A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli

435

Biochimica et Biophysica Acta, 561 (1979) 435--444 © Elsevier/North-Holland Biomedical Press

BBA 99405

A PURIFIED NUCLEOPROTEIN FRAGMENT OF THE 30 S RIBOSOMAL SUBUNIT OF ESCHERICHIA COLI

PNINA SPITNIK-ELSON *, DAVID ELSON and RENNE ABRAMOWITZ

Biochemistry Department, Weizmann Institute of Science, Rehovot (Israel) (Received June 27th, 1978)

Key words: Nucleoprotein fragment; Ribosomal subunit; (Escherichia coli)

Summary A '13 S' nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 30-S ribosomal subunits and purified to gel electrophoretic homogeneity. It contained two polynucleotides, of about 1.1 • l 0 s and 2.5. 104 daltons, which separated when the fragment was deproteinized. The major protein components were $4, $7 and $9/11, with $15, S16, S18, $19 and $20 present in reduced amount.

Introduction

We have recently described a procedure for the production, isolation and characterization of ribosome fragments [1,2]. The method was designed to minimize changes in the composition and physico-chemical behavior of the fragments during their production and subsequent processing. An important feature of the method is the maintenance of an unchanging ionic environment, achieved by using the same buffer in all steps of the procedure, both preparative and analytical When the method was applied to the intact 50 S ribosomal subunit of Escherichia coli, the various nuclease-produced fragments maintained unchanging sedimentation and electrophoretic properties throughout the procedure, making it possible to single out a specific fragment and purify it to gel electrophoretic homogeneity [ 1 ]. The present communication describes the application of the procedure to the intact 30 S E. coli ribosome, the isolation and characterization of a

* To whom correspondence should

be addressed.

436 homogeneous fragment of this subunit, and some general features of the behavior of nuclease-digested 30-S ribosomes under the conditions employed. Materials and Methods

Buffer Fragmentation buffer is 1 mM magnesium acetate/10 mM Tns-acetate (pH 7.8)/20 mM potassium acetate.

Insoluble ribonuclease Insoluble ribonuclease was prepared by covalently binding bovine pancreatic ribonuclease A {Sigma, 5 × crystallized) to cyanogen bromide-activated Sephadex G-25, fine (Pharmacia) as described previously [1]. 1/ag-equivalent of the insoluble enzyme is that a m o u n t that has the same activity as 1 gg of ,:.oluble RNAase.

Preparation o f ribosomes, R N A and proteins The preparation of E. coli MRE 600 70-S ribosomes and 30-S ribosomal subunits, and the purification of RNA and proteins from ribosomes and ribosome fragments was as described elsewhere [1]. The same precautions against contaminating nucleases were observed.

Gel electrophoresis Analytical and preparative electrophoresis of nucleoproteins and RNA was carried out in composite polyacrylamide-agarose (3--0.5%) gels in fragmentation buffer [1]. Analytical gels were stained for RNA with Methylene Blue. The molecular weight of purified RNA was roughly estimated from its mobility in analytical gels [3], using 16 S RNA and tRNA as standards. Purified ribosomal proteins were identified by two-dimensional polyacrylamide gel electrophoresis [4].

Preparation of ribosome fragmen ts 30-S ribosomes in fragmentation buffer (absorbance at 260 nm = 100, 1 cm light path) were stirred 1 h at 0°C in a plastic tube with 0.05 gg-equivalents/ml of insoluble RNAase. After removal of the enzyme by centrifugation, the digest was heated to 57°C in a water bath at a rate of l°C/min, kept at this temperature for 30 min, removed from the bath, and allowed to cool at room temperature. The conditions of enzymic digestion and heating were chosen according to the results of preliminary exploratory experiments. The heated digest was loaded onto a convex 5--20% sucrose gradient in fragmentation buffer in a Beckman Ti-15 zonal rotor and centrifuged at 30 000 rev./min for 16 h at 5°C. 16-ml fractions were collected and their absorbance at 260 nm was measured. Nominal sedimentation, constants were assigned to the various fractions on the assumption t h a t the sedimentation constant is a linear function of the radial distance [5] between the starting position of the ribosomes (tube 11 in Fig. 1, s = 0) and the final position of 30-S ribosomes {tube 60, s = 30) in the sucrose gradient. Since the gradient was n o t isokinetic, those nominal sedimentation constants are only approximations of the true constants.

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F RACTION NUMBER Fig. 1. S u c r o s e g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n a t i o n o f a h e a t e d n u c l e a s e d i g e s t o f 30-S r i b o s o m e s ( 1 1 0 0 A 2 6 0 u n i t s ) . S e e M a t e r i a l s a n d M e t h o d s s e c t i o n f o r details.

Appropriately pooled fractions were concentrated by pressure filtration (Diaflo cells, Amicon) through a filter that retains material of molecular weight 10 000 and higher (filter PM-10, manufacturer's specifications). The concentrated solution was diluted with fragmentation buffer and reconcentrated several times in order to remove the sucrose. Precautions against contamination by extraneous nucleases were observed throughout the procedure [ll. Results

Buffer In order to avoid possible complications arising from changes in the ribosome and its fragments during their equilibration with a new medium, the same buffer was employed throughout the entire procedure. The buffer chosen was 1 mM magnesium acetate/10 mM Tris-acetate (pH 7.8}/20 mM potassium acetate. Subunits are stable and compact in this buffer, sedimenting at 29 S. The buffer's low ionic strength makes it suitable for all the techniques employed, including gel electrophoresis.

Dissociation of digested ribosomes Although its 16 S R N A chain was cleaved, the RNAase-digested 30 S ribosome remained a single particle with the same sedimentation and electrophoretic behavior as the undigested subunit (see Fig. 2 below, see also ref. 6). Heat was employed to dissociate the ribosomal fragments from each other (see Materials and Methods), the conditions of the heat treatment having been worked out in earlier experiments. The digest remained clear during the heat treatment and afterwards.

438 R

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9" 15

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80" 95

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(+) Fig. 2. A n a l y t i c a l gel e l e e t r o p h o r e s i s p a t t e r n s of 30-S r i b o s o m ~ f r a g m e n t s . O R , origin; R, i n t a c t 30 S r i b o s o m e s ; D H , t o t a l n u c l c a s e digest, h e a t e d ; D, total digest, n o t h e a t e d . T h e o t h e r s a m p l e s are p o o l e d a n d c o n c e n t r a t e d n u c l e o p r o t e i n f r a c t i o n s f r o m the s u c r o s e g r a d i e n t of Fig. I a n d are i d e n t i f i e d b y t h e g r a d i e n t f r a c t i o n n u m b e r s . T h e gel w a s s t a i n e d f o r R N A .

Separation and examination of fragments After being heated, the digest was fractionated by sucrose gradient centrifugation. Repeated experiments gave a highly reproducible separation pattern, the only variation being the relative size of the various peaks. Fig. 1 shows the pattern obtained in the experiment described here. It contains four major regions. The first is at the top of the gradient (tubes 1--22), ending in a faint shoulder at a b o u t 8 S. The second (tubes 23--45) contains material sedimenting somewhat more slowly than 30-S ribosomes, with distinct peaks at a b o u t 13 S and 19 S. The material in the third region (tubes 47--68) sediments essentially like 30-S ribosomes. The rest of the gradient comprises the fourth region and contains aggregates sedimenting more rapidly than 30-S ribosomes, including a large peak in the dense cushion layer at the b o t t o m of the gradient. Pooled sucrose gradient fractions were concentrated and examined by analytical gel electrophoresis (Fig. 2). The fractions from each of the four gradient regions displayed relative electrophoretic mobilities consistent with their sedimentation behavior, namely: very fast; slower, but still faster than 30-S ribosomes; the same as 30-S ribosomes; and slower than 30-S ribosomes, with some material failing to enter the gel. Preparative gel electrophoresis was employed to effect further fractionation and purification of three of the pooled sucrose gradient fractions (Fig. 3). Fractions from each preparative electrophoretic run were pooled, concentrated by pressure filtration, and examined by analytical gel electrophoresis. RNA and proteins were prepared from these fractions and also from a fourth fraction,

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FRACTION NUMBER Fig. 3. Preparative gel e l e c t r o p h o r e s i s . P o o l e d a n d c o n c e n t r a t e d s u c r o s e gradient f r a c t i o n s (Figs. 1 a n d 2) w e r e s u b j e c t e d to e l e c t r o p h o r e s i s in an a p p a r a t u s in w h i c h t h e m a t e r i a l t r a v e r s e d a vertical gel c o l u m n a n d was f l u s h e d i n t o a f r a c t i o n c o l l e c t o r as it e m e r g e d (see Materials a n d M e t h o d s , Ref. 1). T h e figure s h o w s three separate runs, e a c h o f a single f r a c t i o n , c a r r i e d o u t w i t h the s a m e gel c o l u m n .

from the bottom of the gradient, and were anfilyzed. Descriptions of these fractions from the four regions of the sucrose gradient are given below.

The fraction from the top of the sucrose gradient (tubes 1--22) This fraction contained about 20% of the total amount of material absorbing at 260 nm that was loaded on the preparative sucrose gradient, and was quite heterogeneous in size {Fig. 2). The material in tubes 3--8 was of molecular weight less than 10 000, and virtually all of it passed through the filter during concentration and was lost (Fig. 2, sample 3--8). That part of the material of tubes 9--22 that was retained by the filter, was fractionated by preparative gel electrophoresis (Fig. 3), and the electrophoretic fractions were examined by analytical gel electrophoresis (Fig. 4). A number of bands were seen, none of w h i c h stained for protein; nor could protein be detected in solution by the usual methods. What is of interest here is the presence of several discrete RNA fragments. Sucrose gradient fraction 23--45 This fraction contained two nucleoprotein fragments (Fig. 2), presumably corresponding to the 13-S and 19-S peaks of the sucrose gradient (Fig. 1). By preparative gel electrophoresis (Fig. 3) the 13 S fragment was purified to electrophoretic homogeneity (Fig. 5, fraction 22--25, RNP). Its purified RNA contained two major components of about 1.1 • l 0 s and 2.5 • 104 daltons, respectively (Fig. 5, fraction 22--25, RNA). Its extracted proteins were found to contain $4, $7 and $9/11 as major components, plus S15, S16, S18, S19 and $20 in reduced amount (Table I). The remaining preparative gel electrophoresis fractions, 26--29 and 30--35 (Fig. 3), contained both the 13-S and the 19-S nucleoprotein fragments

440

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F i g . 4. A n a l y t i c a l gel e l e e t r o p h o r e s i s . S u c r o s e g r a d i e n t f r a c t i t ~ l ~ 2 2 ( F i g . 1) w a s s u b j e c t e d t¢~ p r e p a r a t i v e g e l e l e c t r o p h o r e s i s ( F i g . 3). T h e n u m b e r e d s a m p l e s in t h c f i g u r e a r e f r a c t i o n s f r o m t h e p r e p a r a t i v e gel ( F i g . 3). T h e y c o n t a i n e d n o d e t e c t a b l e p r o t e i n . O R , ~ r i g i n : D H , t o t a l d i g e s t , h e a t e d ; 1~ S R N A , a d e g r a d e d s a m p l e w a s u s e d as m a r k e r . T h e gel w a s s t a i n e d f o r R N A .

(Fig. 5). All of the RNA and protein compoz~ents of the 13 S fragment were present. In addition, a larger RNA component of about 2 - 1 0 s daltons appeared; proteins $15, S16 and $19 became major protein components; and five additional major proteins appeared: $5, $6, $8, S10 and S12 {Table I). These last five proteins are therefore probably associated with the 2 - l 0 s dalton RNA in the 19-S fragment. The results do not 'allow a decision as to whether or n o t the 19 S fragment contains components also present in the 13 S fragment.

Sucrose gradient fraction 5 7--68 As recovered from the sucrose gradient, this fraction consisted of a single nucleoprotein complex with sedimentation and electrophoretic properties

441 22-25 2 6 - 29 30-35 FRACTIONS • RNP RNA RNP RNA RNP RNA (-|

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(÷} Fig. 5. A n a l y t i c a l gel e l e c t r o p h o r e s i s . S u c r o s e g r a d i e n t f r a c t i o n 2 3 - - 4 5 ( F i g . 1) w a s s u b j e c t e d t o p r e p a r a tive gel e l e c t r o p h o r e s i s ( F i g . 3). T h e f r a c t i o n n u m b e r s in t h i s f i g u r e r e f e r to t h e p r e p a r a t i v e e l e c t r o p h o r e s i s f r a c t i o n s of Fig. 3. F o r e a c h f r a c t i o n , t h e w h o l e n u c l e o p r o t e i n ( R N P ) a n d its p u r i f i e d R N A are r u n side b y side. E s t i m a t e d m o l e c u l a r w e i g h t s o f t h e p u r i f i e d R N A : 2 2 - - 2 5 , t h e t w o s t r o n g b a n d s are a b o u t 1.1 • 10 s a n d 2 . 5 • 104 d a l t o n s . 2 6 - - 2 9 a n d 3 0 - - 3 5 , t h e s a m e p l u s a b a n d of a b o u t 2 • 10 $ d a l t o n s . T h e gel w a s s t a i n e d f o r R N A .

similar to those of the 30 S ribosome (Figs. 1 and 2). Preparative gel electrophoresis caused no apparent change, and fractions taken from the peak and the ascending and descending sides of the preparative gel eluate (Fig. 3) showed the same homogeneous nucleoprotein complex. Its purified RNA showed three bands in gel electrophoresis, traces of 16 S RNA and two intense bands of a b o u t 2.2 • l 0 s and 1.5 • 10 s daltons. All the proteins of the 30 S subunit were present, although n o t in the usual relative amounts. This fraction probably consists of traces of undegraded 30-S ribosomes together with large undissociated fragments.

Sucrose gradient fraction 108--112 This fraction sedimented the most rapidly of all the material in the sucrose gradient (Fig. 1) and had the lowest gel electrophoretic mobility (Fig. 2), showing it to be the largest aggregate present. No attempt was made to purify

22--25

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(Approx. molecular weight (daltons)) $5

Proteins present *

FRAGMENTS

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OF RIBONUCLEOPROTEIN

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Preparative gel e l e c t r o phoresis fraction

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CHARACTERIZATION

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443 it further. Its RNA was made up of many small polynycleotides and it contained some but n o t all of the 30-S subunit proteins. The fraction apparently consists of one or more very large aggregates made up of small fragments. Discussion

This communication describes the fragmentation of the 30 S ribosome of E. coli under a specific set of conditions, employing a single buffer throughout the entire procedure and using heat [7] to dissociate the nuclease-digested particles. We have recently described the fragmentation of the 50 S ribosome by means of the same procedure, employing the same buffer (1 mM magnesium acetate/10 mM Tris-acetate (pH 7.8)/20 mM potassium acetate). The results obtained with both subunits were very similar. When the heated nuclease digest was fractionated by preparative sucrose gradient centrifugation, it was found in both cases that about 20% of the total ultraviolet-absorbing material was released in the form of small polynucleotides devoid of protein. This uppermost fraction was followed in the gradient by two or three nucleoprotein fractions smaller than the total subunit and representing fragments of it, two of which (one from each subunit) have been purified to homogeneity (present work and ref. 1). Farther down in the gradient was a large fraction t h a t sedimented with the same velocity as the total subunit or slightly faster. It contained essentially all of the proteins of the subunit together with large RNA components, including traces of intact 16 S or 23 S RNA. This fraction appears to consist of traces of undigested subunits plus large fragments that have n o t dissociated from each other or have aggregated. Finally, both subunits show a large and sharp peak at the b o t t o m of the sucrose gradient. Its sedimentation and gel electrophoretic properties show it to be the largest complex present, much larger than the original subunit and undoubtedly formed by aggregation. Analysis of its components showed it to be made up of a number of small polynucleotides together with proteins from all the other sucrose gradient fractions; but not all of the proteins were present. It is known that some, but not all, of the ribosomal proteins aggregate at neutral pH [8]. We suggest that this large and somewhat selective aggregate may be made up of small protein-rich nucleoprotein fragments which, as their RNA content diminishes, increasingly take on the aggregation properties of their proteins. If so, this may make it difficult to isolate certain small fragments. The RNA of the fragments was extracted with phenol and sodium dodecylsulfate in the presence of Mg2÷ ions, a m e t h o d that does not expose hidden breaks in RNA. All of the fragments we have examined have contained more than one polynucleotide. This shows, therefore, that the parent nucleoprotein contained more then one piece of RNA and that these pieces were held together by proteins, since they came apart when the proteins were removed. This is also true of 30-S subunit fragments examined in other laboratories [7,9--11]. A number of reports have been published on 30-S ribosome fragments [7,9--16]. Our sucrose gradient fractionation pattern is quite similar to those obtained by Schendel et al. [7] and Roth and Nierhaus [10]. The protein c o m -

444

position of our homogeneous 13 S fragment does not correspond exactly to any previously reported, nor do they correspond exactly with each other. [7,10,11,15,16]. This is probably due to the widely different experimental conditions employed in the production and subsequent handling of the fragments. There are, however, a number of similarities. For example, two of the three major proteins present in the 13 S fragment described here, $7 and $9/11, have been found together a number of times [7,10,11,14--16], and their closeness to each other in the ribosome is supported by the fact that they can be crosslinked with bifunctional reagents (see Ref. 17 for summary). The same is true of $5 and $8, found in our 19 S fragment and in others' [7,10,15]. Finally, it should be noted that the fractionation procedure of Roth and Nierhaus [10] also produced a large fraction of small protein-free polynucleotides at the top of the sucrose gradient, similar to that described here. Their m e t h o d differed considerably from ours in the nuclease used, the time of digestion, the way of dissociating the fraction from the digested ribosome, the buffers employed, and the fact that the digest was transferred from one buffer to another during the procedure. This result may reflect a topological feature of the ribosome, suggesting the presence of rather extensive regions of unprotected RNA. In general, in addition to what may be learned from the analysis of individual fragments, information on the structure of the ribosome can be obtained by observing the fragmentation and fractionation processes themselves under various well defined conditions.

Acknowledgements This research was partly supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. References 1 S p i t n i k - E l s o n , P., Elson, D., A b r a m o w i t z , R. a n d Avital, S. ( 1 9 7 9 ) B i o c h i m . Biophys. A c t a , in the press 2 S p i t n i k - E l s o n , P. a n d Elson, D. ( 1 9 7 8 1 in M e t h o d s in E n z y m o l o g y ( M o l d a v e , K. a n d G r o s s m a n , L., eds.), Vol. 59, pp. 4 6 1 - - 4 8 1 , A c a d e m i c Press, N e w Y o r k 3 P e a c o c k , A.C. a n d D i n g m a n , C.W. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 6 6 8 - - 6 7 4 4 Avital, S. and Elson, D. ( 1 9 7 4 1 A n a l y t . B i o c h e m . 57, 2 7 4 - - 2 8 6 5 A n d e r s o n , N.G. and R u t e n b e r g , E. ( 1 9 6 7 1 A n a l y t . B i o c h e m . 21, 2 5 9 - - 2 6 5 6 Cabn, F., S c h a c h t e r , E.M. a n d Rich, A. ( 1 9 7 0 ) B i o e h i m . Biophys. A c t a 209, 512 520 7 S c h e n d e l , P., Maeba, P. and C r a v e n , G.R. ( 1 9 7 2 ) Proc. Nat. Acid. Sci. U.S. 69, 5 4 4 - - 5 4 8 8 S p i t n i k - E l s o n , P. a n d Zingher, S. ( 1 9 6 7 1 B i o c h i m . Biophys. A c t a 133, 4 8 0 - - 4 8 5 9 Sz6kely, M., B r i m a c o m b e , R. and M o r g a n , J. ( 1 9 7 3 1 Eur. J. B i o c h e m . 35, 574 581 10 R o t h , H.E. a n d Nierhaus, K.H. ( 1 9 7 3 1 FEBS L e t t . 31, 3 5 ~ 3 8 11 Y u k i , A. and B r i m a c o m b e , R. ( 1 9 7 5 ) Eur. J. B i o c h e m . 56, 23 34 12 B r i m a c o m b e , R., M o r g a n , J.M. a n d Cox, R.A. ( 1 9 7 1 ) Eur. J. B i o c h e m . 23, 5 2 - - 6 0 13 M o r g a n , J. and B r i m a c o m b e . R. ( 1 9 7 2 1 Eur. J. B i o c h e m . 29, 5 4 2 - - 5 5 2 14 S p i t n i k - E l s o n , P., Z a m i r , A., Miskin, R., K a u f m a n n , Y., Ehrlich, Y.H., G i n z b u r g , I. and Elson, D. ( 1 9 7 2 1 in R N A Viruses, R i b o s o m e s ( B l o e m o n d a l , H., Jaspars, E.M.J., V a n K a m m e n , A. a n d Planta, R.J., eds.), pp, 2 5 1 - - 2 5 9 , Elsevier, A m s t e r d a m 15 M o r g a n , J. a n d B r i m a c o m b e , R. (19731 Eur. J. B i o c h e m . 37, 4 7 2 - - 4 8 0 16 Z i m m e r m a n n , R . A . , M u t o , A. a n d Mackie, G.A. ( 1 9 7 4 ) J. Mol. Biol. 86, 4 3 3 4 5 0 17 E x p e r t - B e x a n q o n , A., B a r r i t a u l t , D., Milet, M., G u e r i n , M.F. and H a y e s , D.H. ( 1 9 7 7 ) J. Mol. Biol. 112, 603--629