Interactions between dextran sulfate and Escherichia coli ribosomes

96

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95661

INTERACTIONS B E T W E E N D E X T R A N COLI RIBOSOMES

SULFATE AND E S C H E R I C H I A

F. MIYAZAWA*, O. R. O L I J N Y K , C. J. T I L L E Y AND T. TAMAOKI

The University of Alberta Cancer Research Unit (McEachern Laboratory) and Department of Biochemistry, Edmonton, Alberta (Canada) (Received January 3oth, 1967)

SUMMARY

Dextran sulfate exhibited dual effects on Escherichia coli ribosomes causing aggregation at low concentrations and dissociation and eventual breakdown at high concentrations. By the use of radioactive dextran sulfate it was found that dextran sulfate, like poly(U) and other synthetic polynucleotides, binds to the 3o-S subunit, but not to the 5o-S subunit, of E. coli ribosomes through a mechanism which is dependent upon the concentration of Mgz+. The addition of dextran sulfate to a ribosome-poly(U) complex resulted in the partial release of bound poly(U) and the formation of ribosomal aggregates. Conversely, dextran sulfate bound to ribosomes could be partially released by the addition of poly(U). From the molar ratios (P/S or S/P) in these experiments, dextran sulfate was estimated as being approximately four times more powerful than poly(U) in releasing ability. Consistent with these observations, it was shown that dextran sulfate inhibited polyphenylalanine synthesis directed by poly(U).

INTRODUCTION

Dextran sulfate, a polyanion with anticoagulant activity, binds to basic proteins 1 and, as a result, exhibits several effects of biological interest ~. For example, dextran sulfate inhibits ribonucleasO -5, and liberates DNA from deoxyribonucleoprotein preparations in a stepwise manner ~. We reported previously that treatment of L cells with dextran sulfate resulted in a rapid lysis of the cells and a complete disappearance of ribosomes 7. Subsequent studies, using isolated ribosomes from mammalian (L cells) and bacterial (Escherichia coli) cells, revealed that the effect of dextran sulfate on ribosomes was complex, causing aggregation or dissociation depending upon the concentration of dextran sulfate 8. Abbreviation: poly(U), polyuridylic acid. * On leave from the National Institute of Hygienic Sciences, Tokyo, Japan.

Biochim. Biophys. Acta, 145 (1967) 96-1o 4

DEXTRAN SULFATE-RIBOSOME INTERACTION

97

The present paper describes further studies on interactions between dextran sulfate and E. coli ribosomes and the consequential effect on protein synthesis in vitro. Evidence is presented that dextran sulfate binds to the 3o-S subunit, but not to the 5o-S subunit, of the ribosome, and that such a binding affects both the messenger RNA-ribosome interaction and protein synthesis in vitro. MATERIALS AND METHODS

Preparation o[ ribosomes. E. coli B was grown in nutrient broth medium supplemented with o.I % glucose. Ribosomes were prepared from cells in exponential growth essentially according to the method of TlSSI~RES et al. 9 using IO mM Tris-HC1 buffer (pH 7.4) containing IO mM MgCI,, 50 mM KC1 and 6 mM fl-mercaptoethanol. For fractionation of 3o-S and 5o-S particles, ribosomes were dissociated by dialysis against I mM Mg*+-5o mM KC1-6 mM fi-mercaptoethanol-Io mM Tris-HC1 buffer (pH 7-4)- The sample was centrifuged through a 5-20 % sucrose gradient; fractions containing 3o-S and 5o-S subunits were pooled separately and dialyzed against appropriate buffers. Treatment o/ribosomes with dextran sul/ate. Concentrated solutions of dextran sulfate were added to ribosome preparations to the final concentrations indicated in each experiment. The mixture was kept in ice for 20 min and then analyzed by sedimentation in a sucrose gradient. Sedimentation analysis in sucrose density gradients. A sample (0.2 to 0.3 ml) was placed on a 5-20 % sucrose gradient (4.5 ml) made with the same buffer as that used for the sample, and centrifuged at 2 to 4 ° at 35 ooo rev./min for lO5 min or 2 h in a Spinco SW39 rotor. Absorbance at 260 m# was determined from the top of the gradient by using an Isco flow cell (Instrumentation Specialties Co., Lincoln, Nebr.) attached to a Gilford spectrophotometer, model 2000, as described previously 1°. For determination of radioactivity, fractions of 0.25 ml each were collected after measurement of absorbance. To each fraction IOO/~g of yeast-soluble RNA and trichloroacetic acid (final conch. 5 %) were added. The precipitates were collected on Millipore filters and the radioactivity was measured in a Nuclear Chicago liquidscintillation counter. Incorporation o/amino acid in vitro. The amino acid-incorporating system was essentially the same as that described by NIRENBERG AND MATTHAE111.The reaction mixture contained the following per ml: Tris-HC1 (pH 7.8), IO/~moles; KCI, 50/~moles; MgCI 2, IO /,moles; [liC]phenylalanine (351 mC/mmole), 0.005 /,mole; tyrosine, o.o25~mole; 17 other amino acids, o.o5/~mole each; ATP, I/~mole; GTP, 0.03 /~mole; CTP, o.o3#mole; UTP, o.o3#mole; fi-mercaptoethanol, 6/~moles; phosphoenol pyruvate, 5 #moles; pyruvate kinase, 20/~g; poly(U), I rag; E. coli ribosome, 3 mg; soluble protein, I mg. The reaction mixture was preincubated at 37 ° for 15 min prior to the addition of [laClphenylalanine. After incubation for 9 ° min at 37 ° the reaction was terminated by the addition of HC10 a (final concn. 0.5 M). The precipitates were dissolved in I M NaOH (I h, 37 °) and reprecipitated with 0.5 M HC104. The precipitates were collected on Millipore filters and the radioactivity was counted in a Nuclear Chicago liquid-scintillation counter. Materials. [14C]Phenylalanine was purchased from New England Nuclear Corp., Boston, Mass., poly(U) and [3H]poly(U) (18.Ol mC/mmole of phosphorus) from Biochim. Biophys. Acta, 145 (1967) 9 6 - 1 o 4

98

F, MIYAZAWA t't a[.

Miles Chemical Co., Elkhart, Ind., amino acids from Nutritional Biochemicals Corp., Cleveland, Ohio, nucleoside triphosphates, phosphoenolpyruvate and pyruvate kinase from Sigma Chemical Co., St. Louis, Mo. Dextran sulfate (sodium dextran sulfate 500, prepared from dextran with tool. wt. 5' lO5, 2.2 sulfate groups per glucose unit) is a product of Pharmaeia, Uppsala, Sweden. IaHlDextran sulfate (7 mC/mlnole of sulfur) was prepared from the above product by a catalytic exchange procedure by New England Nuclear Corp., Boston, Mass. RESULTS

Aggregation and degradation o/ E. coli ribosomes in the presence o/ dextran sul[ate Sedimentation patterns of E. coli ribosomes in the presence of 2 mM Mg ~+ and varying amounts of dextran sulfate are shown in Fig. I. Two main features are noted. First, at low concentrations of dextran sulfate, there was a considerable decrease in the 7o-S fraction due to aggregate formation (Fig. ib and c). As will be shown later (Fig. 3) the 3o-S particles also formed aggregates although this effect

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Biochim. Biophys. Mcta, 145 (1967) 9 6 - 1 o 4

99

DEXTRAN SULFATE--RIBOSOME INTERACTION

is not well-defined in Fig. I. These results suggest that dextran sulfate interacts with 3o-S and 7o-S particles. Further evidence will be presented below. Secondly, with increased amounts of dextran sulfate, 7o-S particles dissociated into the 3o-S and 5o-S subunits (Fig. Id). Further increase in the dextran sulfate concentration resulted in the formation of smaller size particles (Fig. Ie and f), probably due to the removal of some of the component proteins from the ribonucleoprotein particles TM. An increase in the Mg*+concn. in the buffer up to IO mM did not alter the basic pattern described above, although correspondingly higher amounts of dextran sulfate were required to obtain similar effects. The addition of KC1 also interfered with the action of dextran sulfate. In the experiments described below dextran sulfate at less than ioo #g/ml and buffers containing IO mM Mg ~+ and 50 mM KC1 were employed. Under these conditions essentially no dissociation or degradation of ribosomes occurred. 705

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Binding o/ dextran sul/ate to ribosomes When E. coli ribosomes in IO mM Mg~+-5o mM K C I - I o mM Tris-HC1 buffer (pH 7.4) were mixed with radioactive dextran sulfate and analyzed b y zone sedimentation, a pattern such as shown in Fig. 2 was obtained. Radioactive peaks corresponded with the 3o-S and 7o-S fractions, indicating the binding of dextran sulfate to these particles. There was considerably less radioactivity associated with the 5o-S fraction. This selective binding of dextran sulfate is further shown in Fig. 3, in which interactions with individual 3o-S and 5o-S components were examined at two Mg 2+ concentrations. It is seen in Fig. 3 that the binding of dextran sulfate to 3o-S particles occurred at IO mM Mg~+ (Fig. 3a'), but not at o.I mM Mg2÷ (Fig. 3a), indicating that such an interaction depends upon the concentration of Mg2+. In a separate experiment, the minimum requirement of Mg ~+ was established as 0.5 mM. It is also clear from Fig. 3a' that treatment of 3o-S particles with dextran sulfate caused extensive formation of aggregates of these particles. It was found that heparin, another sulfated polysaccharide, also interacted with the 3o-S and 7o-S particles, but not with the 5o-S particles (Fig. 4). It has been reported that poly(U) and some other synthetic polynucleotides Biochim. Biophys. Acta, 145 (1967) 9 6 - 1 o 4

F. MIYAZAWA et al.

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Fig. 3- S e d i m e n t a t i o n analysis of m i x t u r e s of ribosomal s u b u n i t s and radioactive d e x t r a n sulfate a t t w o c o n c e n t r a t i o n s of Mg 2+. (a) A m i x t u r e of 3o-S particles and [3H]dextran sulfate (7 m C / m m o l e of sulfur, final concH. 5 #g/ml) at o.I mM Mg2+; (a') s a m e as (a) except t h a t the Mg*+ concn, w a s io mM; (b) a m i x t u r e of 5o-S particles and ESH]dextran sulfate at o.I mM Mg2+; (b') s a m e as (b) except t h a t the Mg z+ concn, was io raM. S e d i m e n t a t i o n from left to right. - A260 m# in t h e presence of d e x t r a n sulfate; . . . . . , A260 m# in the absence of d e x t r a n sulfatel • - - - • , trichloroacetic acid-precipitable radioactivity. Fig. 4. Binding of [36S~heparin to E. coli ribosomes. E. coli ribosomes, which were once dissociated into 3o-S and 5o-S s u b u n i t s b y dialysis a g a i n s t I mM Mg*+-5o mM KC1-6 mM fl-mercaptoe t h a n o l - i o mM Tris-HC1 buffer (pH 7.4) were dialyzed a g a i n s t the same buffer containing 5 mM Mg*+ to partially r e f o r m 7o-S ribosomes. This p r e p a r a t i o n was mixed w i t h [3bSJheparin (o.8 pC[ mg, Calbiochem, Los Angeles, Calif.) at a final concn, of 1.5 mg/ml, k e p t in ice for 20 rain and t h e n analyzed b y s e d i m e n t a t i o n in a sucrose g r a d i e n t as described in MATERIALS AND METHODS. R a d i o a c t i v i t y of each gradient fraction was a s s a y e d b y collecting r i b o s o m e s on Millipore filters according to NIRENBERG AND LEDER 21. S e d i m e n t a t i o n f r o m left to right. - - - - , A260mtx ; • - - - 0 , radioactivity.

specifically attach to the 3o-S subunit 13. This attachment does not require an external supply of energy 14, but is dependent upon the presence of Mg ~+ (see refs. 13 and 15). The formation of aggregates of 3o-S and 7o-S particles in the presence of poly(U) has also been observed~a,14,16-~s. Thus, there is a similarity between sulfated polysaccharides and polyrmcleotides in their manner of binding to ribosomes.

Release o/ribosome-bound poly (U) by dextran sul/ate and vice versa Since both dextran sulfate and poly(U) have a high affinity for the 3o-S subunit, the possibility was examined that the binding of one compound affects that of the other. In the first experiment, ribosomes were mixed with radioactive poly (U), and to this mixture varying amounts of non-radioactive dextran sulfate were added. As shown in Fig. 5, the amount of radioactive poly(U) that was associated with 7o-S particles decreased with the addition of dextran sulfate. At the same time, there was an increase in radioactivity near the top of the gradient which accounted for the Biochim. Biophys. Acta, 145 (1967) 96-1o 4

DEXTRAN SULFATE--RIBOSOME INTERACTION (a) Control 50s

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Fig. 5- R e l e a s e of [3H]poly(U) f r o m p o l y ( U ) - r i b o s o m e c o m p l e x b y t h e a d d i t i o n of d e x t r a n sulfate. A n E. coli r i b o s o m a l p r e p a r a t i o n in io m M Mgi+-5o m M KC1-6 m M f l - m e r c a p t o e t h a n o l io m M Tris-HC1 b u f f e r (pH 7.4) w h i c h h a d b e e n once d i s s o c i a t e d in low M g 2+ (i mM) buffer, w a s m i x e d w i t h [~H]poly(U) a n d divided into 4 e q u a l portions. O n e (a) s e r v e d as control. T h e o t h e r t h r e e were t r e a t e d w i t h d e x t r a n s u l f a t e a t (b) 20/zg/ml, (c) 4 ° # g / m l , a n d (d) i o o # g / m l , ior 2o m i n a t o ° a n d t h e n a n a l y z e d b y s e d i m e n t a t i o n in sucrose g r a d i e n t s . S e d i m e n t a t i o n f r o m left to right. , A260 my; • - - - • , trichloroacetic acid-precipitable r a d i o a c t i v i t y .

major part of that lost from the 7o-S fraction. This indicates that the bound poly(U) was released from 7o-S particles upon addition of dextran sulfate. (Note also the decrease in the specific activity of the 7o-S fraction in Fig. 7-) It was observed, however, that dextran sulfate did not release all the poly(U) bound to ribosomes. In the presence of excess amounts of dextran sulfate, approx. 35 % of the original poly(U) still remained attached (Fig. 5; see also Fig. 7). It is also seen in Fig. 5 that, concomitant with the release of bound poly(U), there was an extensive formation of aggregates of 3o-S and 7o-S particles. This resulted in pellet formation during sedimentation analysis of a treated sample and a decrease in the ultraviolet-absorbing material which was revealed in the sucrose gradient (25 % decrease in ioo/~g/ml dextran sulfate). Conversely, in the second experiment, non-radioactive poly(U) was added to a sample containing complexes of ribosomes and radioactive dextran sulfate. This resulted in the release of the latter from the complex and the formation of ribosomal aggregates, similar to that described above (Fig. 6). Also, as in the first experiment, such release was not complete, but ceased upon reaching approx. 4 ° % of the initial amount (Fig. 6; see also Fig. 7).

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Effluent volume (ml) Fig. 6. R e l e a s e of [ S H ] d e x t r a n s u l f a t e f r o m d e x t r a n s u l f a t e - r i b o s o m e c o m p l e x b y t h e a d d i t i o n of p o l y ( U ) . A n E. coli r i b o s o m a l p r e p a r a t i o n in io m M Mg2+-5o m M KC1-6 m M f l - m e r c a p t o e t h a n o l - i o m M Tris-HC1 b u f f e r (pH 7.4) w a s m i x e d w i t h [3H~dextran s u l f a t e (final 3/~g/ml), k e p t in ice for 20 m i n a n d t h e n d i v i d e d into 4 e q u a l portions. O n e (a) s e r v e d as control. T h e o t h e r 3 were t r e a t e d w i t h p o l y ( U ) a t (b) 3o/zg/ml, (c) IOO/~g/ml, a n d (d) 4oo/zg/ml, for 20 rain a t o ° a n d t h e n a n a l y z e d b y s e d i m e n t a t i o n in sucrose g r a d i e n t s . S e d i m e n t a t i o n f r o m left to right. , A260 my; • - - - 0 , trichloroacetic acid-precipitable r a d i o a c t i v i t y . Biochim. Biophys. Acta, 145 (1967) 9 6 - 1 o 4

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In order to determine the relative amounts of poly(U) and dextran sulfate which compete with each other, molar ratios of phosphorus/sulfur (in the case of the ribosome-dextran sulfate complex vs. poly(U)) or sulfur/phosphorus (in the case of the ribosome-poly(U) complex vs. dextran sulfate) were calculated. The decrease in specific activity of the 7o-S fraction as a function of these molar ratios is shown in Fig. 7. It can be seen that to achieve the m a x i m u m release of bound poly(U), approx. 5 molar excess dextran sulfate-sulfur was required, whereas to release the m a x i m u m a m o u n t of bound dextran sulfate, approx. 20 molar excess poly(U)-phosphorus was needed. Thus it m a y be said that in the competition between bound poly(U) and dextran sulfate and vice versa, dextran sulfate has 4-fold releasing ability relative to poly(U). TABLE I EFFECT OF I)EXTRAN SULFATE ON PHENYLALANINE INCORPORATION DIRECTED BY POLY(U) The m e t h o d for [14C]phenylalanine i n c o r p o r a t i o n is described in MATERIALS AND METHODS. D e x t r a n sulfate w a s added to the reaction m i x t u r e prior to the addition of poly(U). The total v o l u m e of each reaction m i x t u r e was 0.25 ml, of which a o . i - m l p o r t i o n was used for d e t e r m i n a lion of [14C]polyphenylalanine formed.

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[14C]Phenylalanine incorporated Counts/rain per o.z ml Inhibition

(%)

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Inhibition o/ poly(U)-directcd phenylalanine incorporation by the addition o~ dexlran sul/ate The effect of dextran sulfate on protein synthesis was studied using the poly (U)directed phenylalanine-incorporating system al. Such work m a y reveal whether or not Biochim. Biophys. Acta, 145 (1967) 96-1o 4

:DEXTRAN

SULFATE--RIBOSOME

10 3

INTERACTION

the h e a v y ribosomal aggregates, which are formed in the presence of dextran sulfate and contain 40 % of the initially bound poly(U) (Fig. 5), are functional. It was found t h a t the addition of dextran sulfate at 20, 40 and 60 #g/ml inhibited phenylalanine incorporation b y 20, 45 and 92 °/o, respectively (Table I). Sedimentation analysis of ribosomes in the reaction mixture after phenylalanine incorporation revealed that the formation of aggregates similar to that shown in Fig. 5~took place under these conditions. I t is interesting to note that, at high concentrations ( > I mg/ml), poly(U) inhibited phenylalanine incorporation and that this inhibition was accompanied b y the aggregate formation similar to that caused b y dextran sulfate. Thus, it appears likely that those ribosomal aggregates, formed b y the addition of poly(U) or dextran sulfate, were non-functional in polypeptide synthesis.

DISCUSSION

''

The data presented in this paper provide evidence that a non-specific polyanion, dextran sulfate, can interact with E. coli ribosomes in a manner which, in certain respects, mimics the binding of poly(U). This includes the specificity of such an interaction to the 3o-S subunit 1~, the dependency on the concentration of Mg *+ (see refs. 13 and 15) and the formation of ribosomal aggregates13,14, ~-ls. Preliminary experiments using heparin (Fig. 4), polyvinyl sulfate and L-cell ribosomes indicated t h a t these characteristics m a y be extended to a number of natural and synthetic polyanions and to ribosomes from various sources. These observations suggest that anionic groups (sulfate or phosphate group) of polymers play an important role in establishing association with ribosomes. This is consistent with the mechanism for the messenger RNA-ribosome interaction proposed by MOORE19 that the sugar-phosphate backbone, and not the bases,of messenger RNA is involved in its binding to ribosomes. The present data showed that the addition of dextran sulfate to a ribosomepoly(U) complex could release the bound poly(U) and, conversely, the bound dextran sulfate could be released b y the addition of poly(U). In both cases, however, the release was not complete, but approx. 4 ° °/o of material remained bound. A similar observation has been reported by NAORA AND NAORA~° in a study of a complex of DNA and calf-thymus ribosomes. They have observed that the addition of poly(U) releases the bound DNA from the complex but that 37 % of the bound DNA is never expelled. They are of the opinion that a binding site exists oil a ribosome which is specific for both DNA and messenger RNA but has differential affinities for these macromolecules. In the present experiments, it cannot be decided whether or not dextran sulfate binds at the same site as poly(U). Our observation can be explained either by the direct interaction, or by an indirect interaction, such as electrostatic repulsion or steric effect, between the polyanions bound at two different ribosomal sites. Approaching this problem b y a competition experiment in which phenylalanine incorporation was measured as a function of the relative amount of poly(U) and dextran sulfate was not satisfactory. It was found in such an experiment that the addition of low concentrations of dextran sulfate ( < 20/zg/ml) caused marked enhancement of phenylalanine incorporation. This suggests that dextran sulfate has an effect other than the interference with poly(U)binding and thus such a system is not suitable for the purpose intended. Biochim. Biophys. Acta,

145

(1967)

96-1o 4

104

F. MIYAZAWA C[ aI.

ACKNOWLEDGEMENTS

W e a r e g r a t e f u l t o D r . T. INIHEI for s t i m u l a t i n g d i s c u s s i o n . O u r t h a n k s are also d u e t o Mr. RICHARD F. STORY for e x c e l l e n t t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d b y g r a n t s f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e of C a n a d a a n d M e d i c a l R e s e a r c h C o u n c i l of C a n a d a ( M A - I 9 5 3 ) .

REFERENCES I E. CHARGAFF AND K. B. OLSON, .f. Biol. Chem., 122 (1938) 153.

2 3 4 5 6 7 8

9 io ii 12 13

14 15 16 17 18

19 20 21

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