Binding sites for ribosomal proteins S8 and S15 in the 16 S RNA of Escherichia coli

422

Biochimica et Biophysica Acta, 5 6 3 ( 1 9 7 9 ) 4 2 2 - - 4 3 1 © Elsevier/North-Holland

Biomedical Press

BBA 99485

BINDING SITES FOR RIBOSOMAL PROTEINS $8 AND S15 IN THE 16 S RNA OF E S C H E R I C H I A C O LI *

ROBERT

A. Z I M M E R M A N N

and K A R I N S I N G H - B E R G M A N N

**

Ddpartment de Biologie Mol~culaire, Universitd de Gen~ve, 1211 Gen~ve 4 (Switzerland) and Department o f Biochemistry, University o f Massachusetts, Amherst, MA 01003 (U.S.A.) (Received December 22nd, 1978)

Key words: rRNA ; Ribosomal protein; Pro tein-RNA binding; (Escherichia coli)

Summary A fragment o f the 16 S ribosomal RNA of Escherichia coli that contains the binding sites for proteins $8 and S15 of the 30 S ribosomal subunit has been isolated and characterized. The RNA fragment, which sediments at 5 S, was partially protected from pancreatic RNAase digestion when S15 alone, or $8 and S15 together, were bound to the 16 S RNA. Purified 5 S RNA was shown to reassociate specifically with protein S15 by analysis of binding stoichiometry. Although interaction between the fragment and protein $8 alone could not be detected, the 5 S RNA selectively bound both $8 and S15 when incubated with an unfractionated mixture of 30-S subunit proteins. Nucleotide sequence analysis demonstrated that the 5 S RNA arises from the middle of the 16 S RNA molecule and encompasses approximately 150 residues from Sections C, C'1 and C~. Section C consists of a long hairpin loop with an extensively hydrogen-bonded stem and is contiguous with Section C'~. Sections C'~ and C~, although not contiguous, are highly complementary and it is likely that together they comprise the base-paired stem of an adjacent loop.

Introduction

Proteins $4, $7, $8, S15, S17 and $20 o f the 30 S ribosomal subunit of Escherichia coli have been found to interact individually with specific binding

sites in the 16 S RNA molecule [1--6]. Through the isolation o f RNA fragments that are capable of reassociating with one or m o r e of these proteins, the * Paper No. 5 in t h e s e r i e s 'RN A - P r o t e i n I n t e r a c t i o n s in t h e R i b o s o m e ' . Preceding paper is Mackie, G.A., a n d Z i m m e r m a n u , R.A., (1978) J. Mol. Biol. 121, 17--39. * * P r e s e n t address: I n s t i t u t de B i o c h i m i e , Universit~ de Lausanne, 1011 Lausanne, Switzerland.

423 distribution and properties of their binding sites in the 16 S RNA have been characterized [7--18]. Interpretation of these data is now greatly facilitated by the availability of the complete nucleotide sequence of the small-subunit RNA [19,20]. The binding sites for proteins $4, S8, S15 and $20 were initially located within the 5' half of the 16 S RNA and that for $7 within the 600 3'-terminal residues [7]. More specifically, proteins $4, S17 and $20 have been shown to interact within a 550-nucleotide segment at the 5' end of the RNA molecule [7--11] and the main features of this region have been delineated [12--15]. Portions of the nucleic acid chain that assocfate with protein $7 have been further defined by the analysis of ribonucleoprotein fragments derived from intact 30 S subunits [16,17]. Finally, proteins $8 and S15 have each been demonstrated to interact with sequences from the middle of the 16 S RNA [ 7,9--11,18]. The present report describes the isolation, binding properties and structural characteristics of an RNA fragment containing binding sites for both $8 and S15. Materials and Methods

Ribosomal components. Unlabelled and all-labelled proteins, as well as 14Cand 32P-labelled 16 S RNA, were prepared from 30-S ribosomal subunits of E. coli strain MRE600 as previously described [5]. RNA-protein complexes. Samples of 16 S RNA and protein were added to 100 gl of Tris/Mg/K buffer (0.05 M Tris-HC1 (pH 7.6)/0.02 M MgC12/0.35 M KC1/0.006 M 2-mercaptoethanol) as indicated in the text. The reaction mixtures were incubated for 30 min at 40°C, chilled to 0°C in an ice bath, and treated with pancreatic RNAase A at an enzyme :substrate ratio of 1 : 5 (w : w). Products were separated by centrifugation through 3 to 15% sucrose gradients in Tris/Mg/K buffer for 20 h at 40 000 rev./min in an IEC SB283 rotor. After fractionation of the gradients, the radioactivity in each tube was measured by scintillation counting [5]. Preparation o f 5 S R N A fragment. 14C- and 32P-labelled 5 S RNA f~agments were produced from complexes of proteins $8 and S15 with '4C- and 32p. labelled 16-S RNAs by pancreatic RNAase digestion and isolated by sucrose gradient sedimentation in the manner described above. Fractions containing the protected 5-S RNA fragments were deproteinized by phenol extraction and purified by centrifugation through a second 3 to 15% sucrose gradient in Tris/Mg/K buffer. The fragment was concentrated by ethanol precipitation after the addition of unlabelled carrier tRNA. Saturation curves. The stoichiometry of protein-5-S RNA fragment complexes was determined by incubating a fixed amount of J4C-labelled RNA fragment with increasing amounts of all-labelled protein. The mixtures were fractionated by sucrose gradient centrifugation and the quantities of protein and RNA in the complex were calculated from the 3H and 14C radioactivity in the 5 S peak and from the specific activities of the components, which were approx. 2000 cpm per ~g for the [3H/proteins and 500 cpm per pg for the [ 14C]RNA [5]. Molar ratios were computed using molecular weights of 13 996 for $8 [21], 10 000 for $15 [22], and 45 000 for the 5 S RNA fragment.

424

Chromatographic analysis. Unfractionated 3H-labelied 30-S subunit proteins were incubated with purified ~4C-labelled 5 S R N A fragment for 60 min at 0°C in 300 pl Tris/Mg/K buffer and the mixture was separated on sucrose gradients as described above. Fractions containing the protein-5 S R N A fragment complex were pooled and mixed with 10 mg of unlabelled 30-S subunits. After precipitation of the ribonucleoprotein with 5% trichloroacetic acid (final concentration), the pellet was resuspended in Tris/Mg/K buffer and extracted with 67% acetic acid [9]. Proteins were applied to a 3-mm internal-diameter × 40-cm column of phosphocellulose at PH 6.5 and eluted with a linear NaC1 gradient [ 5]. Labelled and unlabelled proteins in the eluate were assayed and identified as reported earlier [9]. Fingerprinting. 32p-labelled 5 S R N A fragment, as well as sub-fragments derived from it by polyacrylamide gel electrophoresis under denaturing conditions [12], were fingerprinted by standard procedures following complete digestion with T1 RNAase and bacterial alkaline phosphatase [23,24]. The products arising from each of the RNAase TI oligonucleotides after secondary hydrolysis with pancreatic RNAase A or 0.2 N NaOH were also analyzed [23]. Results and Discussion

Digestion of RNA-protein complexes and the reassociation of proteins with the 5 S RNA fragment It was previously reported that pancreatic RNAase digestion of the protein $15-16 S RNA complex at an enzyme : substrate ratio of 1 : 5 produced an RNA fragment of roughly 4 S which retained most of the b o u n d protein [5]. When similar digests were centrifuged for longer periods of time, the slowlysedimenting R N A was resolved into 3-S and 5-S c o m p o n e n t s and, as shown in Fig. 1, S15 was associated exclusively with the larger of the two. Hydrolysis

RNA

o 100 t o

.

Q

.

as 5S

1

< Z c~

~'

.

.

S15

/f

p / /

5o

,'

~

/

.

.

b

.

1 0

o I

o

o/ \ o

i,

.

$8

i

1 ! '

""

.

t

.

.

c

~

$ ~

,,'A

/

~:X f'\

.

$8

,

.

1

d

415o

z

1 1

,

/I

/ !,,

/

i ILl [

~

o ~-

,

I

/

° 'Jl

i, / ~

IO0

J°

L:~. ~_

~u

o

,o

2o

Lo FRACTION

20

,o

2o

,o

to

NUMBER

Fig. 1. P a n c r e a t i c R N A a s e d i g e s t i o n o f 16 S R N A a n d of c o m p l e x e s b e t w e e n 16 S R N A a n d p r o t e i n s $ 1 5 a n d SS. 25 p g o f 1 6 S [ 1 4 C ] R N A w e r e i n c u b a t e d w i t h (a) n o p r o t e i n , (b) 1.1 ~ g o f [ 3 H ] $ 1 5 , (c) 0.8 /Jg of [ 3 H | $ 8 o r (d) 0.8 ~g of [ 3 H ] S S a n d 1.5 ~g o f u n l a b e l l e d $ 1 5 for 3 0 rnin a t 4 0 ° C . T h e m i x t u r e s w e r e t h e n chilled, d i g e s t e d w i t h 5 ~g o f p a n c r e a t i c R N A a s e A for 15 m i n at 0 ° C , and f r a c t i o n a t e d b y s u c r o s e gradient c e n t ~ f u g a t l o n . T h e d o t t e d lines in (b) and ( c ) r e p r e s e n t s e d i m e n t a t i o n p r o f i l e s for p r o t e i n s $ 1 5 a n d $ 8 a l o n e u n d e r the s a m e c o n d i t i o n s .

425 of free 16 S RNA under the same conditions yielded no 5 S R N A fragment, although substantial amounts of 3 S RNA fragment were recovered among the products (Fig. la). Moreover, the sedimentation profile of $15 in the absence of RNA demonstrated that no protein aggregates formed during sample preparation or sucrose gradient centrifugation ( d o t t e d line, Fig. lb). It was therefore concluded that the 5 S R N A fragment was specifically protected from enzymatic degradation by the presence of protein $15. Protein $8 was also f o u n d to remain attached to small R N A fragments following hydrolysis of the $8-16 S R N A complex with pancreatic RNAase. The ribonucleoprotein fragments appeared to be heterogeneous, however, with sedimentation coefficients of 2 S to 4 S, and attempts to resolve them into discrete c o m p o n e n t s were unsuccessful, even after long periods o f centrifugation (Fig. lc). In particular, the 5 S R N A fragment was not recovered under these conditions. Since the binding sites for $8 and S15 were thought to be located in the same region of the R N A molecule [10], it was considered possible that protein S15 would contribute to the stability of the S8-RNA fragment complex. Accordingly, $8 and S15 were simultaneously incubated with 16 S RNA and the resulting complex was digested with pancreatic RNAase. Fig. l d shows that 3H-labelled $8 cosediments with the protected 5 S fragment when S15 was also present. In a parallel experiment, it was demonstrated that $8 did not reduce the amount of 3H-labelled S15 bound to the 5 S fragment complex. The fact that $8 and $15 are retained by the 5 S R N A after RNAase digestion o f the $8, S15-16 S R N A complex provides good evidence that this fragment contains binding sites for both proteins. A more rigorous test consists in determining whether or n o t the isolated fragment is capable of specifically reassociating with the two proteins [9]. When $15 alone was incubated with the 5 S R N A fragment, a stable complex was detected by sucrose gradient centrifugation. From measurements of binding stoichiometry in the presence o f excess protein, it was determined that the complex reached saturation when 0.75 mol protein was b o u n d per mol 5 S R N A (Fig. 2). This result suggests that the fragment contains the entire binding site for S15. Moreover, the molar binding ratio observed is in good agreement with the value obtained for the interaction of S15 with the 12 S R N A fragment [9], which spans the 5' 900 nucleotides of the 16 S R N A molecule, and only slightly lower than that obtained for the binding of S15 to intact 16 S R N A [5]. To facilitate comparison, all three saturation curves are depicted in Fig. 2. Purified $8 did n o t interact with the 5 S RNA, however, either alone or in the presence of S15, nor did any other of the RNA-binding proteins of the 30 S subunit interact individually with this fragment. Nonetheless, since the digestion experiment illustrated in Fig. 1 indicated that the attachment site for $8 was located in the 5 S RNA, the binding properties of the fragment were examined by an alternative technique. Isolated 5 S R N A fragment was incubated with an unfractionated mixture of 3H-labelled 30-S subunit proteins and the resulting ribonucleoprotein complex was separated from u n b o u n d protein by sedimentation. Fragment-bound proteins were then extracted and fractionated by chromatography on phosphocellulose (Fig. 3). The only peaks containing significant amounts o f radioactivity were those corresponding to

426

~

1.5

515

= z

_

"~ 1.0 == /

~_,

. . . . . . . . . . . . . . . . . . . . . .

f

,Y /

...J

""

_

J f

0.5

o

0

-"

I

t

I

2

13

3

4

5

6

MOLESPRDIEIN] MOLES RNA ADDEDTO INCUBAIION Fig. 2. Saturation binding o f p r o t e i n S 1 5 to the i s o l a t e d 5 S R N A fragment. Increasing quantities o f [ 3 HI S 1 5 w e r e i n c u b a t e d w i t h 2 , 5 # g o f 5 S [ 14 C] R N A fragrnent for 60 rain at 0 ° C in 1 0 0 p l Tris]Mg]K buffer. P r o c e d u r e s for the analysis o f t h e p r o t e i n o R N A c o m p l e x e s and for the c a l c u l a t i o n o f m o l a r p r o t e i n : R N A ratios are described in Materials and M e t h o d s . T h e saturation curve o b t a i n e d for p r o t e i n S 1 5 and the 5 S R N A fragment () is c o n t r a s t e d with similar curves for intact 16 S R N A ( . . . . . . ) a n d for the 12 S R N A fragment (-- -- --) p u b l i s h e d earlier [ 5 , 9 ] .

protein $8 and to proteins $14 and S15. Analysis o f a parallel sample by polyacrylamide gel electrophoresis, which resolves S14 from $15 [9], revealed that only the latter protein was present. The molar ratio of $8 and S15 in the 5 S fragment complex was estimated to be approx. 1 : 2 from the amount of radio-

r

06~ 05 ~

400

. . . . . . . . . . . . . .

•

PROTEINS ASSOCIATED WITH 5S RNA FRAGMENT SI

55 SB

S5.SlO 52

$7 5161517

53

$4 SIB,SII,S9 519 S21 514,515 512,513

04 ~

300 I

520

,~

Z

r

12oo o o

03~

o.

~oo ,

0

50

I00

150 FRACTION NUMBER

200

250

C9

300

Fig. 3. C h r o m a t o g r a p h i c analysis o f p r o t e i n s ~ s o c i a t e d w i t h the 5 S R N A fragment. C o m p l e x e s were prep~Lred by i n c u b a t i n g 9 ~g o f 5 S [ 1 4 C ] R N A fragment with 27 ~g of u n f r a c t i o n a t e d 30 S s u b u n i t [ 3 H ] proteins. Proteins associated with t h e R N A w e r e a n a l y z e d by c h r o m a t o g r a p h y o n p h o s p h o c e n u l o s e . E l u t i o n profile for [ 3 H ] p r o t e i n s ( -); e l u t i o n profile for u n l a b e l l e d proteins (- . . . . . ). Protein $ 8 o c c u p i e s a u n i q u e p o s i t i o n in the c h ~ o m a t o g r a m ; p r o t e i n $ 1 5 was distinguished from p r o t e i n $ 1 4 , w h i c h eintes at t h e same p o s i t i o n , by p o l y a c r y l a m i d e gel analysis o f a parallel sample [ 9 ] . Material eluting u n d e r t h e $1 p e a k , w h i c h w a s n o t retained by t h e c o l u m n , failed to c o e l e c t r o p h o r e s e w i t h $1 in the p o l y a c r y l amide gel and m a y consist o f aggregated protein.

427 activity eluting with each peak and the molecular weights of the two proteins [21,22]. These findings demonstrate that the 5 S RNA contains the binding site for $8 as well as for S15, although association o f the fragment with $8 is somewhat less efficient than with S15. When tested under similar conditions, isolated 3 S RNA did not interact with any o f the 30-S subunit proteins. We cannot presently explain why $8 in an unfractionated protein mixture can associate with the 5 S fragment whereas purified $8 fails to do so. It is possible that the interaction of $8 with the fragment is weaker than with intact 16 S RNA and that the fragment complex therefore dissociated during centrifugation. Alternatively, the structure of protein $8 may be altered by purification or storage in such a way that it forms a less stable complex with its binding site in the RNA molecule. Isolated $8 has in fact been found to undergo a reversible confolTnational change when kept for e x t e n d e d periods in high concentrations of urea that leads to a substantial decrease in its ability to bind to 16 S RNA [25]. In the protein mixture, $8 may be protected from such structural alterations. Finally, certain of the unfractionated 30 S proteins m a y facilitate the interaction of $8 with the 5 S fragment without themselves becoming attached. Structure of the 5 S RNA fragment and the binding sites for proteins $8 and $15 The primary structure of the 5 S fragment was investigated by the fingerprinting technique of Sanger and co-workers [23,24]. 3:P-labelled 5 S RNA of high specific activity was prepared from a pancreatic RNAase digest of the protein $8, $15-16 S RNA complex and completely hydrolyzed with RNAase T1 in the presence of alkaline phosphatase. The resulting oligonucleotides were separated by two-dimensional electrophoresis, yielding the fingerprint presented in Fig. 4. The pancreatic RNAase digestion products o f each spot were characterized by electrophoresis at pH 1.9. This procedure was sufficient to identify all of the T1 oligonucleotides observed in the fingerprint by reference to the catalog of T~ products from 16 S RNA [26]. In this way, the 5 S fragment was found to encompass 14 residues of Section C", all of Section C, and most of Sections C'~ and C~ (Fig. 5). The C"-C-C'I Segment comprises a sequence of 110 nucleotides begining at residue 571 of the nucleic acid molecule [19,21]. The portion of Section C~ present in the 5 S RNA contains 42 nucleotides and is separated in the primary structure from the C"-C-C'I sequence by about 40 bases, including the 10 3'-terminal residues of Section C'~ and all of Section K' [19,20], which are missing from the fragment. Fig. 5 illustrates a possible secondary structure for Sections C, C'~ and C~ proposed on the basis of their nucleotide sequences. Section C is thought to be folded into a long, extensively base-paired hairpin loop. Sections C'~ and C~, which are highly complementary, probably form the stem of a second hairpin loop. Thus, despite the absence of Section K', it is likely that Section C~ remains stably hydrogen-bonded to the initial portion of Section C'~. In addition to the excision o f Section K', pancreatic RNAase digestion reproducibly introduces one or more internal scissions in the unpaired loop of Section C (heavy arrow, Fig. 5). Such breaks results in the disappearance of the oligonucleotide C-U-C-A-A-C-C-U-G from the fingerprint and the appearance of a new oligo-

428

5S ,p~ '3.5

• UUUG O AAUUCCAG AUACUG tt

UUAAGQbx QCAUCUG

cuuGl OUCUCG O AAAUCCCCG

0 AAccuGo

AG •

CG

AACUG0 AAGACG CCCCCUGi t t ' ~ C ~ G

, l ' O ~ c.cG

Acu~ t O CUCAG CAAG AueO O uc~,G

O

UAG• O

0 UGO Fig. 4. Fingerprint of 5 S KNA fragment. 5 S [ 3 2 P ] R N A was prepared from t he p r o t e i n $8, S15-[32p]" 16 S RNA c o m p l e x and c o m p l e t e l y h y d r o l y z e d with TI RNAase i n t he presence of bacterial alkaline phosphatase [24]. The digestion products were fractionated by high-voltage electrophoresls on (i) eallulose acetate in 5 % acetic acid-7 M urea, p H 3.5, in the firstdimension and (li)DBAE-paper in 7 % formic acid in the second, and further analyzed to determine their nucleotlde composition and sequence [23]. The spot marked X has the composition (A-U, C, U ) G and m a y represent a variant of either A-U-A-C*U-G or C-A-U-C-U-G [27]. The oligonucleotide A-A-C-C-UoG results from pancreatic R N A a s e hydrolysis w i t h i n the sequence C-U-C-A-A-C-C-U-G at the top of the hairpin loop in Section C (see Fig. 5) during fragment preparation. The p r o m i n e n t spot Y and several fainter spots, i n d i c a t e d by cross-hatching on t he plan, m a y arise from additional pancreatic RNAase seisslons.

nucleotide, A-A-C-C-U-G, which corresponds to its 3'-terminal portion (Fig. 4). As in the case of Sections C'1 and C~, however, the discontinuity is hidden, since the right and left halves of Section C are presumably held together by hydrogen bonds. Polyacrylamide gel electrophoresis of the 5 S RNA in the presence of urea disrupted its secondary structure and resolved the fragment into five main corn-

429

uc A/

C

A

G--c

G U G

G A

C ~G C ~G C

C ~G A

~C A A

C~

G

G~C G .... U C~G C~G

U~ A U~ A A G A G G C A G U .... G

A C AG .... U

U .... G G~C U--A

A~U

G~C , A ~ U 31

r ......

Lc__~__G_=

C '1 ~/x

U--A r- . . . . . .

"t

~,

=G.... :_ u~, AG

U

G

-A-"-- C

G

....

A~U [u :--:_G_i

C G C

U.... GQ

(- . . . . .

C G C

G--C, LU--T----A3 U~A

GA

G

u ~

A

G~

C

U G G

C~ G U~A C~ G

U.... G

U~A

G--C

G~C

C U C G

GG : .... UU GA ~ U G 4C C C I' Fig. 5. N u c l e o t i d e s e q u e n c e a n d possible s e c o n d a r y s t r u c t u r e o f t h e 5 S R N A f r a g m e n t . T h e s e n s e o f t h e s e q u e n c e is 5 ' ( l e f t ) -* 3 ' ( r i g h t ) . T h e s e c o n d a r y s t r u c t u r e w a s d e r i v e d f r o m t h e c o m p l e t e p r i m a r y s e q u e n c e [ 1 9 , 2 0 ] a n d is s i m i l a r b u t n o t i d e n t i c a l to t h a t p r e v i o u s l y s u g g e s t e d [ 2 6 , 2 7 ] . N o t e t h e e x c i s i o n o f t h e s e q u e n c e t h a t links S e c t i o n s C] a n d C~ ( S e c t i o n K ' ) f r o m this f r a g m e n t as well as t h e c u t t h a t o c c u r s in t h e u n p a i r e d l o o p o f S e c t i o n C ( h e a v y a r r o w ) , w h i c h m u s t t h e r e f o r e be accessible t o p a n c r e a t i c R N A a s e in t h e r i h o n u c l e o p r o t e i n c o m p l e x . T h e l i g h t a r r o w m a r k s t h e p o s i t i o n o f a s e c o n d a z y c u t t h a t o c c u r s i n a s m a l l f r a c t i o n o f t h e f r a g m e n t p o p u l a t i o n . As i n d i c a t e d in t h e plan, t h e 5 S R N A f r a g m e n t was u s u a l l y o b s e r v e d t o c o n t a i n o l i g o n u c l e o t i d e s f r o m S e c t i o n C" w h i c h lie a d j a c e n t t o t h e 5' t e r m i n u s o f S e c t i o n C. A r e g i o n o f ' h y p h e n a t e d ' 2-fold s y m m e t r y in S e c t i o n C is e n c l o s e d b y a b o x , as is an u n u s u a l l y l o n g r u n o f c o n s e c u t i v e G • C b a s e p a i r s in t h e C'1-C'2 s t e m .

ponents (Ehresmann, C., personal communication). Two o f these arise from the left side of Section C and differ slightly in the position of the cuts defining their 3'-termini. The positions of the major and minor scissions are indicated in Fig. 5 b y heavy and light arrows, respectively. The largest component, containing 62 nucleotides, encompasses the right side of Section C and most of Section C'~. The remaining two bands derive from Section C~; the 3' terminus of the principal c o m p o n e n t occurs at the point shown in Fig. 5, whereas the second extends another ten residues in the 3' sense. The 5 S RNA is similar in structure to a fragment isolated previously by digestion of the protein S15-16 S R N A complex with RNAase T~ [5,7]. The

430 latter fragment differs from the former in that it (1) interacted specifically with protein $15 b u t not with protein $8, (2) contained the sequence C-U-C-A-A-C-C-U-G from Section C intact, and (3) was n o t protected by bound protein, since an identical fragment could be isolated from T1 RNAase digests of free 16 S R N A [5]. In addition to the 5 S fragment, pancreatic RNAase digestion o f complexes containing $8, S15 and 16 S RNA yielded a 3 S c o m p o n e n t that retained small amounts of $8, but no S15 (Fig. 1). Fingerprints of the 3 S R N A revealed many of the unique oligonucleotides from Sections C, C'l and C'2 which may explain why $8 remained associated with some of this material (see Fig. lb). Several characteristic spots from other portions of the 16 S RNA were present as well, however, and it was concluded that the 3 S RNA consisted of a heterogeneous mixture of relatively RNAase-resistant segments from throughout the molecule. Furthermore, u p o n gel electrophoresis under denaturing conditions, the 3 S R N A was fractionated into many small components, the largest of which did not exceed 30 nucleotides in length. Thus, despite the prominence of oligonucleotides from Sections C, C'1 and C':, the inability of the isolated 3 S c o m p o n e n t to interact with either protein $8 or S15 must be ascribed to its highly degraded state. Since base-paired segment:-, of the nucleic acid chain are more stable to RNAase digestion than single-stranded regions [27], it is likely that the surprisingly large sedimentation coefficient of this R N A results from the persistence o f substantial secondary structure. Portions of the 16 S RNA that interact with either $8 or S15 individually have also been investigated [11,18]. In particular, S8-specific fragments were isolated from pancreatic RNAase digests of the $8-16 S RNA complex by a two-step electrophoretic procedure [11]. The protected R N A was found to consist of two complementary sequences, each 20 nucleotides in length, which comprise the lower half o f the hairpin loop in Section C (see Fig. 5). Similar sequences were recovered in association with $8 by a gel filtration technique [18]. In contrast, protein S15 alone protected the lower portion of the basepaired stem formed by Sections C'~ and C~ as well as the right~hand side of the Section C stem [11]. It is n o t e w o r t h y in this connection that partial RNAase hydrolysis o f the S8-16 S RNA complex from Bacillus stearothermophilus resulted in the protection of segments apparently analogous to the lower portions of Sections C and C'L • C~ in E. coli 16 S RNA [28]. These findings, together with those reported here, raise the possibility that $8 and S15 each make contact with both hairpin structures in the C-C'~ • C~ region. Although it would be premature to speculate u p o n the precise structures within Sections C, C'~ and C~ that directly interact with proteins $8 and $15, several characteristics of their binding sites can be inferred from the available data. ( 1 ) T h e binding sites for $8 and $15 are very closely related to One another in the 16 S RNA primary structure and may partially overlap. (2) The excision of Section K' and the accessibility of the unpaired loop at the t o p o f Section C to pancreatic RNAase suggest that the upper portions o f the two hairpins depicted in Fig. 5 are not in intimate contact with either protein. (3) Since no substantial single-stranded sequences occur among the protected R N A fragments, the association o f $8 and S15 with the R N A must be mediated primarily by the imperfect double-stranded stems of the two hairpin loops

431

within the C-C'I-K'-C~ segment. It is intriguing to note a number of unusual features in the secondary structure o f this region, including a 'hyphenated' 2-fold axis and a run o f stacked G • C base pairs (Fig. 5). Several palindromes occur in the C-C'I-K'-C~ region as well. Although the significance o f such regularities is presently unknown, it is nonetheless tempting to consider them as potential recognition sites for ribosomal proteins. Acknowledgements The authors wish to express their appreciation to Dr. A. Tissi~res for his interest in this work, to Dr. G.A. Mackie for the preparation o f 32P-labelied RNA, to Miss A.-M. Piret for expert technical assistance, and to Dr. C. Ehresmann for analysis o f the fingerprint presented in Fig. 4. This project was supported by a grant from the Fonds National Suisse de la Recherche Scientifique. References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Mizushima, S. and Nomura, M. (1970) Nature (London) 226, 1214--1218 Schaup, H.W., Green, M. and Kurland, C.G. (1970) Mol. Gen. Genet. 109, 193--205 Schaup, H.W., Green, M. and Kurland, C.G. (1971) Mol. GerL Genet. 112, 1--8 Garrett, R.A., Rak, K.H., Daya, L. and StSffler, G. (1971) Mol. Gen. Genet. 114, 112--124 Muto, A., Ehresmanrt, C., Fellner, P. and Zimmermann, R.A. (1974) J. Mol. Biol. 86, 411--432 Held, W.A., Ballou, B., Mizushima, S. and Nomura, M. (1974) J. Biol. Chem. 249, 3103--3111 Z i m m c r m a n n , R.A., Muto, A., FeUner, P., Ehresmann, C. and Branlant, C. (1972) Proe. Natl. Aead. Sci. U.S. 69, 1282--1286 Schaup, H.W., Sogin, M., Woese, C. and Kurland, C.G. (1971) Mol. Gen. Genet. 114, 1--8 Z i m m e r m a n n , R.A., Muto, A. and Mackie, G.A. (1974) J. Mol. Biol. 86, 433--450 Z i m m e r m a n n , R.A., Mackie, G., Muto, A., Garrett, R.A., UngewickeU, E., Ehresmann, C., Stieg)er, P., Ebel, J.-P. and Fellner, P. (1975) Nucleic Acids Res. 2, 279--302 Ungewickell, E., Garrett, R.A., Ehresmann, C., Stiegler, P. and Fellner, P. (1975) Eur. J. Biochem. 51, 165 --180 Mackie, G.A. and Z i m m e r m a n n , R.A. (1975) J. Biol. Chem. 250, 4 1 0 0 - - 4 1 1 2 Barritault, D. and Hayes, D.H. (1977) Biochimie 50, 463- -472 Ungewickell, E., Garrett, R.A., Ehresmann, C., Stiegler, P. and Carbon, P. (1977) FEBS Lett. 81, 193---198 Mackie, G.A. and Zimmerrnann, R.A. (1978) J. Mol. Biol. 121, 17--39 Yuki, A. and Brimacombe, R. (1975) Eur. J. Biochem. 56, 23---34 Rinke, J., Yuki, A. and Brimacombe, R. (1976) Eur. J. Biochem. 64, 77--89 Schaup, H.W., Sogin, M.L., Kurland, C.G. and Woese, C.R. (1973) J. Bact. 115, 82--87 Brosius, J., Palmer, M.L., Kennedy, P.J. and Noner, H.F. (1978) Proc. Natl. Acad. Sci. U.S. 75, 4801-4805 Carbon, P., Ehres~nann, C., Ehresmann, B. and Ebel, J.P. (1978) FEBS Lett. 94, 152--156 Allen, G. and Wittmann-Liebold, B. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1 5 0 9 - - 1 5 2 5 Morinaga, T., Funatsu, G., Funatsu, M. and Wittmann, H.G. (1976) FEBS Lett. 64, 307--309 Sanger, F., Brownlee, G.G. and Barrell, B.G. (1965) J. Mol. Biol. 13, 373--398 Brownlee, G.G. and Sanger, F. (1967) J. Mol. Biol. 23, 337---353 Schulte, C. and Garrett, R.A. (1972) Mol. Gen. Genet. 119, 345--355 Ehres~-nann, C., Stiegler, P., Fellner, P. and Ebel, J.-P. (1975) Biochimie 57, 711--748 Ehresmann, C., Stiegler, P., Fellner, P. and Ebel, J.-P. (1972) Biochimic 54, 901--967 Stanley, J. and Ebel, J.-P. (1977) Eur. J. Biochem. 77, 357--366