A protein factor stimulating binding and translating of polyuridylic acid by Escherichia coli ribosomes

BIOCHIMICA ET BIOPHYSICA ACTA

40I

BBA 96559

A P R O T E I N FACTOR STIMULATING BINDING AND TRANSLATING OF POLYURIDYLIC ACID BY E S C H E R I C H I A C O L I RIBOSOMES* MOSHE SMOLARSKY AND MOSHE TAL

Department o/Chemistry, Technion - Israel Institute o/ Technology, Hai/a (Israel (Received February 9th, 197 o)

SUMMARY

Exposure of Escherichia coli MRE-6oo ribosomes to lowqonic-strength (I mM) Tris-acetate causes release of about IO ~/o of their proteins. Removal of this fraction results in a decrease in binding of poly U and in incorporation of phenylalanine by the ribosomes. Adding this fraction to the depleted ribosomes restores both activities. DEAE-cellulose chromatography reveals that this fraction contains a few acidic proteins. Only the one, which elutes at o.175 M NaC1 enhances binding and translation of poly U by the ribosomes. It also has the ability to bind poly U in the absence of ribosomes. Use of [~HJleucine-labeled factor shows that it interacts with the depleted 3o-S ribosomal subunit. The binding activity of depleted ribosomes supplemented with the protein factor is higher than the binding activities of either the ribosomes or the factor alone. This increase suggests cooperative effect in the reconstituted system. Polyphenylalanine synthesis shows that the binding of poly U took place at the mRNA site on the supplemented ribosomes.

INTRODUCTION

The first step in protein synthesis is binding of mRNA to ribosomes. Association of natural mRNA 1-6 as well as synthetic polynucleotides7-13 with 7o-S ribosomes and 3o-S subunits, has been detected. Several protein factors which take part in binding mRNA to ribosomes have been described. REVEL AND GROS la,15, REVEL ee al. 16,17, HERZBERG et al. TM, and BRAWERMAN et al. 19 have identified several initiation factors, originally located on native ribosomes and promoting binding of T 4 mRNA to ribosomes. IWASAKI et al.6 have described a protein factor, designated as Factor F 3, which promotes binding of Q/~ R N A to ribosomes in the presence of tRNA, but does not stimulate binding of a synthetic m R N A such as ApUpGp(Uo) ~. Another protein factor (Factor 3) which stimulates binding of RI 7 R N A and of ApUpG(pA)4 0 has been described by B R O W N AND D O T Y 2°. These authors have " T h e paper forms part of the first author's M.Sc. Thesis.

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shown that this factor can interact with these polymers even in the absence of ribosomes, but were unable to decide from their experiments whether their factor is required for translation of ApUpG(pU)a0, since their S-Ioo supernatant fraction contained Factor 3 activity. In this article we describe the isolation of a new protein factor which stinlulates binding of poly U to the ribosomal particles and has the ability to bind poly U in the absence of ribosomes. This factor interacts with the 3o-S subunit and enhances binding of poly U to this particle. It can also stimulate poly U-dependent binding of phenylalanyl-tRNA~1and enhances the synthesis of polyphenylalanine directed by poly U. MATERIALS AND METHODS

Bu]/ers "Standard buffer": Tris-acetate (pH 7.4), o.oi M; KC1, 0.05 M; magnesium acetate, o.oi M; fl-mercaptoethanol, 0.006 M. "Dissociation buffer": Tris-acetate (pH 7-4), o.oi M; potassium acetate, 0.05 M; magnesium acetate, o.oooi M. "Tris buffer": Tris HC1 (pH 7.4), o.ooi M. "Binding buffer": Tris-HC1 (pH 7.6), o.oi M; KC1, 0.05 M; MgC12, 0.02 M.

Bacteria and culture conditions E. coli MRE-6oo (ribonuelease I - (ref. 22)) were grown at 37 ° in Tris salt-casanfino medium which contained in I h NaC1, 5 g; KC1, 2 g; NH4C1, I g; MgCI2.6 H20, o.2g; CAC12.2H20, o.oi47 g; MgSO 4, 0.02 g; NaH2PO 4, 0.032 g; gelatine, O.Ol g; casamino acid (vitamin-free), 5 g; glucose, IO g, and Tris, 1.21 g. The pFI was adjusted with HC1 to 7.2. The bacteria were grown up to the middle of the logarithmic phase, harvested, washed once in standard buffer and frozen at --18 ° until use. To prepare ribosomes labeled in [3H~leucine, the bacteria were grown in 5 1 of medium according to DAVIS AND MINGIOL123containing 0. 5 mC of L-IaHlleucine, at 37 ° up to the mid-log phase. The cells were harvested, washed once with standard buffer and frozen at --18 ° until use.

Preparation o/ribosomes Ribosomes were prepared according to the method of TISSII~RES AND WATSON 24 with some modifications. The cells were ground with two weights of alumina and extracted in 4 vol. of Standard buffer. The alumina and cell debris were removed by centrifugation at 15 ooo rev./min in a Servall centrifuge for I h. Ribosomes were sedimented at 15o ooo ×g in a Spinco preparative ultracentrifuge, Model L, for I h. The supernatant (S-I5 o) was frozen in liquid air and stored at --18 ° until use. The ribosomal pellet was suspended in Standard buffer by careful homogenization and the solution was subjected to low-speed centrifugation at 15 ooo rev./min for IO min. The ribosomes were then sedimented at 15o ooo ×g for I h and washed once more under high and low centrifugation in Standard buffer. Ribosomes at this stage were named "ribosomes from Standard buffer".

Isolation o] depleted ribosomes and stimulating ]raction The stimulation factor was stripped from ribosomes under conditions of low ionic strength 2~. A solution of ribosomes from Standard buffer was dialyzed against Biochim. Biophys. Acta, 213 (197 o) 4Ol-416

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

403

ioo vol. of Tris buffer. The solution was changed twice, in 2-h intervals and dialysis was continued overnight, after which the solution was changed once more and dialysis continued for 2 h. Ribosomes at this stage were named "ribosomes from Tris buffer". It should be mentioned here that if the dialysis is prolonged too much the 5o-S subunit is irreversibly converted to a 4o-S particle. The ribosomes were sedimented at 15 ° ooo ×g for 7 h. The upper four-fifths of the supernatant fluid were sucked carefully from the ribosomal pellet. This was named "supernatant fraction", and, as will be shown later, contains a protein component which supports binding of poly U to ribosomes.The supernatant fraction was divided into small portions, frozen in liquid air and kept at --18 °. It was found that this fraction can be stored under these conditions for several months without a significant loss of activity. The ribosomes and the remaining one-fifth of the supernatant fraction were dissolved by careful homogenization in Tris buffer. These ribosomes were named "depleted" ribosomes. All ribosomal solutions were stored in small volumes in sealed ampoules in liquid air and used only once.

Protein concentration Protein was determined by the Folin-Ciocalteu method as modified by LowRY

et al. 26, using a bovine serum albumin Fraction V standard. Calculation o/equivalent amounts o/ the supernatant /raction or o/its protein components "Equivalent volume" is defined as the volume of a supernatant fraction solution which contained, before sedimentation, I A2~0 nm unit of 7o-S ribosomes, 0.33 A260 nm unit of 3o-S, or 0.66 unit of 5o-S subunits. The equivalent volume was calculated by dividing the volume of the original solution of ribosomes from Tris buffer (depleted ribosomes plus supernatant fraction) by the amount of ribosomes present before the sedimentation. The equivalent amount of the protein components isolated from the supernatant fraction was defined according to their proportional amount in the elution profile obtained by DEAE-cellulose chromatography. Accordingly, one equivalent of each component is its amount present in one equivalent volume of supernatant fraction and is added to I A~ 0 nm unit of 7o-S ribosomes, to 0.33 A26o nm unit of 3o-S, or to 0.66 A260nm unit of 5o-S subunits.

Binding o] poly U to ribosomes and to the stimulation ]actor Ribosomes and/or the stimulation factor were mixed with poly U in I ml Binding buffer, in the amounts indicated in the legend to figures and tables, at o ° and incubated at this temperature for 3 min. To the reaction mixture 2.5 ml of Binding buffer were added and the solution was filtered through a millipore filter pretreated with alkali: exposure to 0.5 M KOH at 26 ° for 45 rain, followed by immediate rinsing with water and immersion in o.I M Tris (pH 7.4) for at least 30 rain 27. In some cases binding of poly U to ribosomes was measured according to the method of MOORE11. The results obtained by both methods were essentially the same.

Glycerol gradients To prepare ribosomal subunits, or determine binding of a radioactive material to ribosomes, 5-20 °/o (v/v) glycerol gradients were used 2s. Centrifugation was ear-

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M. SMOLARSKY, M. TAL

ried out at 4 ° in SW-25.I rotor in a preparative ultracentrifuge (Spinco Model L), for 14 h at 18 ooo rev./min in the presence of 7o-S ribosomes, and at 22 ooo rev./min for the same period when only 5o-S and 3o-S subunits were present. For analysis, fractions were collected from the bottom of the tube through a Gilford Model 2000 spectrophotometer, and absorbanee at 260 nm was recorded automatically. For radioactivity measurements, fraction of 0.6 ml were collected directly into vials containing 15 ml of solution according to KINARDeg. In this work glycerol was used instead of sucrose, as it was found that unlike the latter, it is soluble in the solution according to KINARDz9 and does not cause any significant quenching.

Polynucleotide phosphorylase The enzyme was prepared from Micrococcus lysodeikticus according to STEINER AND BEERS3° and purified by (NH4)2SO 4 fractionation.

Preparation o[ poly U Poly U was prepared according to STEINER AND BEERS3° with some modifications. The reaction mixture contained in 5 ml: Tris-HC1, 0.025 M (pH 9.0); KC1, 0.22 M; MgC12, 0.0o25 M; UDP, 3 o m g (Sigma); poly U, o.IoA2,onm/ml as primer, and polynucleotide phosphorylase. The reaction was carried out in a volume of 5 ml in an Ostwald-Fenske No. IOO viscometer at 37 °. When the viscosity reached its maximal value, the reaction was stopped and the poly U isolated ~°. To prepare E3HIuridine-labeled poly U, the reaction mixture contained also o.15 mC of E3HIUDP, 2.35 C/mole (Schwartz). The specific activity of the poly U was 65 500 counts/rain per A26onm unit, and according to this procedure had a high molecular weight, determined by its exclusion by Sephadex G-2oo.

Incorporation o/ [3Hlphenvlalanine Incorporation of [3H]phenylalanine was carried out according to NIRENBERG with some modifications. The reaction mixture contained in I ml: KC1, 0.05 M; NH4C1, 0.02 M; MgC12, o.o18 M; Tris-acetate (pH 7.8), o.I M; fl-mercaptoethanol, 0.002 M; ATP, o.ooi M; GTP, o.oooi M; total E. coli tRNA, 4.6 "/1260 nm units; phosphocreatine, 2 mg (Sigma); creatinephosphokinase, 0.5 mg (Sigma); poly U, 2 A 26onm units; 6/zC E3H]phenylalanine, 750 mC/mmole (Radiochemical Center); S-I5O supernatant 1.5 nag protein; and ribosomes as indicated in the legend to Table I I and to Fig. 6. The reaction mixture was incubated at 37 ° for 20 rain, and polymerization was then terminated by addition of an equal volume of a cold IO % trichloroacetic acid solution containing 0.2 % D,L-phenylalanine. The washing procedure was similar to the method of SIEKEVITZ32. The tubes were then transferred to a boiling water bath for 3 ° rain, and after cooling in ice centrifuged at IO ooo rev./min for IO rain. The pellet was dissolved in I ml 0.2 M K O H by shaking the tube in a Vortex. The protein was reprecipitated by addition of I ml cold IO % trichloroacetic acid solution containing 0.2 % D,L-phenylalanine. After 5 rain in an ice bath, the precipitant was centrifuged at lO ooo rev./min for IO min. This step was repeated 4 times and the sediment was washed once with 95 % ethanol, suspended in an ethanolethyl ether (3 : i, v/v) solution and incubated at 5 °° for 20 rain. The suspension was centrifuged at IO ooo rev./min for 20 min, the pellet was washed once with ethyl ether, dried and dissolved in o.I ml of dry formic acid. I ml of absolute ethanol was added AND MATTHAE131,

Biochim. Biophys. Acta, 213 (197o) 4Ol-416

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

405

and tile solution was transferred to IO ml of scintillation liquid containing 8 g 2,5-diphenyloxazole (PPO) and IOO mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene (dimethyl-POPOP) per 1 toluene. Radioactivity was measured in a Packard Tri-Carb scintillation counter.

Preparation o~ t R N A Total E. colt tRNA was prepared from E. colt MRE-6oo according to the method described by ZUBAY33. The tRNA solution was frozen in liquid air and stored at --18 °. Sedimentation coeHicients Sedimentation coefficients were determined on a Beckman Model E analytical ultracentrifuge equipped with electronic speed control and a photoelectric scanner for ultraviolet optics.

RESULTS

Chemical and physical changes detected in ribosomes exposed to low ionic solution In a previous work 25 it was found that ribosomes exposed to low-ionic-strength solution (Tris buffer) release about I0 % of their proteins. The sedimentation profile of such depleted ribosomes is shown in Fig. Ia. It should be noted that the sedimentation constants of these ribosomes reach normal S values on raising the ionic strength 34. In addition, it should be emphasized that these ribosomes were derived from 70-S particles prepared as described under MATERIALSAND METHODS, and which (as is seen in Fig. Ib) do not contain "native" subunits 35.

Fig. I. Sedimentation profiles of ribosomes. Ribosomes in concentration of i ~42e0 nm/ml from Tris buffer (a) or S t a n d a r d buffer (b). The profiles were recorded on Spinco analytical ultracentrifuge Model ]E e q u i p p e d w i t h scanner and m o n o c h r o m a t o r . Each recording includes the absorbance curve at 260 n m a n d a derivative curve. The n u m b e r s represent sedimentation constants given as s20,w (S).

I3iochim. Biophys. ~Jcta, 213 (197o) 4Ol-416

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M. SMOLARSKY, M. TAL

E//ect o/protein ]raction released/rom ribosomes by low ionic strength on their biological activity Since the supernatant fraction derived from 7o-S ribosomes submitted to extensive dialysis against Tris buffer might contain some of the functional ribosomal proteins, it was interesting to test whether this fraction had any activity in the process of protein synthesis. In such an investigation, several conditions must first be satisfied. It has to be checked whether exposure of ribosomes to Tris buffer results in irreversible changes or in inactivation. Secondly, it is essential that under appropriate conditions it will be possible to reassociate the supernatant fraction with the depleted ribosomes and obtain active reconstituted ribosomal particles. As is shown in Table I, exposure of ribosomes to dialysis against Tris buffer does not impair their ability to bind poly U. It can also be seen that ribosomes from Tris buffer have even higher activity in poly U binding, probably because they do not contain endogenic mRNA. TABLE I EFFECT OF SUPERNATANT FRACTION ON BINDING OF POLY U TO RIBOSOMES The b i n d i n g r e a c t i o n was carried out as d e s c r i b e d u n d e r ~,IATERIALS AND METHODS. The r e a c t i o n m i x t u r e c o n t a i n e d in i ml: i A260nm u n i t of ribosomes, a n e q u i v a l e n t v o l u m e of s u p e r n a t a n t f r actio n w h i c h c o n t a i n e d i o . S / , g p r o t e i n a n d 0.28 A,e 0 nm u n i t of ~H-labeled p o l y U (i 8 24o c o u n t s / r a i n ) . B i n d i n g w a s m e a s u r e d b y f i l t r a t i o n t h r o u g h a n a l k a l i - t r e a t e d filters as de~:cribed u n d e r MATERIALS AND METHODS. The control v a l u e w i t h o u t r i b o s o m e s a n d s u p e r n a t a n t f r a c t i o n w a s 7 ° c o u n t s / r a i n . The g i v e n v a l u e s are after s u b t r a c t i o n of t h e control.

Reaction system

Amount o/ 3H-labeled poly U bound (counts~rain)

R i b o s o m e s from S t a n d a r d buffer R i b o s o m e s from Tris buffer D e p l e t e d ribosonles Supernatant fraction Depleted ribosonles+ supernatant fraction

2968 3274 1825 222 2990

However, removal of the supernatant fraction from the ribosomes causes a decrease of 44 ~o in their ability to bind poly U. Further, when this fraction is added to the depleted ribosomes, their activity in binding poly U is completely recovered. It can also be seen from Table I that the supernatant fraction itself has the ability, to some extent, to bind to poly U in the absence of ribosomes. In addition, it can be seen that the binding activity of depleted ribosomes supplemented with the factor is higher than those of either these ribosomes or the factor alone. This increase suggests that a cooperative effect exists in the reconstituted system. The effect of the supernatant fraction on the ability of ribosomes to incorporate phenylalanine into polyphenylalanine is shown in Table II. It is obvious that exposure to dialysis against Tris buffer does not cause any decrease in their incorporative activity. Removal of the supernatant fraction results in a 5-fold decrease in the amount of phenylalanine incorporated. Readdition of this fraction to the depleted ribosomes restores their activity in polyphenylalanine synthesis. The supernatant fraction, when added to depleted ribosomes, increases the poly U-dependent binding of phenylalanyl-tRNA, as described by PEREK AND TAL21. The maximal enhancement Biochim. Biophys. Acta, 213 (197 o) 4Ol-416

407

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U TABLE If EFFECT

OF S U P E R N A T A N T

F R A C T I O N ON A C T I V I T Y O F R I B O S O M E S I N P O L Y P H E N Y L A L A N I N E

SYNTHE-

SIS

The reaction m i x t u r e contained 20 A =s0 nm units ribosomes a n d / o r 20 equivalent volumes of supern a t a n t fraction t h a t contained 225/,g protein. I n c o r p o r a t i o n was carried out as described u n d e r MATERIALS AND METHODS.

Reaction system

,qmount o/ [aH]phenylalanine

incorporated (counts/rain) Ribosomes from s t a n d a r d buffer Ribosomes from Tris buffer Depleted ribosomes S u p e r n a t a n t fraction Depleted r i b o s o m e s + s u p e r n a t a n t fraction

51 293 53 472 9 996 378 58 163

is obtained when depleted ribosomes are supplemented with one volume equivalent of supernatant fraction.

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Fig. 2. DEAE-cellulose c h r o m a t o g r a p h y of s u p e r n a t a n t fraction. DEAE-cellulose was treated successively with 0.02 M K O H , 0.02 M HC1 and later with distilled w a t e r while r e m o v i n g the small particles b y decantation. Using this ion exchanger, a column of DEAF--cellulose 1. 5 cm × 47 cm was prepared. IOO ml of 3 M NaC1 solution were passed t h r o u g h the column, which was washed afterwards with a large volume of Tris buffer, until the conductivity of the eluted liquid reached t h a t of the buffer. The column was loaded with 15 ml of the supernatallt fraction containing 24 mg protein. C h r o m a t o g r a p h y was performed in steps, using ioo-ml solution of o, o.165, 0.20, 0.25 and 0.300 M NaC1 in Tris buffer. The salt concentration was changed gradually between steps b y allowing the n e x t solution to drop into 15 ml of the previous solution, above the ion exchanger. The remaining material was eluted from the column b y a gradient of salt concentration o.3-1 M NaC1 in Tris buffer. Fractions of 5 ml were collected t h r o u g h Uvicord s p e c t r o p h o t o m e t e r (LKB, Type 47Ol A) and t r a n s m i t t a n c e at 253 u m was recorded automatically. 0.2 ml from each fraction was mixed with 0.28 A 2 6 0 n m unit of 3H-labeled poly U (18 240 counts/min) in i ml of Binding buffer. Binding was measured b y filtration t h r o u g h an alkali-treated filter as described under MATERIALS AND METHODS.The salt concentration in the fractions eluted from the DEAE-cellulose c o l u m n was assayed b y transferring samples of 0.2 ml into 20 ml of Tris buffer. I n the resulting solutions, the c o n d u c t i v i t y was measured b y conductivity bridge Model RC 16132, I n d u s t r i a l I n s t r u m e n t s Inc, Cedar Grove, N.J. The salt concentration was determined with reference to a s t a n d a r d curve p r e p a r e d u n d e r the same conditions from different salt solutions in Tris buffer in t h e range of o - i M NaC1.

Biochim. Biophys. Mcta, 213 (197 o) 4Ol-416

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M. SMOLARSKY, M. TAL

Fractionation o/supernatant/raction and e//ect o/ its di//erent protein components on binding activity o/ribosomes Since the supernatant fraction comprises several proteins, it was of interest to establish which protein is responsible for its ability to bind to poly U and to stimulate binding of this polymer to ribosomes. The supernatant fraction was fractionated on a DEAE-cellulose column. The elution profile in Fig. 2 shows that the supernatant fraction was separated into several protein components, designated from A to I in the order of appearance. Fraction A was not adsorbed on the ion exchanger and was eluted when the column was washed with Tris buffer. Fractions A, H, and I did not appear in most cases, probably due to experimental fluctuation in preparing the ribosomes and the supernatant fraction. For this reason, we were concerned in this study with the two major protein components which appeared consistently, C and E. In the fractions eluted from the column, poly U-binding activity was measured by means of alkali-treated millipore filters 27, in the absence of ribosomes. It is obvious from Fig. 2 that only one of the protein components (Factor E) is capable of binding poly U. This factor is eluted from the column concurrently with the front of one of the main protein fractions. It can be seen from the transmittance in ultraviolet light that Factor E probably does not contain RNA. The effect of the protein components eluted from the DEAE-cellulose column on poly U binding by ribosomes is described in Table I I I . In this experiment, equivalent amounts of the protein fractions were added to depleted ribosomes and their ability to bind poly U was measured. It is clear that Factor E alone can stimulate binding of poly U to ribosomes as the complete supernatant fraction. No significant stimulation of binding poly U to ribosomes b y Fraction C could be detected.

TABLE EFFECT

III OF F R A C T I O N S

ELUTED

FROM DEAE-CELLULOSE

C O L U M N ON A B I L I T Y

OF R I B O S O M E S

TO

BIND POLY U

B i n d i n g was m e a s u r e d b y t h e m e t h o d of MOOREn. I - m l s o l u t i o n s of r i b o s o m e s in B i n d i n g buffer c o n t a i n i n g I A260 nm u n i t of t h e ribosomes i n d i c a t e d a n d e q u i v a l e n t a m o u n t s of t h e p r o t e i n fractions, were p a s s e d t h r o u g h a n i t r o c e l l u l o s e m e m b r a n e a t a r a t e of I ml/5 min. The filters w e re t h e n s a t u r a t e d w i t h a l b u m i n b y passing, a t t h e s a m e rate, 2. 5 ml of f r e s h l y - m a d e b o v i n e s e r u m a l b u m i n ( F r a c t i o n V) s o l u t i o n c o n t a i n i n g 2. 5 m g / m l of p r o t e i n in t h e s a m e buffer. B i n d i n g of p o l y U w a s carried o u t b y p a s s i n g i ml of B i n d i n g buffer c o n t a i n i n g o. 3 A 260 nm a l l - l a b e l e d pol y U w i t h specific a c t i v i t y of 16 ooo c o u n t s / m i n per A,80 nm u n i t a t t h e s a m e rate. The filters were w a s h e d t w i c e w i t h p o r t i o n s of 2 m l B i n d i n g buffer, once a t a slow r a t e a n d once a t a r a p i d r a t e . The filters were d r i e d a n d c o u n t e d as d e s c r i b e d u n d e r MATERIALS AND METHODS.

Reaction system

R i b o s o m e s from S t a n d a r d buffer R i b o s o m e s from Tris buffer Depleted ribosomes Depleted ribosomes + w h o l e supernatant fraction Depleted ribosomes+ Fraction C Depleted r i b o s o m e s + F r a c t i o n E

Biochim. Biophys. Aeta, 213 (197 o) 4 O l - 4 1 6

A mount o/ protein [raetion added (,ug)

--

22.8 lO. 9 lO-4

A mount o/ alllabeled poly U bound (counts/rain) 680 785 347 Soo 420 775

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

409

E/]ect o/ Mg 2+ concentration on binding o/poly U to ribosomes and to Factor E Since it is known that Mg2+ stimulates binding of mRNA to ribosomes 2,5,s,36,37, it was of interest to investigate the influence of Mg2+ concentration on binding of poly U to depleted ribosomes, to Factor E and to depleted ribosomes supplemented with Factor E. It is obvious from the results of an experiment described in Fig. 3 6000

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5000 4000 3000 2000

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Mg•+ CONCENTRATION (M)

Fig. 3- Effect of 1VIg~+ concentration on binding of poly U to ribosomes in presence or absence of F a c t o r E a n d to F a c t o r E alone. Reaction m i x t u r e s contained in I ml: T r i s - H C l (pH 7.6), o.oi M; KC1, o . o s M ; MgC1v as indicted; i A2e0n m unit of ribosomes where indicated; an equivalent a m o u n t of F a c t o r E where indicated, containing 2o/~g protein and 0.28 A~80 nm u n i t of 3H-labeled poly U with specific radioactivity of 65 5o0 counts/miD per A260 nm unit. After incubation for 3 mid at o ° 2. 5 ml of the above buffer with the a p p r o p r i a t e Mg 2+ concentration were added and t h e solutions were filtered t h r o u g h alkali-treated m e m b r a n e s 27, p r e w a s h e d w i t h the same buffers, and the filters were t h e n washed 5 times with the appropriate buffer. The m e m b r a n e s w e r e dried and the radioactivity counted as described u n d e r MATERIALS AND M E T H O D S . O - - O , ribosomes; A - A , F a c t o r E; C)- - -Q), combined values of ribosomes and F a c t o r E. The experimental values of the complete s y s t e m (ribosomes plus F a c t o r E) is given b y Q)-Q).

that maximal binding is obtained at 0.05 M Mg2+. The dashed line in this figure represents the sum of the amounts of poly U bound separately to depleted ribosomes and Factor E. Comparing this line with the binding curve of poly U to depleted ribosomes supplemented with Factor E, it can be seen that in the presence of the factor, enhanced binding sets in at concentrations above 0.o0I M Mg~+. Maximal increase of binding of poly U to the supplemented ribosomes was found at 0.02 M Mg2+, hut significant enhancement can also be detected at 0.003 M Mg2+. Since this experiment was carried out by filtration of the reaction mixtures through alkalitreated nitrocellulose membranes 27, it was checked, as a necessary control, whether the adsorption of ribosomes and Factor E alone are dependent on Mgz+ concentration. The results, shown in Fig. 4, indicate that binding of ribosomes and Factor E to millipore filters is dependent on Mg2÷ concentration. The curves shown in Fig. 3 are after the appropriate corrections.

E//ect o] Factor E on binding o/poly U to ribosomal subunits It is known that mRNA binds and directs the binding of aminoacyl-tRNA to the 30-S subunit s'Ds'39. Since Factor E was found to stimulate binding of poly U to ribosomes, it was of interest to investigate whether Factor E associates with ribosomes. To answer this question, a supernatant fraction was prepared from 3H-labeled riboBiochim. Biophys. Acta~ 213 (197 o) 4Ol-416

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Fig. 4. B i n d i n g of r i b o s o m e s a n d F a c t o r E t o n f i l l i p o r e f i l t e r s as f u n c t i o n of MR 2+ c o n c e n t r a t i o n . i A 280 nm u n i t of 3 H - l a b e l e d r i b o s o m e s o r o n e e q u i v a l e n t of a l l - l a b e l e d F a c t o r E c o n t a i n i n g 16 P'g p r o t e i n w e r e d i s s o l v e d in 3 m l of b u f f e r c o n t a i n i n g : T r i s - H C 1 ( p H 7.6), o . o i M; 1,2C1, o . o 5 M; a n d M g C I 2 a s i n d i c a t e d . T h e s o l u t i o n s w e r e f i l t e r e d t h r o u g h a l k a l i - t r e a t e d m e m b r a n e s °-7 a s d e s c r i b e d i n l e g e n d t o F i g . 3.

somes and chromatographed on a DEAE-cellulose column, and the fractions from Factor E region were pooled. In an experiment described in Fig. 5, the association of Factor E with the ribosomal subunits was examined at three different Mg ~+ concentrations. It is obvious t h a t Factor E is associated only with the 3o-S subunit at Mg 2+ concentrations exceeding o.ooi M. Ribosomes were sedimented in the presence of poly U, and the broad

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FRACTIONNO. F i g . 5. B i n d i n g of 3 H - l a b e l e d F a c t o r E t o r i b o s o m a l s u b u n i t s . R i b o s o m a l s u b u n i t s w e r e p r e p a r e d f r o m d e p l e t e d r i b o s o m e s b y c e n t r i f u g a t i o n in g l y c e r o l g r a d i e n t s in T r i s b u f f e r a s d e s c r i b e d u n d e r MATERIALS AND METHODS. T h e g l y c e r o l r e m o v e d b y d i a l y s i s a g a i n s t T r i s b u f f e r a n d t h e r i b o s o m a l s u b u n i t s w e r e s e d i m e n t e d a t 5 ° o o o r e v . / n l i n f o r 7 h. T h e r i b o s o m a l p e l l e t s w e r e d i s s o l v e d in a n l i n i m a l v o l u m e of T r i s b u f f e r a n d a n e q u i v a l e n t a m o u n t of ~ 3 H ~ l e u c i n e - l a b e l e d F a c t o r E a n d poly U were added. Each ribosomal subunit solution was divided into three portions. Salt conc e n t r a t i o n w a s r a i s e d t o : T r i s - H C 1 ( p H 7.6), o . o i M; KC1, o . o 5 M; a n d MgC12 a s i n d i c a t e d . S a m p l e s f r o m t h e s i x r e s u l t i n g s o l u t i o n s c o n t a i n i n g i o ~4260nrn u n i t s of 3 o - S s u b u n i t s o r 2 o //260 nm u n i t s of 5o S s u b u n i t s , 5 A28o nm u n i t s of p o l y U a n d 67. 5 / i g of [ 3 H ] l e u c i n e - l a b e l e d F a c t o r E ( 7 4 o o c o u n t s / r a i n ) w e r e l a i d o n 5 2o 0~, ( v / v ) g l y c e r o l g r a d i e n t s in t h e s a m e b u f f e r s . T h e s a m p l e s w e r e c e n t r i f u g e d f o r 14 h a t 2o o o o r e v . / m i n a t 4 ° in Sx,V 25.1 r o t o r . T h e g r a d i e n t s w e r e a n a l y z e d a s d e s c r i b e d u n d e r MATERIALS AND METHODS. Biochi~.

Biophys.

Acta,

213 (197 o) 4 O l - 4 1 6

411

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

and double peaks obtained with 3o-S subunits m a y be attributed to formation of polysomes and of a dimer of 3o-S subunit known to be formed at high Mg~+ concentrations ~9. In another experiment (not shown here) the Factor E-supplemented ribosomal subunits were sedimented in the absence of poly U, with similar results. It could be concluded that the association of Factor E to ribosomes is not dependent on the presence of poly U. Is the interaction of Factor E with the ribosomes essential for poly U binding? To study this question, depleted ribosomal subunits in Tris buffer were prepared, associated with Factor E in Binding buffer, and sedimented. The supernatant liquid containing the excess Factor E not bound to the ribosomes was discarded. In the experiment described in Table IV, the ability of these ribosomal subunits to bind TABLE

IV

EFFECT OF FACTOR E ON BINDING OF gI-[-LABELED POLY g TO RIBOSOMAL SUBUNITS D e p l e t e d r i b o s o m a l s u b u n i t s in Tris buffer were p r e p a r e d b y m e a n s of gl yc e rol g r a d i e n t s . E q u i v a l e n t a m o u n t s of F a c t o r E were a d d e d a n d t h e s o l u t i o n s were d i a l y z e d a g a i n s t B i n d i n g buffer. The r i b o s o m a l s u b u n i t s were t h e n s e d i m e n t e d a t 5 ° ooo r e v . / m i n for 7 h. The s u p e r n a t a n t l i q u i d w i t h excess F a c t o r E was discarded. The r i b o s o m a l p e l l e t w a s d i s s o l v e d in a m i n i m a l v o l u m e of B i n d i n g buffer. D e p l e t e d r i b o s o m a l s u b u n i t s , s i m i l a r l y t r e a t e d b u t n o t s u p p l e m e n t e d w i t h F a c t o r E, were also p r e p a r e d . To e x a m i n e a c t i v i t y in p o l y U bi ndi ng, r i b o s o m e s in t h e a m o u n t s indic a t e d were m i x e d in I ml B i n d i n g buffer w i t h 0.28 A~e0n m u n i t of a l l - l a b e l e d p o l y U (18 240 c o u n t s / r a i n ) a n d i n c u b a t e d for 3 m i n a t o °. B i n d i n g was m e a s u r e d as de s c ri be d u n d e r MATERIALS AND METHODS.

Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes

3° 5° 3° 3° 5° 3°

S S S+5o S S +Factor E S +Factor E S+5o S+Faetor E

d mount o/ribosomes added (A~s0nm units)

Amount o/~HNet e//ect labeled poly U o] Factor E bound (counts/min) (counts/rain)

0.33 0.66 0.99 0.33 o.66 o.99

720 302 lO73 2997 726 4516

---2277 424 3443

poly U was tested and compared to the binding activities of subunits similarly treated but not supplemented with Factor E. It is clearly seen from the results shown in Table IV that Factor E can be associated with the 3o-S ribosomal subunit and that this interaction stimulates binding of poly U to this ribosomal particle. The low binding activity of the 5o-S subunit and the weak stimulation of poly U binding to this particle m a y be attributed mainly to slight contamination of the 3o-S subunit.

E/]ect o] Factor E on polyphenylalanine synthesis The effect of Factor E on the activity of ribosomes in phenylalanine incorporation was tested in an experiment described in Fig. 6. At low concentrations of Factor E the incorporation depends almost linearly on the amount of factor added. In saturating amounts, Factor E enhances polyphenylalanine synthesis about 2.5-fold. At higher concentrations of the factor (not shown in Fig. 6), a slight decrease in the amount of polyphenylalanine synthesized was detected. This inhibition m a y be due to excess Factor E being bound to poly U and interfering with migration of the riboBiochim. Biophys. Acta, 213 (197 o) 4 O l - 4 1 6

412

M. SMOLARKSY, M. TAL

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Fig. 6. E f f e c t of F a c t o r E on c a p a b i l i t y of r i b o s o m e s to i n c o r p o r a t e [ ~ H ] p h e n y l a l a n i n e . The r e a c t i o n m i x t u r e c o n t a i n e d i o A~60 nm u n i t s of d e p l e t e d r i b o s o m e s a n d F a c t o r E as i n d i c a t e d . I n c o r p o r a t i o n was carried o u t as described u n d e r MATERIALS AND METHODS.

somes on this mRNA. Factor E in high concentrations m a y also compete with ribosomes in binding poly U. Another possible inhibitory effect of an excess of Factor E m a y also be due to nucleolytic activity, traces of which m a y be present in the purified factor.

DISCUSSION

The main difficulty in investigating the mechanism of action of ribosomes lies in their complex structure. One way to overcome this difficulty is to break down the ribosomal particles partly or completely into their components and reassociate them in a systematic manner in order to get active particles. In contrast to the methods commonly used, according to which some ribosomal proteins are dissociated from ribosomes by high salt concentrations, low ionic strength was used here for this purpose. With this method 25, ribosomes release about IO o/ ,,o of their proteins after dialysis against I him Tris-acetate (pH 7.4). It was thus of interest to examine whether these proteins contribute to ribosomal activity. The present results indicate that exposure of ribosomes to dialysis against Tris buffer does not cause any irreversible changes in respect of the ribosomal functions tested. Removal of the supernatant fraction caused a decrease in their ability to bind poly U and phenylalanyl-tRNA 21 and to synthesize polyphenylalanine. Reassociating this fraction with the depleted ribosomes fully restored their activity. By DEAE-cellulose chromatography, one of the protein components, designated Factor E, was isolated and shown to be responsible for binding of poly U. Factor E is adsorbed at neutral p H to DEAE-cellulose and moves by electrophoresis in polyacrylamide gel at pH 8.3 towards the anode (not shown here). These facts indicate that Factor E is negatively charged under the p H conditions in which its activity was measured. Since ribosomes and poly U are also negatively charged, it is clear why a certain Mg 2+ concentration is obligatory for associating Factor E with ribosomes and binding Factor E to poly U. Mg2+, as a divalent cation, links between Factor E and ribosomes on the one hand, and poly U on the other. It cannot, however, be excluded that other factors (such as other divalent cations, hydrogen 13iochim. Biophys. Acta, 213 (197 o) 4 o i - 4 1 6

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

413

bonds or van der Waals forces) m a y contribute to the interaction of ribosomes, Factor E and poly U. What is the mechanism of action of Factor E in poly U binding? It was found that this factor binds poly U in the absence of ribosomes on the one hand, and associates with ribosomes in the absence of poly U on the other. This fact suggests that Factor E might act by binding poly U at one site and ribosomes at another. Binding activity of depleted ribosomes supplemented with the factor is, however, higher than the sum of the separate activities of the ribosomes and the factor. This increase suggests a cooperative effect in the reconstituted system, and it can be assumed that Factor E has a role in the formation of the ribosomal binding site for poly U and in determining its steric structure. The results described here show that the ability of depleted ribosomes supplemented with the supernatant fraction is not lower than the biological activity of ribosomes from standard buffer. This indicates that under the present experimental conditions (ionic strength, Mg2+ concentration and period of incubation at low temperature) Factor E associates spontaneously with ribosomes. Hence it m a y be presumed that no energy source is needed for this reassociation. Factor E associates with the 3o-S subunit and enhances binding of poly U to this particle. This result is in agreement with those obtained b y o t h e r investigators, that m R N A is bound to the 3o-S subunit 4,7,8,4°. Aminoacyl-tRNA can also be bound to this isolated subunit 38,a9,41, and the initiati:m complex is formed on this particle 42-]4. Still another question is whether Factor E is essential for binding poly U to ribosomes, or only supports it. The residual activities of depleted ribosomes in binding poly U and in polyphenylalanine synthesis m a y be due to the procedure of the preparation of the supernatant fraction where one-fifth of its volume was retained by the ribosomal pellet. The incorporation experiment described in Table II shows that the depleted ribosomes have one-fifth activity compared with the supplemented ribosomes. Comparison of the results in Table I and IV shows that sedimentation of ribosomes through a glycerol gradient in Tris buffer enhances their dependence on the factor. The discrepancy between the incorporation experiment (Table II) and the binding experiments (Tables I and IV) regarding the dependence of the activity on the added fraction m a y be explained by the relatively high proportion of ribosomes still active in binding poly U, compared with those active in protein synthesis. F r o m present results, it can be concluded that Factor E strongly stimulates the activity of ribosomes, but not whether it is essential for binding poly U. It was found that Factor E alone stimulates the activity of depleted ribosomes in poly U binding to the same extent as the whole supernatant fraction, so that the other protein components in this fraction probably play no role in binding this polynucleotide to ribosomes. It was, however, observed that the stimulation detected in the incorporation activity of depleted ribosomes on addition of Factor E alone is lower than that of the supernatant fraction. It is not yet clear whether this is due to partial inactivation of Factor E in the course of its purification, or to absence of the other components of the supernatant fraction. Since poly U binding is only the first step in the process of polyphenylalanine synthesis, the other protein components m a y be necessary for the subsequent steps. The effect of the protein components in different combinations on polyphenylalanine synthesis, as well as on the binding reactions, is now under investigation. Biochirn. Biophys. Acta, 213 {i97 o) 4Ol-416

414

M. SMOLARSKY, M. TAL

Several protein factors taking part in protein synthesis have been described in literature. No direct attampt has yet been made to establish whether Factor E is identical with or at least similar to any of these proteins, but literature data to-date indicate differences between Factor E and the others. TRAUB AND NoMURA4s found that in their split protein fraction obtained after the exposure of the 3o-S subunit to high CsC1 concentration, the acidic proteins slightly enhance poly U binding, while not supporting specific mRNA-directed tRNA binding or amino acid incorporation. As Factor E is detached from the ribosomes at low ionic strength and since it stimulates poly U binding as well as phenylalanyl-tRNA binding and polyphenylalanine synthesis directed by poly U, we believe that our own factor is not identical with any of the acidic proteins mentioned above 45. As described by m a n y investigators, there are several protein factors which play a role in binding mRNA to ribosomes -- Factor C (refs. 1 4 - 1 6 , I 8 ) , Factor F 3 (ref. 6), Factor 3 (ref. 2o) and Factor F I I I (ref. 46). (The last-named resembles Factor C in properties and method of preparation.) Factor C is known to promote binding of T~ m R N A to the 3o-S subunit 16. Factor F 3 appears to be specifically required for the binding of natural m R N A to ribosomes. These two factors are classified as initiation factors. It has been shown by many investigators that the initiation factors do not enhance translation i~ vitro of poly U in the absence of an initiator tRNA, such as N-acetylphenylalanyl-tRNAlm7. By contrast, Factor E stimulates polyphenylalanine synthesis in the absence of N-blocked phenylalanyl-tRNA and unlike the initiation factors, its maximal effect is at high Mg2+ concentration (o.o2 M). Literature data also indicate that initiation factors are located on the native 3o-S subunits, not on 7o-S ribosomes 4°,48-5°. By contrast, it was shown that Factor E is isolated from 7o-S ribosomes. Another difference between factor E and the initiation factors is the method of their preparation. Initiation factors are derived from ribosomes by washing with a solution of high salt concentration, while Factor E is obtained from ribosomes by low-ionic-strength treatment. Factor 3 as described by BROWN AND DOTY2° resembles Factor E in its ability to bind natural mRNA (RI7) as well as synthetic polynucleotide (ApUpG(pA)40) to ribosomes and it also binds these polymers in the absence of ribosomes. However, there is still one difference between Factor E and Factor 3 in the translation in vitro of synthetic mRNA. Since our supernatant fraction enhances polyphenylalanine synthesis 5-fold in the presence of S-I5O solution (enhancement which might be explained by method of preparing of this supernatant fraction, see MATERIALSAND METHODS), it is concluded that S-I5O has very low Factor E activity, if at all. By contrast, S-Ioo solution has high Factor 3 activity 2°. So far, it cannot yet be concluded definitely whether Factor E is different from Factor 3It may be mentioned here that BARONDES AND NIRENBERG 12 a n d MOORE 11 found that for poly U binding to ribosomes, no enzymatic factor present in the S-Ioo supernatant fraction is required. BRAWERMANAND EISENSTADT51 also found that an enzymatic factor in the S-Ioo solution, which stimulates translation of f2 RNA, does not enhance poly U translation. Protein factors which stimulate phenylalanine incorporation are the transfer factors reviewed recently 52. Literature data to-date indicate that none of them is involved in mRNA binding, and the fact that Factor E stimulates polyphenylalanine

Biochim. Biophys. Acta, 213 (197o) 4Ol-416

PROTEIN FACTOR FOR BINDING AND TRANSLATING POLY U

415

synthesis in the presence of the crude S-I5 o fraction (known to contain most of their activity) suggests that it is not identical with them. Since Factor E shows stimulatory effect in binding both poly U and phenylalanyl-tRNA 21, and similarly enhances polyphenylalanine synthesis, we believe that it enhances poly U binding to the mRNA site on the ribosomal particle. Factor E shows significant affinity to poly U, reflected in binding the polymer to ribosomes. An attractive hypothesis, suggesting a biological role for the factor possessing this property, may be derived from the latest findings of ARGETSINGERSTEITZ 53 a n d H I N D L E Y AND STAPLES 54 who demonstrate UUUGA sequences before

the initiation codons for coat and A proteins in RI 7, as well as before the initiation triplet in the coat protein of Q/~ phages. These sequences, in addition to secondary structure of their region, may serve as a specific signals for the formation of the initiation complex with the nearby AUG triplet. Interaction of tile UUUGA sequence, with the ribosome is a prerequisite for initiation. Thus a protein factor which has affinity to poly U may contribute to this interaction. Experiments to verify this hypothesis are now in progress. ACKNOWLEDGMENT

The authors wish to thank Dr. M. Revel for critical reading of this manuscript. We also acknowledge with thanks the support of the Gerard Swope Research Fund. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3°

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