the overwhelming majority of ribosomes isolated and stored in these eolutions do not dissociate into subunits. (d) Sedimentation a&y& The technique of sucrose gradient analysis utilized in these studies was similar to that reported by Britten $ Roberts (1960). Two different sucrose gradients were used (16 to 30% and 6 to 20%) in solutions of varying buffered salt concentrations. Centrifugation was carried out at 37,000 rev./n-&r using a Spinco SW39 rotor at 6°C for varying time intervals. Samples were collected, appropriately diluted and optical densities at 200 rnp were read using a Beckman DU spectrophotometer. Samples in which radioactivity was to be measured were precipitated with cold 7% trichloroacetic acid, poured over membrane filters, washed with cold 7% trichloroacetic acid and fixed to planchets. The samples were then counted in a Nuclear Chicago gas-flow geiger counter. The Beckman model L-ZU ultracentrifuge with a B-IV rotor was used to prepare homogeneous samples of dissociated ribosomal subunits by a modification of the method of Anderson, Barringer, Babelay t Fisher (1904). Subunit suspensions in volumes ranging from 20 to 60 ml. were centrifuged on a 10 to 20% sucrose gradient containing 0.01 M-Tris (pH 7.6), O*OOOl M-MgCl,. The gradient was linear with respect to rotor volume and sedimentation was carried out at 40,000 rev&in for 3.6 hr. (e) Ribonuckase treatment Stock solutions of pancreatic RNase containing 2 mg of RNase/ml. of deionized water were stored at 4°C with no apparent loss of activity. Riboaome suspensions were standardized to contain 60 to 70 o.D.~~~ units/ml. The incubation mixture contained approximately 20 O.D. units of ribosome suspension and 40 pg of pancreatic RNase in a total volume of O-3 ml. This was incubated for 20 min at 23°C. (f) Protein and RNA oMern&atione Suspensions of ribosomal subunits were precipitated by adding l/9 volume 70% trichloroacetic acid. The precipitate was washed 4 times with cold 7% trichloroacetic acid. The washed sediment was resuspended in 0.3 M-KOH and incubated at 37°C overnight. Samples of all solutions were analyzed for RNA and protein. RNA content was determined by the orcinol procedure (Mejbaum, 1939). Protein content was determined by the Lowry method (Lowry, Rosebrough, Farr & Randall, 1961). (g) Ribosowzal binding of 14C-kzbeled polywidylic acid Ribosomes were isolated as subunits from alumina-ground cells in solution B. The ribosomal subunits were prepared for binding by the addition of solution C to a magnesium concentration of 2.6 x 10v3 M, a level found to be optimum for binding of polyuridylic acid. Simultaneous to the adjustment of magnesium concentration, [l*C]polyuridylic acid was added (0.06 PO/ml.) and the mixture was incubated at 0°C for 30 min. After this time, the mixture was divided into 2 parts, one of which was treated with T1 ribonuclease as previously described. The fractions collected after centrifugation were diluted with cold buffer and optical density at 260 rnp determined. They were then adjusted to 7% trichloroacetic acid by the addition of 70% trichloroacetic acid. The precipitates were collected and washed on membrane filters and radioactivity measured in a windowless gas-flow Geiger counter. (h) Chemicals Pancreatic RNase and DNase I were obtained from Worthington Biochemical Company. T1 RNase was obtained from Calbiochem. [14C]Uracil was obtained from New Englsnd Nuclear Company. l*C-labeled poly U was obtained from the Miles Chemical Company.

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3. Results The sedimentation characteristics of ribosomes and ribosomal subunits isolated from anaerobically grown R. spheroides were de6ned by ultraoentrifugal analysis. When the ribosomes were isolated from sonically disrupted cells in lOWaM-Mg2+ (solution A), ultracentrifugal analysis revealed one large peak. The calculated sedimentation coefficient corrected to infinite dilution of this component was 66s. Ribosomal subunits again isolated from anaerobically grown organisms and dialyzed against 10m4 Ed-Mg’+ (solution B) were studied using the same techniques. Two major components were observed and their calculated sedimentation coefficients corrected to infinite dilution were 29s and 45s. Sedimentation coefficients determined at magnesium concentrations (2.5 x10m3 M and 1 x 10m3 M) in which subunits and undissociated ribosomes were present, remained essentially unchanged. The 66s particle evidently corresponds to the 70s ribosome of E. coli and the two subunits, 29s and 45s, to the 30s and 50s subunits. Similar values for the ribosomes of R. palustris and Rhodospirillum rubrum (66s) have been reported by Taylor & Starck (1964). It is of interest that while the 45s subunit differs considerably in its sedimentation behavior from the “normal” 50s particle, the 29s subunit is very close to the value of 30s. (a) RNase activity on ribosomul subunits When ribosomes isolated from R. spheroides grown anaerobically were treated with pancreatic RN&se (an enzyme specific for the site distal to the 3’ phosphate adjacent to pyrimidines), sedimentation analysis demonstrated the 29 s component to be extremely labile to RNase action. Figure l(a) shows a sucrose gradient sedimentation analysis of R. spheroides subunits. The faster sedimenting peak is mainly composed of 45s subunits with some undissociated 66s ribosomes also present. The same sample treated with pancreatic RN&se is shown in Fig. l(b). It may be observed that the RNase treatment has eliminated the 29s component and the 66s material, leaving an apparently intact 45s peak. If the treated samples are studied in the analytical ultracentrifuge, the same change after RNase incubation is observed. The 29s subunit from aerobically cultured R. spheroides shows the same sensitivity to RN&se, while the 45s subunit is similarly resistant. In a similar experiment, E. coli ribosomal subunits were treated with pancreatic RNase. A sucrose gradient sedimentation profile of these treated subunits is shown in Fig. 2. In this case both the 50s and 30s components are identifiable after RNase incubation. It should be noted that the amounts of material in both peaks in the case of E. coli and the 45 s component in the case of R. spheroides are significantly reduced following RNase degradation. Two lower concentrations of pancreatic RNase tested, 13 pg/ml. and l-3 pg/ml., were also active in eliminating the 29s component when incubated under the same conditions. T, ribonuclease, an enzyme whose degradative activity is specific for a different site (distal to the 3’ phosphate of guanosine), was also active in eliminating the 29s R. spheroides ribosomal subunit. In order to eliminate the possibility that the disruptive action of sonication altered the ribosomal subunit structure, making the 29s unit labile to RNase, ribosomal subunits were isolated by osmotic lysis of R. slpheroides spheroplasts. The suspension

RIBOSOMES

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61

of ribosomal subunits obtained was treated with pancreatic RNase as before and analyzed by sucrose gradient sedimentation. The 29s subunit was once again totally degmded by RN&se action while the 45s subunit again remained relatively stable. It is concluded that the observed sensitivity to RN&se is not due to subunit alteration during isolation. 66s45S

29s

0~150

Fraction

Fra. 1. Effect of RNeee treatment on R. spheroidee ribosomal subunits. Ribosomes were extracted from sonically disrupted anaerobically grown cells using 0.01 ad-Tris-HCl (pH 76), O*OOOl M-MgCl,. Ribosomal su&ensions were sedimented at 37,006 rev./min for 100 I& in a gradient of 6 to 20% sucrose 0.01 M-Tris-HCl (pH 76), 0,005 M-M~C~,. (a) Control; (b) riboaomal suspension incubated 20 min at 23°C with 130 pg/ml. pancreatic RN&se.

(b) RNme

activity

on 66s ribosm

The observed differential RN&se sensitivity of R. spheroides ribosomal subunits prompted investigation of the RNase effect on the 66s ribosomes of this organism. Suspensions of 66s ribosomes, from sonically disrupted anaerobically grown cells, were obtained either by isolating ribosomes directly in lOWa M-Mga+ (native ribosomes) or by isolation of subunits in lo-* M-Mga+ and subsequent resssociation by dialysis against lo- a rd-Mg2 + (reassociated ribosomes). When native 66s ribosomes isolated in solution C were treated with pancreatic RN&se there was an observable change in the sedimentation pattern. As shown in Fig. 3, there is a reduction in the 66 s peak and a concomitant elevation of the 45 s component. Similar RNase treatment of reassociated 66s ribosomes @rat isolated in solution B and then re~.sociated by dialysis against solution C) yielded a significantly different sedimentation pattern. In Fig. 4 are shown the reassociated ribosomes with and without RNase digestion. Clearly, the RNase treatment reduced the 66s component and again there

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Fraction

FIG. 2. Effect of RNase treatment on E. coli ribosomel subunits. Ribosomes were extracted from sonically disrupted E. c&i in 0.01 M-Trk-HCl (pH 7*6), O*OOOlm-MgCl,. Sedimentation in 15 to 30% sucrose gradient containing 0.01 X-Tris-HCl, 0.005 nr-MgC1, at 37,000 rev./min for 150 min. (a) Control; (b) ribosomal suspension incubated 20 min at 23°C with 130 pg/ml. pancreatic RN-.

of the 45s component. If Figs 3 and 4 are compared, it is evident that after RNase incubation a significantly greater proportion of the native 66s ribosomes remain unaffected as compared to similarly treated reassociated ribosomes. Since it has been reported (T’so, Bonner & Vinograd, 1958) that cations other than Mg2+ play a role in ribosome association and binding, similar RNase studies were conducted using O-06 M-KC1 in the solutions. Native ribosomes were isolated by sonication in solution A and then treatedwith pancreatic RN&se. Sedimentation analysis (Fig. 5(a) and (b)) showed that in this case the 66s component remained relatively intact. However, there was a significant increase in materirtl at the top of the gradient.

is an elevation

RIBOSOMES

2 0 .z “a 0

OF

R.

SPHEROIDES

- ---

0. IS0 ;

o*ioo t

0,050 c

FIO.

4

Fraction

Fro. 3, Effeot of RNase treatment on undissociated R. s&%videe riboeomes. Ribosomea were isolated from sonically dhupted anaerobically grown R. epheroidas using 0.01 X-Tris-HCl (pH 74), 0.01 M-MgCl,. Sediment&ion in 15 to 30% sucrose gradient containing 0.005 M-Tris-HCl (pH 7*6), 0.01 u-MgCl,, 0.06 ad-KC1 at 37,000 rev./min for 160 min. (a) Control; (b) riboaomal suspension incubated 20 min at 23°C with pancreatic RN-6 (130 pg/ml.). FIG. 4. prepared for 12 hr. 23% with

Effect of RNase treatment on reassociated as in Fig. 1. The suspension was dialyzed Sedimentation as in Fig. 3. (a) Control; pancreatic RNaee (130 pg/ml.).

R. spheroides ribosomes. Ribosomal extract against 0.01 M-Tris-HCl (pH 76), 0.01 M-MgCls (b) ribosomal suspension incubated 20 min at

When ribosomal subunits isolated by sonication in solution B were reassociated by dialysis against solution A, the reassociated ribosomes had observable RNase sensit.ivity. It is clear from Fig. 6(a) and (b) that RNase has a degrading effect on these ribosomes although there is still material present in the 66 s region. (c) Effect of KC1 in the gradient on sedimentation of ribosonm An interesting corollary of these studies was the effect of KC1 during centrifugation of ribosomes. When ribosome suspensions reassociated in solution C were digested with pancreatic RNase, sedimentation analysis demonstrated an elimination of the 66 s component and an elevation of the 46 s component (Pig. 4). However, this ohange was only observed if the sucrose gradients contained KCl at a concentration near

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tractloll FIG. 5. Effect of RNase on undieaociated R. a@e&des ribosomes isolated in the presence of KCl. Ribosomal extract waa prepared from sonicelly disrupted anaerobically grown R. spheroide-s using 0.01 Y-‘&is-HCl (pH 74), 0.01 M-MgC& O-06 X-KCI. Sedimentation as in Fig. 3. (a) Control; (b) ribosomal suspension incubated 20 min at 23°C with pancreatic RNaae (130 pg/ml.).

0.06 M. If KC1 was omitted a broad peak sedimenting at the 60s to 80s region was observed (Fig. 7(a)). The dependence of this phenomenon on KCl concentration was evidenced by the failure of O*dOS~-Kc1 and 0.0006 M-KC1 to eliminate the disperse peak. Ribosomes reassociated in the presence of 0.06 ~-Kc1 (dialyzed against solution A), treated with RNase and analyzed in gradients lacking KCl contained this same 50s to 80s component. In order to determine whether this broad peak was due to complexing of ribosomal units after RNase treatment, dissociated ribosomes were incubated with pancreatic RNase. Under these conditions virtually all of the 29s subunits are known to be degraded and only the 45s subunit remains structurally intact. The ribosomal suspension was then adjusted to 10ea Y in Mga+ and subsequently centrifuged in a sucrose gradient without KCl. The hetero-disperse band was again present (Fig. 7(b)). This suggested that the RNase indeed degraded the 29s subunits, but in the absence of KCl in the gradient, complexes of 45s subunits formed. It appears that KC1 inhibits aggregation of the 46s subunit under these conditions.

RIBOSOMES

OF

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61

Fraction

FIG. 6. Effect of RNaee on R. sphe~oides ribosomes reassociated in the presence of KCl. Ribosomal extract wea prepared from sonically disrupted anaerobically grown R. sphemida using 0.01 Ilr-Tris-HCI (pH 74), O-0001 M-MgCl,. The extract was dialyzed against O-01 ra-Tris-HCl (pH 7.6). 0.01 na-MgCl,, 0.06 ~-Kc1 for 12 hr. Sedimentation ae in Fig. 3. (a) Control; (b) ribosomal suspension incubated 20 min at 23°C with pancreatic RNaae (130 pg/ml.).

(d) RNA and protein content of R. spheroides subunits Tissi&es, Watson, Sohlessinger & Hollingsworth (1959) reported the composition of E. coli 70s ribosomes and 50s and 30s subunits to be the same; 63% RNA and 37% protein. In light of the differential effect of RNase on the structural integrity of R. s~?~~oidas ribosomal subunits, the RNA and protein composition of these subunits was determined. Ribosomal subunits were prepared and analyzed as described. The results in Table 1 demonstrate that the 45 s subunit contains 62% RNA and 38% protein while the 29s subunit contains 51% RNA and 49% protein. These latter values are significantly different from those found for the organism’s 45s particle and those reported for the subunits of E. coli ribosomes. (e) Ribosomul bound Polyuridylic acid It has been shown that synthetic polynucleotides are bound to the 30s subunit of E. wli ribosomes (Takanami & Okamoto, 1963). In an attempt further to characterize the subunit as well as the site of messenger binding in R. spherook, the

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e 2 %

: 0”

0.200-

Fraction

FIQ. 7. Effect of absence of KC1 in sucrose gradient on sedimentation of RNase treated R. spheroidea riboeomes. (a) Ribosomal extract was prepared and dialyzed aa in Fig .4. The extract was incubeted 20 min at 23°C with pancreatic RNaee (130 pg/ml.). Sedimentation in 5 to 20% sucrose 0.005 X-Trie-HCl pH 7.6, 0.01 M-MgCl,, at 37,000 rev./min for 120 min. (b) Ribosomal extract wae prepared as in Fig. 1 and treated with RNase aa in (a). The extract was made 0.01 x in MgClp and sedimented in 16 to 30% sucrose 0.006 M-Tkie-HCl pH 7.6, 0.01 x-MgCl,, at 37,000 rev./min for 150 min.

stabilizing effect of poly U on ribosome structure was studied (Allen & Zamecmk, 1963). Because T, RN&se will effectively degrade 29s subunits and is not active on poly U, this enzyme was uniquely suited for studying the stabilizing ability of the synthetic messenger. At the magnesium concentration found optimum for poly U binding (2.5 x 10 - 3M), association of the subunits is incomplete. Figure 8(a) shows a broad optical density peak which comprises the 66 s, 46 s end 29 s psrticles. The larger fractions which were collected in this experiment obscured the usual separation into three discrete peaks. The poly U may be observed to sediment in association with eech of the ribosomal particles. A further peak of unbound poly U is present in the light sedimenting region. Analysis of the specifiu activity reveals a large heavy sedimenting peak. This material probably represents aggregates of 66 s monomers with poly U.

RIBOSOMES

OF

03

22. SPHEROIDES

TABLE 1

RNA and protein composition of ribosomal subunits Ribosomal

RNA (%I

Protein (%)

RNA/protein

(8)

particle

29 45

51 (f2) f32(321)

49 (f2) 38 (fl)

0.96 1.63

Ribosomes were isolated from anaerobically grown cells in 0.01 Ma-Trie-0.0001 M-MgCl,. The 29 B and 45 s subunits were isolated by eucroee gradient sedimentation in a zonal ultraoentrifuge ee described. Portions of the separated subunits were precipitated and washed in cold 7% triobloroaoetic acid, dieeolved in 0.3 N-KOH for RNA and protein determination. The results are exp-d a~ percentages of total material and are averages of 3 separate isolations and determinations. Standard deviations are given in parentheses.

0400

0.300

0.200 3 ,’% 0.100 t3 .ex 2

0 I

0

.!A 0.400 ‘i. 0t

66S45s 2;s

ct) .*

i

i

I

Fraction

FIG. 8. T1 RNase treatment of ribosomal suepensions pre-incubated with polyuridylic aoid. Ribosomal extra& was isolated from alumina-ground, anaerobically grown R. spheroides in 0.01 M-Trie-HCl (pH 7.6), O+OOl ad-MgCl,. The extra& wae adjusted to 2.5 x 10” M-MgCl, and 0.05 &ml. W-labeled polyuridylic aoid added. The mixture was incubated at 4°C for 30 min. A portion of the mixture wasp then adjusted to 600 units/ml. in T1 RNase and inoubated at 23°C for 30 min. The remainder of the mixture was incubated in the same manner without the addition of nucleaae. (a) Control; (b) T1 RNaae-treated. -X-XSpeoiflc aotivity (cts/min/o.n.aeo); optical density at 260 mp; -O-O-, radioactivity (cts/min). --O-O--t

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Figure 8(b) represents a sedimentation analysis of the poly U-treated ribosomal suspension analyzed in Fig. S(a) which was further incubated with T, RNase. The optical density profile reveals that a considerable amount of material which sedimented at a higher value than 66s has been eliminated by the T, treatment. Poly U is still associated with the 66 and 45 s ribosomes but a relatively greater amount is present with the 29s subunit. The specific activity of the 29s region is greatly elevated over that in Fig. 8(a). It would appear that binding of poly U has protected the structural integrity of the 29s particle against the action of T, RNase.

4. Discussion The 29s ribosomal subunit of R. spheroides differs from the 45s subunits in two important ways; it has a smaller RNA to protein ratio, and its structure is labile to RNase. The latter observation indicates that structurally important parts of the RNA of the 29s subunit are exposed to nuclease action. It has also been shown that some of the RNA of the 45s subunit is degraded by RNase, but evidently not enough to destroy the structural integrity of this particle. The studies involving 66s ribosomes indicate that the structural integrity of 29s subunits can, in certain cases, be protected from RNase degradation by association with 45 s particles. In other caseswe have shown that this association is not protective. A possible explanation of this apparent ambiguity could be that the protective effect is mediated through a firmer binding between subunits. Even when 66s ribosomes maintain their sedimentation characteristics after RN&se incubation, there is an increase in the amount of material at the top of the gradient. This material is presumably due to degradation of the 66s ribosomes and more specifically to degradation of the 29s moiety. The crucial question is whether associated 29s particles are totally destroyed by RNase or whether a specific association with the 45 s subunit can (under certain conditions) maintain enough of the 29s structure so that the altered ribosome will sediment at 66s. Studies on ribosomes isolated from E. coli indicate that the ribosomal RNA can be altered without significantly changing the sedimentation pattern, Santer (1963) has studied the effect of pancreatic RNase on the ribosomes isolated from E. COG.Sedimentation analysis revealed.that following RNase treatment there is a significant increase in material at the top of the gradient. The 70s ribosomes as well as the 50s and 30s subunits maintained their sedimentation chara&ristics although there was a reduction in amounts of these components. Further, it was found that the RNA isolated from these treated particles was altered and did not show the usual sedimentation patterns. The author concluded that ribosomal particles still maintain their sedimentation characteristics even though the RNA was altered by RNase. Jacob (1961) reported the experiments of Darnell in which ribosomes heavily labeled with 32P and sustaining multiple breaks in the ribosomal RNA not only maintained their sedimentation characteristics, but were biologically active. When the RNA isolated from these ribosomes was analyzed by sedimentation it was found to be unstable and not of normal size. This evidence firmly indicates that ribosomes can maintain their sedimentation characteristics even though there is partial degradation of ribosomal RNA. In the case of R. spheroidt~, observation of the sedimentation patterns of native and reassociated ribosomes following RNase digestion (Figs 5(b) and 6(b)) indicate a similar phenomenon. In both instances, following RNase treatment, relatively the same large amount of

RIBOSOMES

OF R. SPHEROIDES

65

material appears at the top of the gradient. Degradation of the reassociated ribosomes has resulted in a decrease of the 66s component and, as previously noted, a large increase in the 46s subunit. Apparently the material at the top of the gradient comprises degraded 29 s subunits which have been dislodged from 66s ribosomes yielding small light sedimenting fragments and 45~ ribosomal subunits. Native ribosomes, on the other hand, show a relatively small increase in the 45s component following enzymic degrrtdation. This disparity between the amount of m&erial in the respective 45s components and the similarity of the peaks at the top of the gradient suggest that although some degradation of native ribosomes occurred, sedimentation behavior was relatively unaffected. The firmer binding which leads to an apparent RN&se-resistant ribosome is favored by the presence of KC1 end the isolation of ribosomes without preliminary dissociation into subunits (native ribosomes). The strongest inter-subunit binding is observed when native ribosomes are isolated in the presence of KCl. When native ribosomes are isolated without KC1 or subunits are reassociated in the presence of KCl, there would appear to be a heterogeneous mixture of binding states. The weakest intraribosomal binding is observed when reassociation takes place in the absence of KCI. Teshiro & Siekevitz (1966) reported that in the presence of KC1 small subunits of ribosomes isolated from guinea-pig liver form dimers, an observation consistent with our findings. Although intra-ribosomal binding is stronger in the presence of KC1 it appears that KC1 is necessary in the gradient solvent in order to block the aggregation of RNase-treated 45s particles during centrifugation. This evidence indicates that KC1 may also interfere with non-specific ribosome aggregation. The difference in RNA and protein content of the two ribosomal subunits of R. S$.OYY&S suggests possible explanations for the difference in RNase sensitivity, The 29s particle may have a larger percentage of RNA susceptible to RN&se attack, and further, this RNA may be critical in maintenance of the subunit structural integrity. Although RNase can degrade 4Eis particles, the bulk of these subunits maintain their sedimentation characteristics after nuclease attack. In this c&se it would appear that some RNA may be degraded without altering subunit structure significantly. The experiments utilizing poly U and T, RNase indicate that the RNA which is essential for maintaining 29s structural integrity is either closely associated with or possibly part of the messenger binding site. We have observed, following T, RN&se digestion of ribosomal subunits previously incubated with poly U, that while there is a reduction of the poly U sedimenting with other components, there is no decrease in the poly U sedimenting at 29s. Analysis of the specific activities amplifies this observation. A large increase in specific activity is present at 29 s following T, RN&se treatment. Evidently the binding of poly U allows these 29s particles to maintain their sedimentation characteristics. Those 29s subunits not binding poly U, representing the bulk of this material, were degraded by the enzyme. The specifio activity also revealed a large peak sedimenting in the region expected for ribosomal eggregates. Following T, RN&se digestion, this peak remained, though broadened, suggesting that 66s ribosomes which bind poly U to form aggregates have increased structural stability in the presence of RN&se. Apparently poly U binding to 29s subunits is similar whether the subunit is in an associated or non-associated state. In both cases the synthetic messenger maintains the structure of the 29s particle in the presence of T, RNase. The action of poly U may be effeoted through protection of b

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E. D. GRAY

structurally important RNA at the messenger binding site. An alternative possibility is that poly U does not protect the subunit from enzymic attack but, in some fashion, maintains the integrity of the altered particle. We thank Mm M. L. Anderson for excellent technical assistance. The work was supported by grants from the National Institutes of Health and the National Science Foundation. One of us (D. I. F.) is a U.S. Public Health Service Post Doctoral Fellow; another (E. D. G.) is supported by a U.S. Public Health Service Cardio-vascular Program Project grant (HE-06314). REFERENCES Allen, D. W. & Zamecnik, P. C. (1963). Biochem. Biophys. Res. Comm. 11, 294. Anderson, N. G., Barringer, H. P., Babelay, E. F. & Fisher, W. D. (1964). LifeSciencea, 3, 667. Britten, R. J. & Roberts, R. B. (1960). Science, 131, 32. Cohen-Bazire, G., Sistrom, W. R. & Starrier, R. Y. (1957). J. Cell. Camp. Physiol. 49, 251. Gilbert, W. (1963). Cold Spr. Harb. Syrnp. Quant. Biol. 28, 289. H-B, E. L. & Horn, R. (1964). J. Mol. BioZ. 10, 541. Jacob, F. (1901). Cold Spr. Harb. Symp. Quant. Biol. 26, 109. Lamfrom, H. & Glowacki, E. R. (1962). J. Mol. BioZ. 5, 97. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265. Mejbaum, W. J. (1939). 2. physiol. Chem. 258, 117. Santer, M. (1963). Science, 141, 1049. Takanami, M. & Okamoto, T. (1963). J. Mol. BioZ. 7, 323. Tashiro, Y. & Siekevitz, P. (1965). J. Mol. BioZ. 11, 149. Taylor, M. M. & Starck, R. (1964). Proc. Nat. Acud. Sci., Waeh. 52, 968. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingsworth, B. R. (1969). J. Mol. BioZ. I, 221. T’so, P. 0. P., Bonner, J. I% Vinograd, J. (1958). Biochim. biophye. Acta, 30, 570. T’ao, P. 0. P. & Vinograd, J. (1961). Biochim. biophye. Acta, 49, 113.