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Formation in vitro of a 5.8-S-26-S sea urchin rRNA complex
BIOCHIMICA ET BIOPHYSICA ACTA
517
BBA 96706
FORMATION I N VITRO OF A 5.8-S-26-S SEA URCHIN rRNA COMPLEX JOSE SY* AND KENNETH S. McCARTY** Department o/ Biochemistry, Duke University Medica 7, Center, Durham, N.C. 27706 (U.S.A.)
(Received July 2oth, 197o)
SUMMARY Sea urchin 26-S r R N A has been d e m o n s t r a t e d to contain a h y d r o g e n - b o n d e d 5.8-S r R N A t h a t is dissociated b y h e a t w i t h a T,~ of 5 o°, or c o m p l e t e l y dissociated a t 22 ° in t h e presence of 5 M u r e a w i t h a salt c o n c e n t r a t i o n of 0.0375 M NaCl-o.oo375 M t r i s o d i u m c i t r a t e (pH 7.3). The R N A is c o m p o s e d of 15o nucleotides, contains no m e t h y l groups, a n d has a high G + C composition t h a t resembles t h a t of t h e 18 S r R N A . Tile conditions h a v e been d e t e r m i n e d b o t h for t h e release as well as t h e reassociation of 5.8-S r R N A from its 26-S r R N A using u r e a a n d s a l t solutions as denaturants.
INTRODUCTION Sea urchin 26-S r R N A 1 has been d e m o n s t r a t e d to c o n t a i n a h y d r o g e n - b o n d e d 5.8-S r R N A ~, A similar t y p e of R N A was also observed to be associated w i t h a large r R N A of H e L a cellsa, chicken fibroblasts a, Novikoff ascites t u m o r s 4 a n d o t h e r eucar y o t i c cells 2,~,5. These R N A ' s are c o m p o s e d of 14o-15o nucleotides 2,8,n, c o n t a i n no m e t h y l group 2,~,5,~ a n d have a high G + C c o m p o s i t i o n ~,3,~. I n H e L a cells ~ t h e 45-S r i b o s o m a l precursor R N A has also been shown to be the precursor of 5.5-S r R N A as well as 18- a n d 28-S r R N A , a l t h o u g h at this stage t h e 5.5-S R N A is c o v a l e n t l y l i n k e d to t h e 45-S precursor R N A . PEN~ et al. 3 p r o p o s e d t h a t t h e cleavage a n d format i o n of t h e 5.5-S r R N A occurs d u r i n g t h e t r a n s f o r m a t i o n of t h e 32-S r i b o s o m a l precursor R N A into 28-S nucleolar r R N A a. SY AND MCCARTY2 h a v e shown t h a t t h e t e m p e r a t u r e for t h e release of 5.8-S r R N A is salt c o n c e n t r a t i o n d e p e n d e n t , as w o u l d be e x p e c t e d for a p a i r of h y d r o g e n b o n d e d p o l y n u c l e o t i d e chains. W e wish to r e p o r t here the conditions for b o t h the release, as well as t h e reassociation of 5.8-S r R N A from 26-S r R N A using u r e a a n d s a l t solutions as d e n a t u r a n t s .
MATERIALS AND METHODS Sea urchins (arbacia p u n c t u l a t a ) were o b t a i n e d from the D u k e Marine L a b o r a t o r y , Beaufort, N.C. * Part of this communication was submitted in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry, Duke University, June 12, 197o. Present address: Rockefeller University. *~ Send reprint requests to Dr. K. S. McCarty. Biochim. Biophys. Acta, 228 (1971) 517-525
518
j . sY, K. S. MCCARTY
Eggs and embryo The techniques of handling sea urchin eggs and embryos, as well as isotopic labeling of plutei embryos have been described ~. Plutei embryos were labeled with 5 #C/ml of carrier free 3~P1 (New England Nuclear) for 16 h and were followed by a chase of 4 h with o.oi M Na2HP Q at p H 7. Isolation o/total r R N A and 5.8-S r R N A rRNA's were extracted from whole unfertilized eggs or plutei embryos by room temperature phenol extraction has been described 2. Phenol extraction at 55 ° was used to provide 26-S rRNA stripped of its 5.8-S rRNA. All 3*P-labeled RNA's were treated with cetyltrimethylammonium bromide as described by RALPH AND BELLAMY7. The 5.8-S rRNA's were obtained by polyacrylamide gel electrophoresis as described previously 2. Dissociation o/5.8-S r R N A The dissociation of 5.8-S rRNA from 26-S rRNA was affected either b y heat or urea, For heat dissociation, RNA samples were dissolved in the desired buffer and placed in a water bath for IO rain at the required temperature. The samples were then rapidly chilled in ice and immediately electrophoresed in either 2.4, 7.5 or io % gels. For urea dissociation, calculated volumes of either 1.8 M NaCl-o.i8 M trisodium citrate (pH 7.3), or 0. 9 M NaCl-o.o 9 IV[ trisodium citrate (pH 7.3) and io M urea were added to RNA samples dissolved in o.I M NaC1, o.oi M sodium acetate (pH 5.1) to produce the desired salt and urea concentration. These solutions were then incubated at the desired temperature for a minimum of I h layered on acrylamide gels and electrophoresed. The dissociated 5.8-S rRNA's were measured either by 260 m/, absorption scanning or for ~2P-labeling RNA b y scintillation counting of I.O m m sliced gels as described previously ~. Reassociation o/5.8-S r R N A with 26-S r R N A 32P-labeled 5.8-S rRNA and 26-S rRNA's were mixed with the proper amount of either 1.8 M NaC1--o.I8 M trisodium citrate or 0. 9 M NaCl-o.o 9 M trisodium citrate and io M urea to produce the desired salt and urea concentration and incubated at 24 °. At selected incubation times, samples were electrophoresed on IO % gels containing a 2,4 % spacer gel. The radioactivity of the eluted RNA's were monitored with a Packard scintillation spectrophotometer. Electrophoresis and sucrose density gradients Acrylamide gel electrophoresis was performed as described by LOENINGs and BISHOP et al. 9 Isokinetic sucrose density gradients were performed as described b y MCCARTY et al. 1° in a SW 25.3 rotor at 5 °. Scintillation counting Gel slices were dried on filter paper discs. The ~2p radioactivity was then measured in a toluene scintillation solution with a packard Tri-Carb scintillation spectrometer as described previously.
Biochim. Biophys. Acta, 228 (1971) 517-525
PROPERTIES OF 5.8-S-26-S rRNA
519
RESULTS
Fig. I shows the specificity of the release of 5.8-S rRNA from 26-S rRNA in the plesence of 8 M urea and 0.022 M Na + concentration for IO rain at room temperature. The RNA's were immediately electrophoresed in 2.4 % gels. Analogous with previous studies using heat treatment s only 26-S rRNA released the 5.8-S RNA, Figs. I a and lb. In contrast, the I8-S rRNA did not yield any such RNA fragment under these conditions, Fig. IC.
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I. R e l e a s e of 5 . 8 - S R N A f r o m 2 6 - S 1RNA b y u r e a . R N A s a m p l e s w e r e m i x e d w i t h IO M t o m a k e a f i n a l c o n c e n t r a t i o n of 8 M u r e a a n d o . o 2 2 M i n N a + c o n c e n t r a t i o n . I n c u b a t i o n a t r o o m t e m p e r a t u r e f o r i o r a i n . T h e i n c u b a t e d s a m p l e s w e r e e l e c t r o p h o r e s e d i n 2. 4 % g e l s w e r e s c a n n e d a t 2 6 0 m/~. a. C o n t r o l 2 6 - S R N A w i t h o u t u r e a t r e a t m e n t , b . 2 6 - S l Z N A t r e a t e d u r e a . c. I 8 - S R N A t r e a t e d w i t h u r e a .
The dependency on urea concentration for the release of 5.8-S rRNA is shown in Fig. 2. The sodium ion concentration was maintained at 0.0375 M NaCl-o.oo375 M trisodium citrate and the temperature of incubation at 22 ° and the urea concentration varies from 1-5 M (Figs. 2a-2d). It should be noted that 5.8-S rRNA was released from the 26-S rRNA at a urea concentration of 5 M (Fig. 2d), although some hint of this dissociation could be detected at 4 M urea (Fig. 2c). In contrast, the 5.8-S rRNA was not released by 5 M urea at 22 ° when the salt concentration was increased to o.o75 M NaCl-o.oo75 M trisodium citrate (Fig. 2e). Thus, a higher concentration of urea is required for dissociation to occur at this salt concentration. It may be concluded, therefore, that a delicate balance of both urea and salt concentration governs the denaturation of this RNA complex. The temperature of incubation is also a critical factor in the dissiociation process. Although 5 M urea in the presence of 0.0375 M NaCl-o.oo375 M trisodium citrate at room temperature was sufficient to dissociate the 5.8-S rRNA from 26-S rRNA (Fig. 2d), the dissociation phenomena did not occur when the incubation was conducted at 4 ° as shown in Fig. 2f. The conditions necessary for reassociation were determined by electrophoresis in IO % acrylamide gels, scanned at 260 m/z. These experiments demonstrated that the dissociated 5.8-S rRNA and 26-S rRNA in 8 M urea which was evident at a salt B i o c h i m . B i o p h y s . A c t a , 2 2 8 (1971) 5 1 7 - 5 2 5
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Fig. 2. Conditions for the release of 5.8-S R N A by urea. 2a, b, c, d. Unfertilized egg R N A ' s (22/~g each) were treated overnight with various concentrations of urea (a, I M: b, 2 M: c, 4 M; d, 5 M) at 22 ° while maintaining salt concentration at 0.0375 M NaCI-o.oo375 M trisodium citrate, e. 26-S R N A (22/~g) similarly treated as in d except salt concentration was o.075 M NaCl-o.oo75 M tris(:dium citrate instead of 0.0375 M NaCl-o.oo375 M trisodium citrate, f. 26-S R N A (23 fig) similarly treated as in d except t e m p e r a t u r e of incubation was at 4 ° instead. Samples were e/ectrophoresed in 7-5 °/o gels and the gels were scanned at 260 m/~. Fig. 3. Complex formation of 5.8-S R N A and 26-S RNA. Stripped 26-S R N A (33 /zg), 14oo c o u n t s / m i n of s2P-labeled 5.8-S R N A were incubated in 7 M urea and 0.255 M NaCl-o.o255 M trisodium citrate at room t e m p e r a t u r e for 20 h. Sample was then electrophoresed in 2. 4 °/o gel. The gel was frozen and sliced for counting in Toluene scintillation fluid as described in MATERIALS AND METHODS. /~- - - & , control before incubation; O - O , after incubation.
concentration of 0.0375 M NaCl-o.oo375 M trisodium citrate at 22 ° was readily reassociated when the salt concentration was increased to 0.255 M NaCl-o.o255 M trisodium citrate. Since only the dissociated 5.8-S rRNA was demonstrable using the io °/o acrylamide gel electrophoresis, the specificity of this association was also demonstrated using 2. 4 % polyacrylamide gels as shown in Fig. 3The time course of z*P-labeled 5.8-S rRNA and stripped 26-S rRNA reassociation was measured in 5 M urea with various final salt concentrations, Fig. 4. Equilibrium was attained in about 25 h when the incubation was in the presence of 5 M urea and 0.075 M NaCl-o.oo75 trisodium citrate. Approx. 62 % of the input 5.8-S rRNA was bond to the stripped 26-S rRNA, whereas at higher salt concentrations as o.1125 M NaCl-o.olI25 M trisodium citrate and o15 M NaCl-o.oI5 M trisodium citrate, the equilibrium was more rapidly attained giving values of 15 and 4 h as shown in Figs. 4 b and 4c. Table I shows the urea requirement and the fact that the efficiency of formation of this complex was much less at 5 ° than at 24 ° as shown in Expts. 1- 7 and that unstripped 26-S rRNA hybridized to 5.8-S rRNA to a maximum extent of 1/3 that Biochim. Biophys..4cta, 228 (1971) 517-525
PROPERTIES OF 5 . 8 - S - 2 6 - S r R N A
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Fig. 4. Time courses of 5.8-S RNA and 26-S RNA reassociation in 5 M urea. a. Stripped 26-S RNA (33 #g) and 32P-labeled 5.8-S RNA (0.o 5 ktg) were incubated in 5 M urea and o.o75 Mi NaC1-o,oo75 M trisodium citrate at room temperature for various times as indicated before electrophoresis in io % gels. b, Stripped 26-S 1RNA (43/*g) and 8*P-labeled 5.8-S RNA (zooo counts/rain) were incubated in 5 M urea and o.ii25 M NaCl-o.oli25 M trisodium citrate at room temperature and were analyzed as in a. c. Stripped 26 S RNA (53/~g) and [8'P]5.8-S RNA (o.o6/zg) were incubated in 5 M urea and o.15 ~ NaCl-o.oi 5 M trisodium citrate at room temperature and were analyzed as in a. Radioactivities associated with 26-S RNA were then obtained from scintillation counting of gel slices. TABLE I REASSOCIATION OF
rRNA
COMPLEX
Reassociation was performed using purified 3iP-labeled 5.8-S rRNA and the substrates as indicated. The RNA's were incubated in 5 M urea, o.o75 NI NaCl-o,oo75 IV[ trisodium citrate for 18 h at the temperatures indicated. The samples were electrophoresed in io % gels containing a 2. 4 % spacer gel. Gels were frozen, sliced and counted as described in MATERIALS AND METHODS. Experiments R N A
I. 2. 3. 4. 5. 6, 7. 8. 8a.
26-S z6-S 26-S 26-S 26-S 26-S 26-S I8-S I8-S
rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA
Substrate
Native Stripped Native Stripped Native Stripped Stripped -(Molar ratio)
Conditions
Reassociation
Urea
Temps.
%
Counts/rain
--+ + + + + + +
24 24 5 5 24 24 24 24 24
o 4 8 14 24 62 69 33 17
(--/553) (I9/519) (6o/742) (9o/64o) (i68/7o4) (44o/714) (689/994) (323/956)
of t h e s t r i p p e d 26-S R N A a t 24 °, E x p t s . 5 - 7 . I t is r e a s o n a b l e t o p o s t u l a t e t h e n t h a t b i n d i n g m a y b e d u e e i t h e r t o a n e x c h a n g e of b o u n d 5.8-S r R N A for free 5.8-S r R N A or t h a t t h e c o m p l e x is r a n d o m a n d l a c k s s p e c i f i c i t y . T h e I 8 - S r R N A u n d e r i d e n t i c a l c o n d i t i o n s b o u n d o n l y I / 2 as m u c h 5.8-S r R N A as d i d t h e s t r i p p e d 26-S r R N A , which when calculated in equimolar concentrations revealed that the I8-S rRNA b o u n d o n l y i / 4 a s m u c h 5.8-S r R N A a s d i d t h e 26-S r R N A . S i n c e i t c o u l d b e s h o w n t h a t p r e p a r a t i o n s of s t r i p p e d 26-S R N A s t i l l r e t a i n e d a f r a c t i o n of t h e b o u n d 5.8-S r R N A , i t is r e a s o n a b l e t o a s s u m e t h a t t h e a m o u n t of [8~P]5.8-S r R N A t h a t a n n e a l s t o p a r t i a l l y s t r i p p e d 26-S r R N A is p r o b a b l y less t h a n o p t i m u m . N o r e a s s o c i a t i o n of r R N A o c c u r r e d i n t h e a b s e n c e of u r e a . Biochim. Biophys. Acta, 228 (1971) 517-525
522
j. sY, K. S. MCCARTY
F u r t h e r details of t h e n a t u r e of the association of the r R N A ' s is shown in Fig. 5. These e x p e r i m e n t s utilize c o m p e t i t i o n of u n l ab el ed r R N A ' s with 32P-labeled 5.8-S r R N A . U n l a b e l e d 5.8-S r R N A ' s were isolated from unfertilized eggs an d their a b i l i t y to c o m p e t e with szP-labeled 5.8-S r R N A from plutei stages were tested. No differences were f o u n d to exist b e t w e e n the R N A e x t r a c t e d from these two stages. I n addition, the Escherichia coli 4-S a n d 5-S r R N A m i x t u r e s did not c o m p e t e with sea urchin s ' P - l ab el e d 5.8-S r R N A u n d e r similar conditions, which d e m o n s t r a t e the dissimilarity of their sequence with that of 5.8-S r R N A from sea urchins.
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Fig. 5. Competitive hybridization of 5.8-S RNA's. Stripped 26-S RNA (io/,g) and 3~P-labeled 5.8-S RNA were mixed with various amounts of unlabeled 5.8-S R2qA (unfertilized eggs RNA) or E. coli 4-S and 5-S RNA's. Samples were incubated in 5 M urea, o.o75 M NaC1 o.0075 M trisodium citrate at room temperature for 20 h and were then electrophoresed in I o % gels containing a 2. 4 % acrylamide spacer gel. Gels were sliced and counted as described in MATERIALSAND METHODS.Radioactivity associated with 26-S 1RNA,in sample where no unlabeled RNA was added was used as ioo %. 0 - 0 , E. coli 4-S and 5-S RNA's; 0 - 0 , unlabeled 5.8-S RNA from unfertilized egg RNA, Fig. 6. Complex of s2P-labeled 5.8-S ]~NA and unlabeled 26-S RNA. a. Stripped 26-S RNA (24 absorbance units) and a2P-labeled 5.8-S RNA (2.7/,g) were incubated in 5 .V£ urea, o.15 M NaCl o.o15 M trisodium citrate at room temperature for I6 h. The incubated sample was diluted with o.15 M NaC]-o.oI 5 M trisodium citrate and run in a 5-28.7 % sucrose gradient in SW 25. 3 for 29 h at 2o ooo rev./min. Gradient fractions were then counted with Triton-toluene scintillation fluid, b. 26-8 1RNA fractions were pooled (arrow) and the RNA precipitated by addition of ethanol (I8-S RNA was used as carrier). The pooled 26-S rR2XTAwas then dissolved in o.~ IV[ NaC1, o.oi M sodium acetate pH 5.z buffer, and a portion of the sample was run in 2. 4 % gel. The gel was sliced and counted as described in MATERIALSAND METHODS.
To s t u d y the specificity of the reassociated complex, t h e r m a l d e n a t u r a t i o n profiles of the 3~P-labeled 5.8-S r R N A - u n l a b e l e d stripped 26-S r R N A c o m p l e x were d e t e r m i n e d . Th e c o m p l e x was isolated from a p r e p a r a t i v e sucrose d e n s i t y g r a d i e n t as shown in Fig. 6a (arrow). T h e control sample was electrophoresed in 2.4 % gels to show the absence of u n b o u n d 5.8-S r R N A c o n t a m i n a t i n g these preparations, Fig. 6b. The d e n a t u r a t i o n profiles of the n a t i v e a n d reassociated complexes were exam i n e d in Figs. 8a an d b. The t e m p e r a t u r e at which the s2P-labeled 5 .8 - S- u n l ab el ed 26-S r R N A complexes a n d th e ~2P-labeled n a t i v e 26-S r R N A released their 32P-labeled 5.8-S r R N A was m e a s u r e d in o.I M NaC1, a n d o . o i M sodium a c e t a t e p H 5.1, indicates t h a t b o t h these R N A samples gave similar m e l t i n g t e m p e r a t u r e s , 5o-52°. In view of the fact t h a t some of t h e c o m p l e m e n t a r y sequences for 5.8-S r R N A m a y also be r e d u n d a n t in the I8-S R N A , e x p e r i m e n t s were p r ef o r m ed with the co m p l ex p r e p a r e d from 32Pqabeled 5.8-S a n d I8-S r R N A . These complexes were isolated from Biochim. Biophys. Acta, 228 (1971) 517-525
PROPERTIES
OF 5.8-S-26-S rRNA
523
sucrose density gradients as shown in Fig. 7- The thermal denaturation profiles of the native and reassociated 26-S complex was determined as shown in Fig. 8c. Release of the unlabeled 5.8-S rRNA from carrier 26-S rRNA was determined in gels by scanning at 26o m# prior to slicing of the gels for counting of the 3~P-labeled 5.8-S rRNA released from the s2P-labeled 5.8-S-I8-S rRNA complex. It should be noted that the melting temperature profiles for this complex extended over a broader range than that of the 5.8-S-26-S rRNA complex formed in vitro Fig. 8c versus Fig. 8b or that of native 5.8-S-26-S rRNA complex Fig. 8a. These experiments suggest therefore that the hydrogen-bond of the I8-S-5.8-S rRNA complex is thermodynamically less stable than the native or in vitro reassociated 26-S-5.8-S rRNA complex. Q 200( Z
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Fig. 7. C o m p l e x of s'P-labeled 5.8-S R N A a n d I8-S R N A . I8-S R N A (I74/zg) a n d s ' P - l a b e l e d 5.8-S R N A (3.2 ]~g) were i n c u b a t e d o v e r n i g h t in 5 M urea, o.15 IV[ N a C l - o . o i 5 1V[ t r i s o d i u m cit r a t e a t r o o m t e m p e r a t u r e . T h e s a m p l e s were d i l u t e d w i t h o.15 M N a C l - o . o I 5 1V[ t r i s o d i u m cit r a t e a n d were f r a c t i o n a t e d b y sucrose d e n s i t y g r a d i e n t s in S W 25. 3 a t 20 ooo r e v . / m i n for 31 h. F r a c t i o n s f r o m sucrose g r a d i e n t were c o u n t e d in T r i t o n - t o l u e n e scintillation fluid. Arrows rep r e s e n t pooled f r a c t i o n s f r o m similar g r a d i e n t s for t h e r m a l d e n a t u r a t i o n profile s t u d y in Fig. 8c. Fig. 8. T h e r m M d e n a t u r a t i o n profile of s~P-labeled 5.8-S R N A - 2 6 - S R N A a n d 3~P-labeled 5.8-S R N A - I 8 - S R N A complex. S a m p l e s of s'P-labeled 5.8-S R N A - 2 6 - S R N A derived as described in Fig. 6 were dissolved in o.i !V[ NaC1, o.oi M s o d i u m acetate, p H 5.1. T h e labeled c o m p l e x w a s h e a t e d to v a r i o u s t e m p e r a t u r e s as i n d i c a t e d for 5 min. T h e s a m p l e s were r a p i d l y chilled a n d elect r o p h o r e s e d in io °/o gels c o n t a i n i n g a 2. 4 % spacer gel. Similarly, s a m p l e s of s2P-labeled n a t i v e 26-S R N A were h e a t e d a n d electrophoresed, a. 32P-labeled 5.8-S R N A . b. s2P-labeled 5.8-S R N A 26-S R N A complex. Gels were sliced a n d r a d i o a c t i v i t y in 5.8-S R N A region w a s t h e n m e a s u r e d . T o t a l c o u n t s / r a i n in 32P-labeled 5.8-S R N A - 2 6 - S R N A c o m p l e x was d e s i g n a t e d as ioo °/o. c. T h e r m a l d e n a t u r a t i o n profile of s ' P - l a b e l e d 5.8-S R N A - I S - S R N A complex. Pooled s a m p l e s (as s h o w n in Fig. 7) were c o n c e n t r a t e d b y e t h a n o l precipitation u s i n g u n l a b e l e d 26-S R N A as carrier. Samples, in o.i M NaC1, o.oi M s o d i u m acetate, p H 5,I, were h e a t e d to v a r i o u s t e m p e r a t u r e s as i n d i c a t e d a n d were chilled r a p i d l y before electrophoresis in io % gels. Gels were sliced a n d c o u n t e d as described in MATERIALS AND METHODS. R a d i o a c t i v i t y in 5.8-S R N A region was t h e n m e a s u r e d . T o t a l c o u n t s / r a i n of t h e i n p u t c o m p l e x was d e s i g n a t e d as IOO %.
DISCUSSION
The extension of transition in the thermal denaturation curve of rRNA representing a 44 ° range for rat liver 11 versus about 3 °0 range for E . col# ~ and has been postulated as due to: (I) relatively short helices, (2) a distribution of helix length, Biochim. Biophys. Acta, 228 (i97 I) 517-525
524
J. sY, K. S. MCCARTY
(3) variation in average helix stability due to variations in the proportion of stronger base pairs and (4) variations in the proportion of miss-matched pairs incorporated in the helical regions n. The limited sharp transition for the release of 5.8-S rRNA, Figs. 8a and 8b m a y be explained then as either a 5.8-S rRNA with a very specific hydrogen-bonding site with the 26-S rRNA or the fact that 5.8-S rRNA chain of 15o nucleotides permits less variation in this hydrogen-bonded complex. When thermal denaturation profiles from ~2P-labeled 5.8-S rRNA-stripped 26-S rRNA complex and 3zP-iabeled 5.8-S r R N A - I 8 - S rRNA complexes were compared with that of native complexes under identical conditions, the sharpness of the native complex denaturation curve was immediately evident. The native complex has a transition breadth of about IO° while that of the reassociated complex has a transition breadth of about 15 ° . In contrast the thermal denaturation profile of the artifactual reassociated 5.8-S-I8-S complex extends over a range of at least 25 °. Thus one m a y conclude that both the native and renatured 5.8-S-26-S rRNA complex produces a more specific hybrid than that of 5.8-S-I8-S complex formed in vitro. It is reasonable to postulate that the capacity of the I8-S rRNA to bind some 5.8-S rRNA, although at a much reduced efficiency m a y be due to short stretches of similarity in base sequence between I8-S and 26-S rRNA. This is not completely clear, however, since there are conflicting reports as to the capacities of I8-S and 28-S rRNA to compete with each other in D N A - R N A hybridization experiments la,14. If the ability of I8-S rRNA to bind a small proportion of 5.8-S rRNA is due to a lack of specificity, then even more stringent conditions than used here will be required for its detection. It is of course essential that the complete nucleotide sequence be known before these questions can be firmly resolved. We would like to suggest that the in vivo specificity of 5.8-S rRNA binding to 26-S rRNA is intimately related to the known processing of precursor rRNA's. As partial evidence for this, it should be noted that kinetic measurements have been made which demonstrate that a 45-S precursor RNA also gives rise to the 5.5-S rRNA2s 3. The formation of 5.5-S RNA28 in other eucaryotic cells appears to involve the step of transformation of 32-28-S rRNA by some mechanism of cleavage a. It seems reasonable that a cleavage at this stage would insure the binding of 5.5-S rRNA~8 with its 28-S rRNA monomer. The processing of large precursor molecules in eucaryotic cells is becoming increasingly evident as, for example, in the formation of insulin from proinsulin 15 and it seems reasonable to postulate that the 32-S structure could possibly prescribe the proper sites for a 5.8-S rRNA-26-S rRNA interaction prior to cleavage. We would like to suggest then that the 5.8-S rlRNA2e or the 5.5-S rRNA2s is involved in maintaining the correct 3-dimensional configuration of either the 28-S or 26-S rRNA necessary for its proper interaction with proteins in ribosome maturation. At the time of the preparation of this manuscript a report appeared by KING AND GOULD1~ which confirms some of the results reported here. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant No. 128o505, American Cancer Society P363 D, and Du Pont. Biochim. Biophys. Acla, 228 (197 I) 517-525
PROPERTIES OF 5.8-S-26-S rRNA
525
REFERENCES i 2 3 4 5 6 7 8 9 to II 12 13 14 15 16
J. SY AND K. S. IV]~CCARTY, Biochim. Biophys. Acta, 166 (1968) 571. J. SY AND K. S. McCARTY, Bioehim. Biophys. Acta, I99 (197 o) 86. J. J. PEN~, E. KNIGHT, JR. AND J. E. DARNELL, JR., J. Mol. Biol., 33 (1968) 609. A. W . PRESTAYKO, M. TONATO AND H. BUSCH, J. Mol. Biol., 47 (197 °) 505 • R. A. VqEINBERG AND S. PENMAN, J . Mol. Biol., 38 (1969) 289. W . F. ZAPISEK, A. G. SAPONARA AND M. D. ENGER, Biochemistry, 8 (1969) 117o. R. K. RALPH AND A. R. BELLAMY, Biochim. Biophys. Acta, 87 (1964) 9U. E. LOENING, Biochem. J., lO2 (1967) 251. D. H. L. BISHOP, J. R. CLAYBROOK AND S. SPIEGELMAN, J. Mol. Biol., 26 (1967) 273. K. S. MCCARTY, D. STAFFORD AND O. BROWN, Anal. Biochem., 24 (1968) 314. P. DUTY, H. BOEDTKER, J. R. FRESCO, R. HASELRORN AND M. LITT, Proc. Natl. Acad. Sci. U.S., 45 (1959) 482. A. RODGERS, Biochem. J., IOO (1966) lO2. S. B. WEISS, W. T. HsU, J. \V. FOFT, AND N. H. SCHERBERG, Proc. Natl. Acad. Sci. U.S., 61 (1968) 114. J. G. ZEIKUS, M. W . TAYLOR AND C. A. BUCK, Exptl. Cell Res., 57 (1969) 74. A. K. TUNG AND C. C. YIP, Proc. Natl. Acad. Sei. U.S., 63 (1969) 442. H. W . S. KING AND H. COULD, J. Mol. Biol., 51 1197 o) 687.
Biochim. Biophys. Acta, 228 (1971) 517-525
517
BBA 96706
FORMATION I N VITRO OF A 5.8-S-26-S SEA URCHIN rRNA COMPLEX JOSE SY* AND KENNETH S. McCARTY** Department o/ Biochemistry, Duke University Medica 7, Center, Durham, N.C. 27706 (U.S.A.)
(Received July 2oth, 197o)
SUMMARY Sea urchin 26-S r R N A has been d e m o n s t r a t e d to contain a h y d r o g e n - b o n d e d 5.8-S r R N A t h a t is dissociated b y h e a t w i t h a T,~ of 5 o°, or c o m p l e t e l y dissociated a t 22 ° in t h e presence of 5 M u r e a w i t h a salt c o n c e n t r a t i o n of 0.0375 M NaCl-o.oo375 M t r i s o d i u m c i t r a t e (pH 7.3). The R N A is c o m p o s e d of 15o nucleotides, contains no m e t h y l groups, a n d has a high G + C composition t h a t resembles t h a t of t h e 18 S r R N A . Tile conditions h a v e been d e t e r m i n e d b o t h for t h e release as well as t h e reassociation of 5.8-S r R N A from its 26-S r R N A using u r e a a n d s a l t solutions as denaturants.
INTRODUCTION Sea urchin 26-S r R N A 1 has been d e m o n s t r a t e d to c o n t a i n a h y d r o g e n - b o n d e d 5.8-S r R N A ~, A similar t y p e of R N A was also observed to be associated w i t h a large r R N A of H e L a cellsa, chicken fibroblasts a, Novikoff ascites t u m o r s 4 a n d o t h e r eucar y o t i c cells 2,~,5. These R N A ' s are c o m p o s e d of 14o-15o nucleotides 2,8,n, c o n t a i n no m e t h y l group 2,~,5,~ a n d have a high G + C c o m p o s i t i o n ~,3,~. I n H e L a cells ~ t h e 45-S r i b o s o m a l precursor R N A has also been shown to be the precursor of 5.5-S r R N A as well as 18- a n d 28-S r R N A , a l t h o u g h at this stage t h e 5.5-S R N A is c o v a l e n t l y l i n k e d to t h e 45-S precursor R N A . PEN~ et al. 3 p r o p o s e d t h a t t h e cleavage a n d format i o n of t h e 5.5-S r R N A occurs d u r i n g t h e t r a n s f o r m a t i o n of t h e 32-S r i b o s o m a l precursor R N A into 28-S nucleolar r R N A a. SY AND MCCARTY2 h a v e shown t h a t t h e t e m p e r a t u r e for t h e release of 5.8-S r R N A is salt c o n c e n t r a t i o n d e p e n d e n t , as w o u l d be e x p e c t e d for a p a i r of h y d r o g e n b o n d e d p o l y n u c l e o t i d e chains. W e wish to r e p o r t here the conditions for b o t h the release, as well as t h e reassociation of 5.8-S r R N A from 26-S r R N A using u r e a a n d s a l t solutions as d e n a t u r a n t s .
MATERIALS AND METHODS Sea urchins (arbacia p u n c t u l a t a ) were o b t a i n e d from the D u k e Marine L a b o r a t o r y , Beaufort, N.C. * Part of this communication was submitted in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry, Duke University, June 12, 197o. Present address: Rockefeller University. *~ Send reprint requests to Dr. K. S. McCarty. Biochim. Biophys. Acta, 228 (1971) 517-525
518
j . sY, K. S. MCCARTY
Eggs and embryo The techniques of handling sea urchin eggs and embryos, as well as isotopic labeling of plutei embryos have been described ~. Plutei embryos were labeled with 5 #C/ml of carrier free 3~P1 (New England Nuclear) for 16 h and were followed by a chase of 4 h with o.oi M Na2HP Q at p H 7. Isolation o/total r R N A and 5.8-S r R N A rRNA's were extracted from whole unfertilized eggs or plutei embryos by room temperature phenol extraction has been described 2. Phenol extraction at 55 ° was used to provide 26-S rRNA stripped of its 5.8-S rRNA. All 3*P-labeled RNA's were treated with cetyltrimethylammonium bromide as described by RALPH AND BELLAMY7. The 5.8-S rRNA's were obtained by polyacrylamide gel electrophoresis as described previously 2. Dissociation o/5.8-S r R N A The dissociation of 5.8-S rRNA from 26-S rRNA was affected either b y heat or urea, For heat dissociation, RNA samples were dissolved in the desired buffer and placed in a water bath for IO rain at the required temperature. The samples were then rapidly chilled in ice and immediately electrophoresed in either 2.4, 7.5 or io % gels. For urea dissociation, calculated volumes of either 1.8 M NaCl-o.i8 M trisodium citrate (pH 7.3), or 0. 9 M NaCl-o.o 9 IV[ trisodium citrate (pH 7.3) and io M urea were added to RNA samples dissolved in o.I M NaC1, o.oi M sodium acetate (pH 5.1) to produce the desired salt and urea concentration. These solutions were then incubated at the desired temperature for a minimum of I h layered on acrylamide gels and electrophoresed. The dissociated 5.8-S rRNA's were measured either by 260 m/, absorption scanning or for ~2P-labeling RNA b y scintillation counting of I.O m m sliced gels as described previously ~. Reassociation o/5.8-S r R N A with 26-S r R N A 32P-labeled 5.8-S rRNA and 26-S rRNA's were mixed with the proper amount of either 1.8 M NaC1--o.I8 M trisodium citrate or 0. 9 M NaCl-o.o 9 M trisodium citrate and io M urea to produce the desired salt and urea concentration and incubated at 24 °. At selected incubation times, samples were electrophoresed on IO % gels containing a 2,4 % spacer gel. The radioactivity of the eluted RNA's were monitored with a Packard scintillation spectrophotometer. Electrophoresis and sucrose density gradients Acrylamide gel electrophoresis was performed as described by LOENINGs and BISHOP et al. 9 Isokinetic sucrose density gradients were performed as described b y MCCARTY et al. 1° in a SW 25.3 rotor at 5 °. Scintillation counting Gel slices were dried on filter paper discs. The ~2p radioactivity was then measured in a toluene scintillation solution with a packard Tri-Carb scintillation spectrometer as described previously.
Biochim. Biophys. Acta, 228 (1971) 517-525
PROPERTIES OF 5.8-S-26-S rRNA
519
RESULTS
Fig. I shows the specificity of the release of 5.8-S rRNA from 26-S rRNA in the plesence of 8 M urea and 0.022 M Na + concentration for IO rain at room temperature. The RNA's were immediately electrophoresed in 2.4 % gels. Analogous with previous studies using heat treatment s only 26-S rRNA released the 5.8-S RNA, Figs. I a and lb. In contrast, the I8-S rRNA did not yield any such RNA fragment under these conditions, Fig. IC.
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I. R e l e a s e of 5 . 8 - S R N A f r o m 2 6 - S 1RNA b y u r e a . R N A s a m p l e s w e r e m i x e d w i t h IO M t o m a k e a f i n a l c o n c e n t r a t i o n of 8 M u r e a a n d o . o 2 2 M i n N a + c o n c e n t r a t i o n . I n c u b a t i o n a t r o o m t e m p e r a t u r e f o r i o r a i n . T h e i n c u b a t e d s a m p l e s w e r e e l e c t r o p h o r e s e d i n 2. 4 % g e l s w e r e s c a n n e d a t 2 6 0 m/~. a. C o n t r o l 2 6 - S R N A w i t h o u t u r e a t r e a t m e n t , b . 2 6 - S l Z N A t r e a t e d u r e a . c. I 8 - S R N A t r e a t e d w i t h u r e a .
The dependency on urea concentration for the release of 5.8-S rRNA is shown in Fig. 2. The sodium ion concentration was maintained at 0.0375 M NaCl-o.oo375 M trisodium citrate and the temperature of incubation at 22 ° and the urea concentration varies from 1-5 M (Figs. 2a-2d). It should be noted that 5.8-S rRNA was released from the 26-S rRNA at a urea concentration of 5 M (Fig. 2d), although some hint of this dissociation could be detected at 4 M urea (Fig. 2c). In contrast, the 5.8-S rRNA was not released by 5 M urea at 22 ° when the salt concentration was increased to o.o75 M NaCl-o.oo75 M trisodium citrate (Fig. 2e). Thus, a higher concentration of urea is required for dissociation to occur at this salt concentration. It may be concluded, therefore, that a delicate balance of both urea and salt concentration governs the denaturation of this RNA complex. The temperature of incubation is also a critical factor in the dissiociation process. Although 5 M urea in the presence of 0.0375 M NaCl-o.oo375 M trisodium citrate at room temperature was sufficient to dissociate the 5.8-S rRNA from 26-S rRNA (Fig. 2d), the dissociation phenomena did not occur when the incubation was conducted at 4 ° as shown in Fig. 2f. The conditions necessary for reassociation were determined by electrophoresis in IO % acrylamide gels, scanned at 260 m/z. These experiments demonstrated that the dissociated 5.8-S rRNA and 26-S rRNA in 8 M urea which was evident at a salt B i o c h i m . B i o p h y s . A c t a , 2 2 8 (1971) 5 1 7 - 5 2 5
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Fig. 2. Conditions for the release of 5.8-S R N A by urea. 2a, b, c, d. Unfertilized egg R N A ' s (22/~g each) were treated overnight with various concentrations of urea (a, I M: b, 2 M: c, 4 M; d, 5 M) at 22 ° while maintaining salt concentration at 0.0375 M NaCI-o.oo375 M trisodium citrate, e. 26-S R N A (22/~g) similarly treated as in d except salt concentration was o.075 M NaCl-o.oo75 M tris(:dium citrate instead of 0.0375 M NaCl-o.oo375 M trisodium citrate, f. 26-S R N A (23 fig) similarly treated as in d except t e m p e r a t u r e of incubation was at 4 ° instead. Samples were e/ectrophoresed in 7-5 °/o gels and the gels were scanned at 260 m/~. Fig. 3. Complex formation of 5.8-S R N A and 26-S RNA. Stripped 26-S R N A (33 /zg), 14oo c o u n t s / m i n of s2P-labeled 5.8-S R N A were incubated in 7 M urea and 0.255 M NaCl-o.o255 M trisodium citrate at room t e m p e r a t u r e for 20 h. Sample was then electrophoresed in 2. 4 °/o gel. The gel was frozen and sliced for counting in Toluene scintillation fluid as described in MATERIALS AND METHODS. /~- - - & , control before incubation; O - O , after incubation.
concentration of 0.0375 M NaCl-o.oo375 M trisodium citrate at 22 ° was readily reassociated when the salt concentration was increased to 0.255 M NaCl-o.o255 M trisodium citrate. Since only the dissociated 5.8-S rRNA was demonstrable using the io °/o acrylamide gel electrophoresis, the specificity of this association was also demonstrated using 2. 4 % polyacrylamide gels as shown in Fig. 3The time course of z*P-labeled 5.8-S rRNA and stripped 26-S rRNA reassociation was measured in 5 M urea with various final salt concentrations, Fig. 4. Equilibrium was attained in about 25 h when the incubation was in the presence of 5 M urea and 0.075 M NaCl-o.oo75 trisodium citrate. Approx. 62 % of the input 5.8-S rRNA was bond to the stripped 26-S rRNA, whereas at higher salt concentrations as o.1125 M NaCl-o.olI25 M trisodium citrate and o15 M NaCl-o.oI5 M trisodium citrate, the equilibrium was more rapidly attained giving values of 15 and 4 h as shown in Figs. 4 b and 4c. Table I shows the urea requirement and the fact that the efficiency of formation of this complex was much less at 5 ° than at 24 ° as shown in Expts. 1- 7 and that unstripped 26-S rRNA hybridized to 5.8-S rRNA to a maximum extent of 1/3 that Biochim. Biophys..4cta, 228 (1971) 517-525
PROPERTIES OF 5 . 8 - S - 2 6 - S r R N A
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Fig. 4. Time courses of 5.8-S RNA and 26-S RNA reassociation in 5 M urea. a. Stripped 26-S RNA (33 #g) and 32P-labeled 5.8-S RNA (0.o 5 ktg) were incubated in 5 M urea and o.o75 Mi NaC1-o,oo75 M trisodium citrate at room temperature for various times as indicated before electrophoresis in io % gels. b, Stripped 26-S 1RNA (43/*g) and 8*P-labeled 5.8-S RNA (zooo counts/rain) were incubated in 5 M urea and o.ii25 M NaCl-o.oli25 M trisodium citrate at room temperature and were analyzed as in a. c. Stripped 26 S RNA (53/~g) and [8'P]5.8-S RNA (o.o6/zg) were incubated in 5 M urea and o.15 ~ NaCl-o.oi 5 M trisodium citrate at room temperature and were analyzed as in a. Radioactivities associated with 26-S RNA were then obtained from scintillation counting of gel slices. TABLE I REASSOCIATION OF
rRNA
COMPLEX
Reassociation was performed using purified 3iP-labeled 5.8-S rRNA and the substrates as indicated. The RNA's were incubated in 5 M urea, o.o75 NI NaCl-o,oo75 IV[ trisodium citrate for 18 h at the temperatures indicated. The samples were electrophoresed in io % gels containing a 2. 4 % spacer gel. Gels were frozen, sliced and counted as described in MATERIALS AND METHODS. Experiments R N A
I. 2. 3. 4. 5. 6, 7. 8. 8a.
26-S z6-S 26-S 26-S 26-S 26-S 26-S I8-S I8-S
rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA
Substrate
Native Stripped Native Stripped Native Stripped Stripped -(Molar ratio)
Conditions
Reassociation
Urea
Temps.
%
Counts/rain
--+ + + + + + +
24 24 5 5 24 24 24 24 24
o 4 8 14 24 62 69 33 17
(--/553) (I9/519) (6o/742) (9o/64o) (i68/7o4) (44o/714) (689/994) (323/956)
of t h e s t r i p p e d 26-S R N A a t 24 °, E x p t s . 5 - 7 . I t is r e a s o n a b l e t o p o s t u l a t e t h e n t h a t b i n d i n g m a y b e d u e e i t h e r t o a n e x c h a n g e of b o u n d 5.8-S r R N A for free 5.8-S r R N A or t h a t t h e c o m p l e x is r a n d o m a n d l a c k s s p e c i f i c i t y . T h e I 8 - S r R N A u n d e r i d e n t i c a l c o n d i t i o n s b o u n d o n l y I / 2 as m u c h 5.8-S r R N A as d i d t h e s t r i p p e d 26-S r R N A , which when calculated in equimolar concentrations revealed that the I8-S rRNA b o u n d o n l y i / 4 a s m u c h 5.8-S r R N A a s d i d t h e 26-S r R N A . S i n c e i t c o u l d b e s h o w n t h a t p r e p a r a t i o n s of s t r i p p e d 26-S R N A s t i l l r e t a i n e d a f r a c t i o n of t h e b o u n d 5.8-S r R N A , i t is r e a s o n a b l e t o a s s u m e t h a t t h e a m o u n t of [8~P]5.8-S r R N A t h a t a n n e a l s t o p a r t i a l l y s t r i p p e d 26-S r R N A is p r o b a b l y less t h a n o p t i m u m . N o r e a s s o c i a t i o n of r R N A o c c u r r e d i n t h e a b s e n c e of u r e a . Biochim. Biophys. Acta, 228 (1971) 517-525
522
j. sY, K. S. MCCARTY
F u r t h e r details of t h e n a t u r e of the association of the r R N A ' s is shown in Fig. 5. These e x p e r i m e n t s utilize c o m p e t i t i o n of u n l ab el ed r R N A ' s with 32P-labeled 5.8-S r R N A . U n l a b e l e d 5.8-S r R N A ' s were isolated from unfertilized eggs an d their a b i l i t y to c o m p e t e with szP-labeled 5.8-S r R N A from plutei stages were tested. No differences were f o u n d to exist b e t w e e n the R N A e x t r a c t e d from these two stages. I n addition, the Escherichia coli 4-S a n d 5-S r R N A m i x t u r e s did not c o m p e t e with sea urchin s ' P - l ab el e d 5.8-S r R N A u n d e r similar conditions, which d e m o n s t r a t e the dissimilarity of their sequence with that of 5.8-S r R N A from sea urchins.
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Fig. 5. Competitive hybridization of 5.8-S RNA's. Stripped 26-S RNA (io/,g) and 3~P-labeled 5.8-S RNA were mixed with various amounts of unlabeled 5.8-S R2qA (unfertilized eggs RNA) or E. coli 4-S and 5-S RNA's. Samples were incubated in 5 M urea, o.o75 M NaC1 o.0075 M trisodium citrate at room temperature for 20 h and were then electrophoresed in I o % gels containing a 2. 4 % acrylamide spacer gel. Gels were sliced and counted as described in MATERIALSAND METHODS.Radioactivity associated with 26-S 1RNA,in sample where no unlabeled RNA was added was used as ioo %. 0 - 0 , E. coli 4-S and 5-S RNA's; 0 - 0 , unlabeled 5.8-S RNA from unfertilized egg RNA, Fig. 6. Complex of s2P-labeled 5.8-S ]~NA and unlabeled 26-S RNA. a. Stripped 26-S RNA (24 absorbance units) and a2P-labeled 5.8-S RNA (2.7/,g) were incubated in 5 .V£ urea, o.15 M NaCl o.o15 M trisodium citrate at room temperature for I6 h. The incubated sample was diluted with o.15 M NaC]-o.oI 5 M trisodium citrate and run in a 5-28.7 % sucrose gradient in SW 25. 3 for 29 h at 2o ooo rev./min. Gradient fractions were then counted with Triton-toluene scintillation fluid, b. 26-8 1RNA fractions were pooled (arrow) and the RNA precipitated by addition of ethanol (I8-S RNA was used as carrier). The pooled 26-S rR2XTAwas then dissolved in o.~ IV[ NaC1, o.oi M sodium acetate pH 5.z buffer, and a portion of the sample was run in 2. 4 % gel. The gel was sliced and counted as described in MATERIALSAND METHODS.
To s t u d y the specificity of the reassociated complex, t h e r m a l d e n a t u r a t i o n profiles of the 3~P-labeled 5.8-S r R N A - u n l a b e l e d stripped 26-S r R N A c o m p l e x were d e t e r m i n e d . Th e c o m p l e x was isolated from a p r e p a r a t i v e sucrose d e n s i t y g r a d i e n t as shown in Fig. 6a (arrow). T h e control sample was electrophoresed in 2.4 % gels to show the absence of u n b o u n d 5.8-S r R N A c o n t a m i n a t i n g these preparations, Fig. 6b. The d e n a t u r a t i o n profiles of the n a t i v e a n d reassociated complexes were exam i n e d in Figs. 8a an d b. The t e m p e r a t u r e at which the s2P-labeled 5 .8 - S- u n l ab el ed 26-S r R N A complexes a n d th e ~2P-labeled n a t i v e 26-S r R N A released their 32P-labeled 5.8-S r R N A was m e a s u r e d in o.I M NaC1, a n d o . o i M sodium a c e t a t e p H 5.1, indicates t h a t b o t h these R N A samples gave similar m e l t i n g t e m p e r a t u r e s , 5o-52°. In view of the fact t h a t some of t h e c o m p l e m e n t a r y sequences for 5.8-S r R N A m a y also be r e d u n d a n t in the I8-S R N A , e x p e r i m e n t s were p r ef o r m ed with the co m p l ex p r e p a r e d from 32Pqabeled 5.8-S a n d I8-S r R N A . These complexes were isolated from Biochim. Biophys. Acta, 228 (1971) 517-525
PROPERTIES
OF 5.8-S-26-S rRNA
523
sucrose density gradients as shown in Fig. 7- The thermal denaturation profiles of the native and reassociated 26-S complex was determined as shown in Fig. 8c. Release of the unlabeled 5.8-S rRNA from carrier 26-S rRNA was determined in gels by scanning at 26o m# prior to slicing of the gels for counting of the 3~P-labeled 5.8-S rRNA released from the s2P-labeled 5.8-S-I8-S rRNA complex. It should be noted that the melting temperature profiles for this complex extended over a broader range than that of the 5.8-S-26-S rRNA complex formed in vitro Fig. 8c versus Fig. 8b or that of native 5.8-S-26-S rRNA complex Fig. 8a. These experiments suggest therefore that the hydrogen-bond of the I8-S-5.8-S rRNA complex is thermodynamically less stable than the native or in vitro reassociated 26-S-5.8-S rRNA complex. Q 200( Z
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Fig. 7. C o m p l e x of s'P-labeled 5.8-S R N A a n d I8-S R N A . I8-S R N A (I74/zg) a n d s ' P - l a b e l e d 5.8-S R N A (3.2 ]~g) were i n c u b a t e d o v e r n i g h t in 5 M urea, o.15 IV[ N a C l - o . o i 5 1V[ t r i s o d i u m cit r a t e a t r o o m t e m p e r a t u r e . T h e s a m p l e s were d i l u t e d w i t h o.15 M N a C l - o . o I 5 1V[ t r i s o d i u m cit r a t e a n d were f r a c t i o n a t e d b y sucrose d e n s i t y g r a d i e n t s in S W 25. 3 a t 20 ooo r e v . / m i n for 31 h. F r a c t i o n s f r o m sucrose g r a d i e n t were c o u n t e d in T r i t o n - t o l u e n e scintillation fluid. Arrows rep r e s e n t pooled f r a c t i o n s f r o m similar g r a d i e n t s for t h e r m a l d e n a t u r a t i o n profile s t u d y in Fig. 8c. Fig. 8. T h e r m M d e n a t u r a t i o n profile of s~P-labeled 5.8-S R N A - 2 6 - S R N A a n d 3~P-labeled 5.8-S R N A - I 8 - S R N A complex. S a m p l e s of s'P-labeled 5.8-S R N A - 2 6 - S R N A derived as described in Fig. 6 were dissolved in o.i !V[ NaC1, o.oi M s o d i u m acetate, p H 5.1. T h e labeled c o m p l e x w a s h e a t e d to v a r i o u s t e m p e r a t u r e s as i n d i c a t e d for 5 min. T h e s a m p l e s were r a p i d l y chilled a n d elect r o p h o r e s e d in io °/o gels c o n t a i n i n g a 2. 4 % spacer gel. Similarly, s a m p l e s of s2P-labeled n a t i v e 26-S R N A were h e a t e d a n d electrophoresed, a. 32P-labeled 5.8-S R N A . b. s2P-labeled 5.8-S R N A 26-S R N A complex. Gels were sliced a n d r a d i o a c t i v i t y in 5.8-S R N A region w a s t h e n m e a s u r e d . T o t a l c o u n t s / r a i n in 32P-labeled 5.8-S R N A - 2 6 - S R N A c o m p l e x was d e s i g n a t e d as ioo °/o. c. T h e r m a l d e n a t u r a t i o n profile of s ' P - l a b e l e d 5.8-S R N A - I S - S R N A complex. Pooled s a m p l e s (as s h o w n in Fig. 7) were c o n c e n t r a t e d b y e t h a n o l precipitation u s i n g u n l a b e l e d 26-S R N A as carrier. Samples, in o.i M NaC1, o.oi M s o d i u m acetate, p H 5,I, were h e a t e d to v a r i o u s t e m p e r a t u r e s as i n d i c a t e d a n d were chilled r a p i d l y before electrophoresis in io % gels. Gels were sliced a n d c o u n t e d as described in MATERIALS AND METHODS. R a d i o a c t i v i t y in 5.8-S R N A region was t h e n m e a s u r e d . T o t a l c o u n t s / r a i n of t h e i n p u t c o m p l e x was d e s i g n a t e d as IOO %.
DISCUSSION
The extension of transition in the thermal denaturation curve of rRNA representing a 44 ° range for rat liver 11 versus about 3 °0 range for E . col# ~ and has been postulated as due to: (I) relatively short helices, (2) a distribution of helix length, Biochim. Biophys. Acta, 228 (i97 I) 517-525
524
J. sY, K. S. MCCARTY
(3) variation in average helix stability due to variations in the proportion of stronger base pairs and (4) variations in the proportion of miss-matched pairs incorporated in the helical regions n. The limited sharp transition for the release of 5.8-S rRNA, Figs. 8a and 8b m a y be explained then as either a 5.8-S rRNA with a very specific hydrogen-bonding site with the 26-S rRNA or the fact that 5.8-S rRNA chain of 15o nucleotides permits less variation in this hydrogen-bonded complex. When thermal denaturation profiles from ~2P-labeled 5.8-S rRNA-stripped 26-S rRNA complex and 3zP-iabeled 5.8-S r R N A - I 8 - S rRNA complexes were compared with that of native complexes under identical conditions, the sharpness of the native complex denaturation curve was immediately evident. The native complex has a transition breadth of about IO° while that of the reassociated complex has a transition breadth of about 15 ° . In contrast the thermal denaturation profile of the artifactual reassociated 5.8-S-I8-S complex extends over a range of at least 25 °. Thus one m a y conclude that both the native and renatured 5.8-S-26-S rRNA complex produces a more specific hybrid than that of 5.8-S-I8-S complex formed in vitro. It is reasonable to postulate that the capacity of the I8-S rRNA to bind some 5.8-S rRNA, although at a much reduced efficiency m a y be due to short stretches of similarity in base sequence between I8-S and 26-S rRNA. This is not completely clear, however, since there are conflicting reports as to the capacities of I8-S and 28-S rRNA to compete with each other in D N A - R N A hybridization experiments la,14. If the ability of I8-S rRNA to bind a small proportion of 5.8-S rRNA is due to a lack of specificity, then even more stringent conditions than used here will be required for its detection. It is of course essential that the complete nucleotide sequence be known before these questions can be firmly resolved. We would like to suggest that the in vivo specificity of 5.8-S rRNA binding to 26-S rRNA is intimately related to the known processing of precursor rRNA's. As partial evidence for this, it should be noted that kinetic measurements have been made which demonstrate that a 45-S precursor RNA also gives rise to the 5.5-S rRNA2s 3. The formation of 5.5-S RNA28 in other eucaryotic cells appears to involve the step of transformation of 32-28-S rRNA by some mechanism of cleavage a. It seems reasonable that a cleavage at this stage would insure the binding of 5.5-S rRNA~8 with its 28-S rRNA monomer. The processing of large precursor molecules in eucaryotic cells is becoming increasingly evident as, for example, in the formation of insulin from proinsulin 15 and it seems reasonable to postulate that the 32-S structure could possibly prescribe the proper sites for a 5.8-S rRNA-26-S rRNA interaction prior to cleavage. We would like to suggest then that the 5.8-S rlRNA2e or the 5.5-S rRNA2s is involved in maintaining the correct 3-dimensional configuration of either the 28-S or 26-S rRNA necessary for its proper interaction with proteins in ribosome maturation. At the time of the preparation of this manuscript a report appeared by KING AND GOULD1~ which confirms some of the results reported here. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant No. 128o505, American Cancer Society P363 D, and Du Pont. Biochim. Biophys. Acla, 228 (197 I) 517-525
PROPERTIES OF 5.8-S-26-S rRNA
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Biochim. Biophys. Acta, 228 (1971) 517-525