Eut J Biochem 123. 95 -98 (1982) *( FEBS 1982

Implication of Arginyl Residues in Aminoacyl-tRNA Binding to Ribosomes

F t m c i x o HERNANDEZ. Abelnido LOPEZ-RIVAS, .lose Antonlo PINTOR-TOR0 ‘rntl Enr ique PALACIAN

lnsti tulo dc Bioquimicu de Macromolkculas. Centro de Biologin Molecular. Consejo Superior dc lnvestigaciones Cientiticas and Universidad Autbnoma de Mudi-id

(Rcceived Novciiihel- 20, 198 1 )

Modification of the 50-S subunits of l~.rchcric/ziu c d i ribosomes with the arginine reagent phenylglyoxal pro- duces inactivation of peptidyl transferase and inhibition of the binding of C(U)-A-C-C-A-LeuAc, phenylalnnyl- tRNA and N-acetylphenylalanyl-tRNA to the ribosome. Hybridization experiments, using 1.25 M LiCl core particles and the corresponding split proteins from unireated and phenylglyoxal-treated 50-S subunits, indicate that inactivation and inhibition of binding are the effects of modification of a protein fraction, the functionality of the RNA moiety being unaffected by the reagent. The split proteins from phenylglyoxal-modified 50-S sub- units are incorporated to 1.25 M LiCl core particles as well as those obtained from unmodified subunits, thus excluding the failure to bind as the cause of inactivation. In agreement with the general role played by the arginyl residues as positive binding sites for anionic ligands, the present results indicate that the arginyl residues or a protein fraction from 5 0 3 subunits might be important in the binding of aminoacyl-tRNA and peptidyl-tRNA to ribosomes.

The wide use, during the last decade, of reagents specific for the chemical modification of arginyl residues in proteins has allowed the role played by these residues as anionic bind- ing sites to be established [l]. A large variety of enzymes are inactivated by modification with arginine reagents, inactiva- tion being correlated with the loss of the capacity for binding a negatively charged substrate or cofactor [l]. Arginyl residues provide positively charged recognition sites for the binding of anionic ligands containing phosphate or carboxylate groups.

Although ribosomes interact with anionic substrates, only recently have the arginyl residues been implicated in their binding. Modification with phenylglyoxal of only a few pro- teins from the 30-S subunit of Escliericlzia coli ribosonies pro- duces inhibition of poly(U) binding and inactivation of poly- peptide synthesis [2] , indicating the involvement of arginyl residues in the binding of mRNA.

The present paper shows that modification of a protein fraction from 50-S ribosomal subunits with phenylglyoxal is accompanied by both inactivation of peptidyl transferase and inhibition of aminoacyl-tRNA binding to ribosomes. These results seem to indicate that arginyl residues are also impli- cated in the binding of aminoacyl-tRNA.

MATERIALS AND METHODS

Modificntioi~ of’ 7 0 3 Rihosonics and 51)-S Subunits ~t.i!li PhcnjYgljmxal

Ribosomes and ribosomal subunits from Esdicrichiu c d i MRE 600 were prepared as described previously [3]. 70-S ribo- somes and 50-S subunits were treated with phenylglyoxal at 25 ’C for 30 min as described previously [2], the reaction being stopped by addition of an excess of arginine. The modi- fied particles and the untreated control were precipitated with 1 vol. of cold ethanol, collected by centrifugation, and suspended in 1 0 mM Tris/HCI (pH ?.8), 10 mM MgClz and 50 mM NH4CI. In the reconstitution experiments (Fig. 3 - 5)

the ribosomal particles were separated in a different way: the preparations were directly centrifuged at 35000 rev. h i i n for 4 h in a Beckman 65Ti rotor and suspended in the above- mentioned butrer solution. The supernatants from both the modified subunits and the untrealed control contained less than IO:, of the total protein. These protein fractions were combined with the corresponding proteins released by the 1.25 M LiCl treatment, to be used in the reconstitution experi- ments.

PI-clpurution of Rihosonzul ‘Cows’ arid Split Proteins

Protein-deficient particles and the corresponding split proteins were obtained from native and modified 5 0 3 sub- units by treatment with 1.25 M LiCl [4]. 5 0 3 subunits (1.5 mg/ml) in 10 mM TrisiHCI (pH 7.4) and 10 mM MgClz were incubated with 1.25 M LiCl at 0 ‘C for 4 h. This incuba- tion was followed by centrifugation at 48000 rev.:min and 2 ‘ C for 3.5 h in a Spinco 65Ti rotor. The pellet, with thc sedi- mented ribosomal ‘cores’, was suspended in 10 m M Tris. HCI (pH 7.4), 50 mM NH4CI and I0 niM MgC12 t o a ribosomal concentration of 14 mg/ml. The supernatant containing the released proteins was combined with thc equivalent amount of the proteins separated from the ribosomal particles by centrifugation after phenylglyoxal treatment. This combined protein fraction was dialyzed against 20 m M Tris/HCI (pH 7.4), 400 mM NH4CI, 20 mM MgC12 and 5 mM 2-mer- captoethanol, at 0- 5 ‘ C for 16 h. After dialysis, the prepara- tion was concentrated with Ayuacide I1 (Calbiochem) and dialyzed a second time under the same conditions (final pro- tein concentration 2.0-2.4 mghil).

Pwtiul Rwonstitution 0fSO-S Subunits

Reconstitution of 5 0 3 subunits was carried ou t from ribosomal ‘cores’ (1.25 M LiCI) and the combined split pro- tein fraction obtained, after phenylglyoxal modification and LiCl treatment, from native and phenylglyoxal-tr-eated sub- units. Ribosomal ‘cores’ (7 pmoi) were incubated at 37 C

96

for 5 min with different amounts of split proteins, in 20 mM Tris/HCl (pH 7.4), 350mM NHdCI, 20mM MgC12 and 2.5 mM 2-mercaptoethanol (final volume 25 pl), this incuba- tion being followed by a second one at 50°C for 30 min.

100

50 Binding of Split Proteins to Ribosomal ‘Cores’

The proteins released from phenylglyoxal-modified and unmodified preparations were labeled by reductive methyla- tion with [14C]formaldehyde [5]. Protein samples (125 pg protein) were dissolved in 88 pl of 0.1 M sodium borate (pH 8.0) and treated with 60 pl 75 mM [‘4C]formaldehyde (10 mCi/mmol) at 0”. After 3 min, four 10-pI aliquots of sodium borohydride (5 mgiml) were added at 30-s intervals followed 1 min later by one 2 0 4 aliquot. The preparation obtained in this way was dialyzed overnight at 0‘C against 1 0 m M TrisIHCl (pH 7 . Q 50 mM NH4Cl and 10 mM MgC12. Different amounts of this protein preparation were added to 159 pmol of 1.25 M LiCl ‘cores’ from native 50-S subunits in 0.5 ml of 20 mM Tris/HCl (pH 7.4), 350 mM NH4CI, 20 mM MgClz and 2.5 mM 2-mercaptoethanol. The mixture was incubated at 50 “C for 30 min, and the ribosomal particles were precipitated with 1 vol. of cold ethanol, and resuspended in 10 mM Tris/HCI (pH 7 . Q 50 mM NH4CI and 10 mM MgC12. 10 p1 of bovine serum albumin (1 mg/ml) were added to 10-pI aliquots of ribosomal particles. After addition of 2 ml of 50 trichloroacetic acid, the samples were filtered through Whatman GFiA filters which were then dried and assayed for radioactivity.

A

A A

- \*

Func t ional A .ssay.s

Poly(U)-directed polyphenylalanine synthesis was per- formed in a crude system containing E. coli extract (1 00000 x g supernatant) [3]. Peptidyl transferase was estimated by the ‘fragment reaction’ assay, using C(U)-A-C-C-A-[3H]LeuAc and puromycin as substrates [6] . Binding of the acetylated ‘fragment’ to 70-S ribosomes or 50-S subunits was carried out in the presence of 10 pM sparsomycin as described previ- ously [7]. Non-enzymatic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to ribosomes, and the enzy- matic binding of phenylalanyl-tRNA, were performed as previously described [8]. The assay mixtures included 20 mM MgCI2 for non-enzymatic binding. To determine enzymatic binding, the MgCll concentration was decreased to 10 mM, and elongation factor T (25 pg/ml) and GTP (150 pM) were also included.

RESULTS

Inactivution of‘ Peptidyl Transferuse and Inhibition of C/ U ) - A-C-C-A- LeuA c Binding by Phenylglyoxal Modification

Modification of 70-S ribosomes or 50-S subunits with the arginine reagent phenylglyoxal produces inactivation of peptidyl transferase as well as loss of the capacity for binding C(U)-A-C-C-A-LeuAc. Fig. 1 shows the effect of modifica- tion of 70-S ribosomes and 50-S subunits, at different molar ratios of reagent to ribosomal particle, on the peptidyl trans- ferase activity determined by the ‘fragment reaction’ assay. In both cases, modification is accompanied by inactivation. With 50-S subunits, inactivation takes place at lower molar ratios of reagent to particle than with 70-S ribosomes, which may be a consequence of the larger number of residues sus- ceptible to modification in 70-S ribosomes as compared with

I 1

0 2500 5000 Reagent 150- S

subunits (rnol/mol)

Fig. 1 , E/fecfec./.s of’ p/wn,/g/joxu/ nzociificution of 70-S ribosomes ( A ) trntl 50-,5‘ \ i i / x i t i i / . \ i 111 on p p / i ( l i . / r,nn.vfivwsr ac.tivirj, and bindrrig of tlw i I w r -

j,/trrrt/ ‘ /w,qni i ,nt . 70-S ribosomes and 50-S subunits were treated with phenylglyoxal a t llic indicated molar ratios of reagent to ribosomal particle. Peptidyl transferase activity and binding of the acetylated ‘frag- ment’ were assayed in aliquots of the preparations. Results are expressed as percentages of the activity and binding of the corresponding control subjected to the same treatments as the modified preparation but in the absence of phenylglyoxal. The control preparations sqnthesized 0.05 tinol acetylleucyl-puromycin min-’ (pmol70-S ribosomes)-’ (A) and 0.09 fmol acetylleucyl-puromycin min-’ (pmol 50-S subunits)-’ (B), and bound 4 (A) and 2 (B) fmol acetylated ‘fragment’ (pmol ribosomal particles)-’

50-S subunits. The capacity of both ribosomal particles for binding the acetylated fragment, C(U)-A-C-C-A-LeuAc, is also eliminated by phenylglyoxal modification (Fig. 1). The parallel loss of the two functional properties agrees with pep- tidy1 transferase inactivation being the result of the lack of binding of the acetylated fragment.

Inhibition ? f Phcnylalunyl-tRNA and N- Acetylphenylalunyl-t R N A Binding by Mod(fication qf’50-S Subunits

In addition to the acetylated fragment, modification of 50-S subunits also prevents the binding of the more physio- logical substrates phenylalanyl-tRNA and N-acetylphenyl- alanyl-tRNA (Fig.2). Binding was assayed in the presence of native 3 0 3 subunits under enzymatic and non-enzqniatic conditions. These results point to aininoacyl-tRNA binding as the primary step affected by phenylglyoxal modification and as responsible for the inactivation.

Reconstituted Ribosomal Particles Containing Components fkom Phenyl~lyosal- Treated and Untreated 50-S Subunits

The inactivation of ribosomes by treatment of 50-S sub- units with phenylglyoxal is produced by modification of the protein moiety of the particle as indicated by reconstitution experiments. The 1.25 M LiCl ‘cores’ obtained from modified 50-S subunits can reconstitute 50-S particles active in poly- phenylalanine synthesis upon incubation with split proteins obtained from untreated subunits (Fig. 3). However, the split proteins obtained from the modified preparation are unable to reconstitute active particles when incubated with LiCl ‘cores’ from untreated subunits. These results indicate that the proteins released from 50-S subunits upon phenylglyoxal modification and subsequent treatment with 1.25 M LiCl are those the modification of which is responsible for the observed

I

0 2000 4000 R e a g e n t l 5 0 - S subunits (mollrnol)

Fig. 2. Inhibition of' tlic liindiiig qf' phen~~lulanyl-tRNA und N-uc~ezyl- pllenjkulunyk-rRNA /?J, nzodificurion 0fSO-S suhiinits. 50-S ribosomal sub- units were treated with phenylglyoxal at the indicated molar ratios of reagent to subunit. Non-enzymatic binding of phenylalanyl-tRNA (0) and N-acetylphenylalanyl-tRNA (A), and enzyinatic binding of phenyl- alanyl-tRNA (O), were determined in aliquots of the preparations. In every case, native 30-S subunits were included in the assay mixture (molar ratio of 30-S to SO-S subunits cquals 1.5). Results are expressed as percent- ages of the binding of the corresponding substrate to a control subjected to the same treatments as the modified preparations but in the absence of phenylglyoxal. The control preparation bound 0.14 pmol phenyl- alanyl-tRNA/pmol 50-S subunits enzymatically, and 0.1 3 pmol phenyl- alanyl-t RNA and 0.09 pmol N-acetylphenylalanyl-tRNA non-enzyma- tically

0 5 10 15 Split proteins added ( K g )

Fig. 3. PoI~phr~i I~ lr ikr i i i~ i i~ . s ~ ~ n f h c . s i . r hj. riho.soiiir,s c'onfuining c~oinponerits ,fi.om p / i e i ~ ~ / g / ~ o s r ~ i - r rea red and ii iztr~u I cti 50- S .xuhun its. 5 0- S subunits we re modified with phenylglyoxal at a molar ratio of reagent to subunit equal to 3600. Ribosomal cores (1.25 M LiCI) and the corresponding split proteins were obtained from native and phenylglyoxal-treated subunits. To reconstitutc SO-S particles, the ribosomal cores obtained from native subunits werc incubated with split proteins from naiive (0) and phenyl- glyoxal-treated subunits (m), and the cores from phenylglyoxal-treated subunits with split proteins from native (0) and phenylglyoxal-treated subunits (O), as described under Materials and Methods. The rccon- stituted particles were supplementcd with native 30-S subunits (molar ratio of 30-S subunits to 50-S cores cquals 1.5) and their polyphcnyl- alanine-synthetic capacity assayed

inactivation, while the corresponding residual core retains the capacity to reconstitute active particles. Similar results were obtained when determining peptidyl transferase activity by the 'fragment reaction' assay, and C(U)-A-C-C-A-LeuAc and phenylalanyl-tRNA binding (Fig. 4).

-0 12 24 0 12 24 0 12 24 Split proteins added ( K g )

Fig. 4. Pq~ticl j~l trrin.sfi~ru.sr tic,iit'irj, unrl i i ~ t m i i ~ ~ i o n ( Y I / J U ( i / i ' of r.ihowmcii

50-S .sirhuniis. Pcptidyl translewse (A) . binding of tlic iicct) Iated Img- incnt' (B) and non-enzymatic binding o f phenqlalanyl-tRNA (C') were assayed in SO-S subunits reconstituted from phenylglyoxal-treated and untreated componcnls as indicated in the legcnd to Fig. 3. The binding of phcnylalanyl-tRNA was assayed in the presence of added 3 0 4 sub- units (molar ratio of 30-S subunits to SO-S 'cores' equals 1.5). The 100",, valuc for activity or binding corresponds to 0.04 fniol acctylleucyl- puroniycin synthesized inin-' (pniol 50-S core)-' (A), 0.01 5 pmol acctylatcd 'fragment' bound pmol 50-S core (€3). and 0.32 piiiol phenyl- alanyl-tRNA bound.pmol 50-S core (C)

[XLr/ic'/l'.S CO?lfUl?liPl,r ~ ' O ~ ? , f 7 ~ J l 7 ~ ~ i l ~ \ fV (J i7 l / J / i ( ' i l l'/q/l'/l V ~ / / - l l ~ l ' ( l l O l / i l l i l / l l ~ ~ / l ~ l ~ ~ l l l Y /

Protein added(Kg)

Fig. 5. Binding to 1.2.5 M LK'l core purriclcJ.s of .split ~~rcitcii7.~,fr.oiiz rnotlific~tl und unniorlifkd 50-S .suhiinit.s. The indicatcd amounts of split proteins from phenylglyoxal-treated (A) and untreatcd SO-S subunits (0) which had been labeled with ['4C]formaldehyde wei-e incubated with IS9 pinol of 1.25 M LiCl core particles obtained from native 50-S subunits and the incorporated radioactivity determined. as indicated under Material5 and Methods

The failure of the split protein fraction from the modified subunits to reconstitute active particles upon addition to 1.25 M LiCl 'cores' is not the result of deficient binding. since this modified protein fraction is incorporated into ribosomal particles as well as the one obtained from unmodified sub- units (Fig. 5) .

DISCUSSION

Modification of lkher-ichia c d i ribosomes with the argi- nine reagent phenylglyoxal is accompanied by inactivation, this being produced by modification of either the 30-S subunits [2] or the 50-S subunits (present results). Both the RNA and the protein moieties of 30-S subunits are modified by phenyl- glyoxal, and modification of either of them produces inacti- vation 121. However, the inhibited step is not the same in both cases : modification of the protein moiety is accompanied by

loss of poly(U) binding capacity. while that of RNA is not. Treatment of 50-S subunits with phenylglyoxal produces inhibition of aminoacyl-tRNA binding, this effect being the result of modification of a protein fraction. In contrast with the 30-S subunits, the RNA moiety of 50-S subunits treated with phenylglyoxal does not lose its functionality, since the 1.25 M LiCl 'cores' obtained from treated subunits are able to reconstitute active particles when supplemented with unmodified split proteins.

I t has been speculated that the low arginine content of proteins, in spite of the existence of six different codons specifying this amino acid, has been probably selected to make the anions binding role of arginine more specific [I 1. The high arginine content of ribosomal proteins (approxi- tnately 8.5'7; arginine residues in E. coli ribosomes [9] as compared with an average of 4.4':<, in a total of 208 different proteins tabulated by Reeck [lo]) might be related to the large number of specific interactions that take place in the ribo- some between proteins and anionic molecules, rRNA, mRNA and tRNA.

The results shown i n this paper seem to indicate lhc in- volvement of arginine residues in the binding of aminoacyl- tRNA. However, this does not mean they are the only binding points for aminoacyl-tRNA. In addition to the possible implication of other basic amino acid residues [ l l , 121, inter- actions between tRNA and rRNA are undoubtedly also important for the binding of these anionic substrates. Multiple RNA-protein and RNA-RNA interactions should be required

to form a stable binding between a substrate as large as aminoacyl-tRNA and the ribosome.

We thank Prof. D. Vizquez for advice and encouragement. This work was supported in part by grants from Foritlo cle fni'c..s/iguc,ionc,.v Smii /urias and Comi.sibn A.~r~.soru tit' ~tivestigacicin Cientifrctr Tlcnicu (Spain).

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Palacihn. E. (1 080) Eur, J . Bioc/7~n7. 108, 137 - 141. 3. L6pez-Rims. A, . Vizquez, D. & Palacihn. E. (1978) EIN. J . Bio-

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F. Hernindez. A. Lope7-Rivaa, J . A. Pintor-Toro, and E. Palaciitn. Instittito de Bioqtiimica d e Macronioli.culas. Cenlro d e Biologia Molecular. Consejo Superior de Investigacionc\ Cientificas y Universidad Autbnoma de Madrid. Facultad de Ciencias. Universidad AutOnonia de Madrid. Canto Blanco, Madrid-34. Spain