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Biochimica et Biophysics Acta, 395 (1975) 146-151 @ Elsevier Scientific Publishing Company, Amsterdam -Printed in The Netherlands

BBA 98320

AFFINITY-LABELLING OF ESCHERZCHZA COLZ RIBOSOMES BY A DERIVATIVE OF PHENYLALANYL-tRNA

A CRITICAL TEST FOR THE SPECIFICITY

K. BAUER*, A.P. CZERNILOFSKY and E. KUECHLER**

Institut fib Biochemie der Uniuersitiit Wien, Vienna (Austria)

(Received December 18th, 1974)

Summary

Under nonenzymatic binding conditions p-nitrophenoxycarbonyl-phenyl- alanyl-tRNA can be bound to the donor as well as the acceptor site on the Escherichia coli ribosome. Precharging ribosomes with phenylalanyl-tRNA in the presence of elongation factor Tu and GTP blocks both the donor- and the acceptor site against affinity labelling by p-nitrophenoxycarbonyl-phenylala- nyl-tRNA.

Introduction

Derivatives of aminoacyl-tRNAs containing reactive substituents have been employed as affinity labels for the Escherichia coli ribosome [l-6] . They can be bound nonenzymatically at high Mg2+ concentration and react with ribosomal proteins and/or RNA under formation of covalent bonds.

It is well established that polyphenylalanine synthesis at high Mg” con- centration is initiated by a mechanism which is different from natural initia- tion. One can therefore not necessarily assume that affinity labelling with p-nitrophenoxycarbonyl-phenylalanyl-tRNA (PNPC-Phe-tRNA) under nonen- zymatic binding conditions occurs at the same site on the ribosome as enzy- matic binding of aminoacyl-tRNA. Binding of p-nitrophenoxycarbonyl-methio- nyl-tRNAy et (PNPC-Met-tRNAy et ) at 5 mM Mg” concentration in the pre-

* Present address: 1. Medizinische Universitiitsklinik, Vienna, Austria. ** To whom reprint requests should be addressed.

Abbreviations: PNPC-Phe-tRNA, p-nitrophenoxycarbonyl-phenylalanyl-tRNA: PNPC-Met-

tRNAyet, p-nitrophenoxycarbonyl-methionyl-tRNA~et (formylatable. methionine specific): poly(U). poly(uridylic acid): EFTu. EFTs and EFG. elongation factors Tu. Ts and G respectively; EFT, mixture of EFTu and EFTs.

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sence of natural mRNA is dependent on initiation factors [7] . It is strongly inhibited by puromycin, indicating that the site of the affinity labelling reac- tion is the ribosomal donor site. However enzymatic binding of aminoacyl- tRNA at the acceptor site by elongation factor EFTu requires the presence of a free a-amino group on the aminoacyl-moiety [8] ; acetylation of the amino group completely abolishes binding by EFTu [8] . Due to the presence of a blocked a-amino group in PNPC-Phe-tRNA, EFTu-dependent binding cannot be expected to occur. We have therefore studied the effect of precharging ribosomes with Phe-tRNA in the presence of EFTu and GTP on the affinity labelling reaction with PNPC-Phe-tRNA. The experiments indicate that these precharged ribosomes are completely blocked against affinity labelling with PNPC-Phe-tRNA. This block can be partially overcome by addition of elonga- tion factor EFG.

Materials and Methods

Ribosomes from E. coli D10 were purified by spinning twice through buffer containing 0.5 M NH4 Cl [ 31. Charging of the tRNA with radioactive phenylalanine and synthesis of PNPC-[’ H] Phe-tRNA was carried out as de- scribed previously [ 3,5] . [ 3 H] Phenylalanine (12.8 Ci/mmol) was obtained from New England Nuclear Corp. [’ 4 C] Phenylalanine (522 Ci/mol) was pur- chased from Radiochemical Centre Amersham; it was diluted to a spec. act. of 104 Ci/mol with cold phenylalanine. EFT (mixture of EFTu and EFTs) and EFG were purified according to Gordon et al. [9]. Binding buffer was 60 mM KCl, 20 mM Hepes pH 7.2, 5 mM magnesium acetate and 0.02 mM dithio- erythritol.

Binding of (’ 4 C] Phe-tRNA Solution A contained in 30 ~1: 65 ,ug (=24 pmol) ribosomes, 5 pug poly(U),

10 pg uncharged tRNA and 7 pg GTP in binding buffer. It was incubated for 10 min at 37°C. Solution B contained in 20 ~1: 0.6 ,ug EFT, 0.3 pg GTP and varying amounts of [’ ’ C] Phe-tRNA (9.4 * lo4 cpm/mg tRNA) as indicated in Fig. 1. It was incubated for 5 min at 37°C. Solutions A and B were then mixed and incubated at 20°C for 20 min. The samples were subsequently diluted with 0.5 ml of cold binding buffer and filtered through Millipore filters [lo]. Filters were counted in a liquid scintillation counter.

Affinity labelling of precharged ribosomes Incubation mixtures for the EFT-dependent binding of Phe-tRNA were

prepared by a modification of the method of Gupta et al. [ll] . Solution A contained in 0.6 ml: 4.0 mg ribosomes, 0.1 mg poly(U), 0.2 mg uncharged tRNA and 0.13 mg GTP in binding buffer. It was incubated for 10 min at 37°C. Solution B contained in 0.4 ml: 1.5 mg Phe-tRNA, 0.012 mg EFT and 0.006 mg GTP in binding buffer. It was incubated for 5 min at 37°C. Solutions A and B were mixed and incubated at 20°C for 20 min. After chilling to 0°C the Mg2+ concentration was raised to 20 mM. 0.37 mg PNPC-[” H] Phe-tRNA (spec. act. 5.6 * 10” cpm/mg tRNA) was added and the mixture incubated for 90 min at 37°C. 50-S subunits were then isolated and the ribosomal proteins

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prepared as described previously [3,5]. In the control experiment with EFG, the incubation following mixing of solutions A and B was shortened to 10 min; 0.05 mg of EFG was subsequently added and the incubation continued for 20 min at 20°C. The sample was then chilled and magnesium acetate was added to raise the concentration to 20 mM. The incubation with PNPC-[” H] Phe-tRNA and all subsequent steps were carried out as described above.

Electrophoresis on polyacrylamide gels 170 pg of protein was used per gel. Electrophoresis on 10% polyacryl-

amide gels was carried out in 8 M urea at pH 4.5 at 2.5 mA per tube for 7 h. Gels were stained with amido black and counted in Toluene-Triton-X100 scin- tillator [3] .

Results

Washed ribosomes were incubated with increasing amounts of [ ’ 4 C] Phe- tRNA in the presence of a constant amount of EFT (mixture of EFTu and EFTs), GTP and uncharged tRNA at 5 mM Mg*+ concentration. Reaction mix- tures were subsequently filtered through Millipore filters and the radioactivity determined. As can be seen in Fig. 1, saturation is obtained when about 75 pmol of Phe-tRNA are used in the incubation mixture. Approx. 2 pmol of Phe-tRNA are bound to 24 pmol of ribosomes at saturation, indicating that only about 8% of the ribosomes are active in this preparation.

In order to investigate the effect of enzymatically bound Phe-tRNA on the affinity labelling reaction a saturating concentration of Phe-tRNA was em- ployed, as indicated by the arrow in .Fig. 1. Ribosomes were incubated with Phe-tRNA in the presence of EFT and GTP at 5 mM Mg2+ concentration. The magnesium was then raised to 20 mM. PNPC-[ 3 H] Phe-tRNA was added and the incubation continued. Ribosomal proteins were isolated as described pre- viously [3,5]. They were separated on polyacrylamide gels in 8 M urea at pH 4.5 and the radioactivity was determined. The result is shown in Fig. 2a. There is essentially no incorporation of radioactivity into any ribosomal protein.

0 50 100 150

pMOLES (14C) PHE tRNA ADDED

Fig. 1. Saturation of ribosomes with [ 14C1 Phe-tRNA under enzymatic binding conditions. Ribosomes

(24 pmol) were incubated with increasing amounts of [ “ClPhe-tRNA in the presence of EFT (0~)

or in the absence of EFT ( ,A ).

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1

L15.Ll6 L27 L32,L33 1

b

0 2 4 6

DISTANCE FROM ORIGIN (cm)

Fig. 2. Identification of ribosomal proteins labelled with PNPC-[jHI Phe-tRNA by electrophoresis on polyacrylamide gels. (a) Ribosomes were precharged with cold Phe-tRNA. EFT and GTP in the presence of poly(U) before incubation with PNPC-[3HlPhe-tRNA. (b) Ribosomes not precharged before labelling with PNPC-[3H]Phe-tRNA. (c) Same as (b) but omitting poly(U). (d) Same as (a) but EFG was added to precharged ribosomes before incubation with PNPC-[3HlPhe-tRNA. Details of the incubation are de-

scribed in Materials and Methods.

Control experiments were carried out in parallel. In the first control Phe-tRNA and EFT were omitted during incubation at 5 mM Mg*’ concentration. The Mg2 + concentration was then shifted to 20 mM, PNPC-[3 H] Phe-tRNA was added and the mixture incubated as before. The pattern of labelled proteins is shown in Fig. 2b. There are two large peaks of radioactivity corresponding to proteins L15-L16 and L27 as demonstrated previously [3,5]. The second con- trol was carried out identically to the first control except that poly(U) was also omitted from the incubation mixture (Fig. 2~). As expected no affinity label- ling of proteins was observed. In the third control elongation factor EFG was added, subsequent to precharging the ribosomes with Phe-tRNA in the presence of EFT and GTP. Incubation was carried out for 20 min to allow synthesis of polyphenylalanine. Subsequently the concentration of Mg*+ was raised to 20 mM, PNPC-[” H] Phe-tRNA was added and the incubation continued. The pat- tern of labelled proteins obtained from these ribosomes is shown in Fig. 2d. There is a small amount of radioactivity incorporated, indicating that the block on the ribosomes can be reversed by the addition of EFG. The pattern shows a small additional peak of radioactivity in the area of proteins L32 and/or L33. Whether or not this incorporation is caused by affinity labelling with PNPC- oligophenylalanyl-tRNA (carrying the PNPC-groups on the terminal Phe residue) formed during the incubation, is presently not clear.

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Discussion

Several experiments have been reported to prove the specificity of the affinity labelling with derivatives of Phe-tRNA. (1) Affinity labelling has been shown to be dependent on the presence of messenger RNA [l-6]. (2) The extent of labelling by PNPC-Phe-tRNA is reduced by puromycin [5] and la- belled puromycin can be bound covalently to ribosomal proteins via its reac- tion with bromoacetyl-Phe-tRNA [ 41. (3) PNPC-Met-tRNAy et is recognized by initiation factor IF 2 and can be bound to the ribosomal donor site at 5 mM Mg2+ concentration in the presence of R 17 RNA as messenger [ 71. Stimula- tion by IF 2 of the affinity labelling reaction with PNPC-Phe-tRNA at 5 mM Mg*’ concentration has also been observed (our unpublished results).

The present investigation extends this line of experiments. Shorey et al. [12] have shown previously that Phe-tRNA bound to ribosomes in the pre- sence of EFTu does not react with acetylphenylalanyl-tRNA added subsequent- ly to the incubation mixture. Here we have demonstrated that ribosomes pre- charged with Phe-tRNA at the acceptor site are refractory to affinity labelling with PNPC-Phe-tRNA. Both results could be due to the fact that access to the donor site on a 70 S ribosome is only possible through an open acceptor site. Alternatively, EFTu-catalysed binding of Phe-tRNA to the acceptor site might require an uncharged tRNA molecule at the donor site and hence both sites would be already blocked [ 131.

The partial reactivation of the blocked ribosomes, by addition of EFG to the system, indicates that the inhibition is readily reversible. The presence of all components required for protein synthesis will thus lead to polyphenylalanine synthesis and to an opening-up of tRNA binding sites. Whether binding of PNPC-Phe-tRNA requires complete run off with loss of the polypeptide chain or just a free acceptor site is presently unknown.

The labelled ribosomal proteins have been identified both by 2-D gel electrophoresis and by immunological techniques, employing antibodies directed against individual ribosomal proteins [ 51. The major protein labelled under nonenzymatic conditions using PNPC-Phe-tRNA is L 27 which is also found in the initiation factor dependent affinity labelling reaction with PNPC- Met-tRNAr ft [ 71. This protein is therefore labelled from the ribosomal donor site. On the other hand, protein L 16 is labelled in the nonenzymatic reaction with PNPC-Phe-tRNA [5] but not in the initiation factor dependent reaction with PNPC-Met-tRNAp “t [ 7 J . L 16 has also been identified as the chloram- phenicol binding protein [ 141. It is therefore considered to be labelled from the acceptor site. This allocation has been confirmed recently by studies em- ploying uncharged tRNA to competitively inhibit the affinity labelling of the donor site [15-171.

The experiments demonstrate that nonenzymatic binding of PNPC-Phe- tRNA at high MgZf concentration occurs both at the donor and the acceptor site of the ribosome.

Acknowledgement

We would like to thank Dr H. Tuppy for discussions and suggestions. This

151

work was supported by a grant from the “Fonds zur Fijrderung der wissen- schaftlichen Forschung.”

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