Eur. J. Biochem. 108, 137-141 (1980) by FEBS 1980 Implication of Arginyl Residues in mRNA Binding to Ribosomes Francisco HERNANDEZ, Abelardo LOPEZ-RIVAS, Jose Antonio PINTOR-TORO, David VAZQUEZ, and Enrique PALACIAN Instituto de Bioquimica de Macromoleculas, Ccntro de Biologia Molecular, Madrid (Received October 8, 1979/April 11, 1980) Modification of Esclzevichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)- dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands. The chemical modification of the amino acid residues of proteins with specific reagents has been extensively used to study the active sites of enzymes and the relationships between structure and function in proteins. Treatment of enzymes that bind anionic substrates or cofactors with reagents specific for arginine is frequently accompanied by an inactivation which is correlated with the loss of the capacity for binding the negatively charged ligand [l]. These results indicate that arginyl residues play a general role in the binding of anionic ligands, containing phosphate or carboxylate moieties, by providing a positively charged recognition site [I 1. Since ribosomes bind mRNA and aminoacyl-tRNA during protein synthesis, and these molecules are negatively charged, arginyl residues might also play a fundamental role in their binding. We have studied the effects of modification of ribosomes with the arginine reagents phenylglyoxal and butanedione on poly(U)-directed polyphenyl- alanine synthesis and poly(U) binding. The results seem to indicate that arginyl residues are implicated in the binding of mRNA to the 30-S ribosomal subunit. MATERIALS AND METHODS Muterials Ribosomes and ribosomal subunits were prepared from Esclzerichiu coli MRE 600 as described previously [2]. Split proteins from 30-S subunits were obtained by dialysis of the subunits against 1 mM Tris-HC1 (pH 7.8) and 10 mM MgC12, and separation of the re- leased proteins by centrifugation [3]. 16-S RNA and total proteins were obtained from 30-S subunits according to Traub el al. [4]. Phenylglyoxal and butanedione were obtained from ECA-Chemie (Stein- heim/Albuch). The radioactive compounds mere pur- chased from the Radiochemical Centre (Amcrsham). Modiji'cation w,itlz Plzenylglyoxul and Butunedione Prior to modification, 70-S ribosomes, ribosomal subunits and split proteins were dialyzed against 100 mM NaHC03 (pH 8.0) and 10 mM MgC12 (modi- fication with phenylglyoxal), or 50 niM sodium borate (pH 8.0) and 10 mM MgC12 (modification with bu- tanedione). Borate was used in the modification with butanedione because of its known stimulatory effect on modification [5]. These preparations (2 mg ribosomal particles or split proteins/ml) were in- cubated with phenylglyoxal (added as a 100mM solution) or butanedione (added directly) at 25 "C for 30 min. The reaction was stopped by addition of an excess of arginine (10 mM). The modified particles were precipitated with one volume of cold ethanol, collected by centrifugation, and the supernatant discarded. The precipitated particles were suspended in 10 mM Tris-HC1 (pH 7.8), 10 mM MgC12 and
Transcript
Eur. J. Biochem. 108, 137-141 (1980) by FEBS 1980
Implication of Arginyl Residues in mRNA Binding to Ribosomes Francisco HERNANDEZ, Abelardo LOPEZ-RIVAS, Jose Antonio PINTOR-TORO, David VAZQUEZ, and Enrique PALACIAN
Instituto de Bioquimica de Macromoleculas, Ccntro de Biologia Molecular, Madrid
(Received October 8, 1979/April 11, 1980)
Modification of Esclzevichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)- dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.
The chemical modification of the amino acid residues of proteins with specific reagents has been extensively used to study the active sites of enzymes and the relationships between structure and function in proteins. Treatment of enzymes that bind anionic substrates or cofactors with reagents specific for arginine is frequently accompanied by an inactivation which is correlated with the loss of the capacity for binding the negatively charged ligand [l]. These results indicate that arginyl residues play a general role in the binding of anionic ligands, containing phosphate or carboxylate moieties, by providing a positively charged recognition site [I 1. Since ribosomes bind mRNA and aminoacyl-tRNA during protein synthesis, and these molecules are negatively charged, arginyl residues might also play a fundamental role in their binding.
We have studied the effects of modification of ribosomes with the arginine reagents phenylglyoxal and butanedione on poly(U)-directed polyphenyl- alanine synthesis and poly(U) binding. The results seem to indicate that arginyl residues are implicated in the binding of mRNA to the 30-S ribosomal subunit.
MATERIALS AND METHODS Muterials
Ribosomes and ribosomal subunits were prepared from Esclzerichiu coli MRE 600 as described previously
[2]. Split proteins from 30-S subunits were obtained by dialysis of the subunits against 1 mM Tris-HC1 (pH 7.8) and 10 mM MgC12, and separation of the re- leased proteins by centrifugation [3] . 16-S RNA and total proteins were obtained from 30-S subunits according to Traub el al. [4]. Phenylglyoxal and butanedione were obtained from ECA-Chemie (Stein- heim/Albuch). The radioactive compounds mere pur- chased from the Radiochemical Centre (Amcrsham).
Modiji'cation w,itlz Plzenylglyoxul and Butunedione
Prior to modification, 70-S ribosomes, ribosomal subunits and split proteins were dialyzed against 100 mM NaHC03 (pH 8.0) and 10 mM MgC12 (modi- fication with phenylglyoxal), or 50 niM sodium borate (pH 8.0) and 10 mM MgC12 (modification with bu- tanedione). Borate was used in the modification with butanedione because of its known stimulatory effect on modification [ 5 ] . These preparations (2 mg ribosomal particles or split proteins/ml) were in- cubated with phenylglyoxal (added as a 100mM solution) or butanedione (added directly) at 25 "C for 30 min. The reaction was stopped by addition of an excess of arginine (10 mM). The modified particles were precipitated with one volume of cold ethanol, collected by centrifugation, and the supernatant discarded. The precipitated particles were suspended in 10 mM Tris-HC1 (pH 7.8), 10 mM MgC12 and
138 Implication of Arginyl Residues in mRNA Binding to Ribosomes
50 mM NH4CI. The modified split proteins were dialyzcd against the same buffer solution.
Functionul Assays
Poly(U)-directed polyphenylalanine synthesis was performed in a crude system containing E. coli extract (100000 x g supernatant) [6].
The binding of poly(U) was determined as de- scribed previously [7]. The ribosomal preparation was incubated with ['H]poly(U) (25 - 50 nCi ; 25 - 50 Ci/ mmol) at 0°C for 10 min in 50 PI 50 mM Tris-HC1 (pH 7.8), 10mM MgC12, 60mM KCI and 5 mM 2-mercaptoethanol. The reaction was ended by addition of 2 mI of the same buffer solution, and the mixture was filtered through nitrocellulose filters previously treated with alkali. The filters were dried and the radioactivity was measured.
Total Reconstitution of' 30-S Ribosomal Subunits
30-S ribosomal subunits were reconstituted from 1 6 3 RNA and the corresponding proteins obtained from untreated and phenylglyoxal-treated 3 0 3 sub- units as described by Traub et al. [4].
RESULTS
lnuctivution u j ' Polypeptide Polymeri,ut ion hi' Modifi'cution qf 7 0 3 Ribosomes or 30-S Subunits
Modification of ribosomes with the arginine re- agents phenylglyoxal and butanedione is accompanied by inactivation of poly( U)-dependent polyphenyl- alanine synthesis. Fig. 1 shows the polyphenylalanine synthesis obtained with ribosomal preparations treated with phenylglyoxal and butanedione at different molar ratios of reagent to ribosomes. Both reagents pro- duced inactivation, phenylglyoxal being the most effective. At a molar ratio of reagent to ribosomes equal to 1000, phenylglyoxal caused almost complete inactivation, while a molar ratio equal to 5000 was required to obtain 75 ''i inactivation with butanedione.
Modification of 30-S subunits with any of the two reagents produces inactivation of polyphenylalanine synthesis, while modification of 50-S subunits takcs place without any significant change in activity (Table 1) . Activity was assayed in the presence of the complementary untreated subunit.
Pol>*( U ) Binding to Modified 7 0 3 Ribosomes and 30-S Subunits
Modification of 70-S ribosomes or 3 0 3 subunits with butanedione is accompanied by loss of the poly( U)-binding capacity. Fig. 2 shows this effect for ribosomal particles treated at two different molar
0 L 0
-1 \.-, I I xx)O 4ooo 6ooo
[ ~ e a g e n t ] / [ i ~ ~ ritmsomes](mo~/rno~)
Fig. 1. EjJect of'modijkariyon o/ 7 0 3 r i lmwma 011 polyp,/imnylalaninr syrztheslv. Ribosomes were treated with phenylglyoxal (0) and bu- tanedioue (A) at the indicated molar ratios of reagent to ribosomes. Polyphenylalanine synthesis was assayed in aliquots of the prep- arations. Activities are expressed as percentages of the activity of a control subjected to the same treatments as the modified prep- arations but in the absence of reagent. The untreated control incorporated in 30 min 1.83 pmol phenylalanine/prnol ribosomes
Table 1. Efrccts qf' treu(rnent with phenylglyyoxal and huruneciione qf 50-S and 30-S suhunits on polyph~v~ylalanine .synrhesi.c Ribosomal subunits were separately treated with phenylglyoxal (molar ratio of reagent to subunit equal to 2000) or butanedione (molar ratio equal to 2500). Polyphenylalanine synthesis was determined in the presence of the complementary subunit (ratio of 30-S to SO-S subunits equal to 1). The unt-eated control in- corporated in 30 rnin 2.8 prnol phenylalanine/pmol 50-S subunits
Ri bosomdl preparation Complementary Polyphenylalanine subunit synthesis (relative
ratios of reagent to particles. To obtain the same degree of inactivation the molar ratio of reagent to particle for the 70-S ribosomes was twice as large as that for the 30-S subunits.
Efrects of Modification of'. Protein Fruction from 30-S Subunits on Poly( Or) Bindin,q to this Fruction
The split proteins obtained from 3 0 3 subunits by prolonged dialysis against a low-ionic-strength buffer are able to bind poly(U) (Fig. 3), as reported previously [ 3 ] . Modification of this fraction, which contains pro- tein S1, with phenylglyoxal and butanedione is accompanied by inhibition of poly(U) binding (Fig. 3). This inhibition increases with the concentration of reagent used in the modification, phenylglyoxal being
P. Hernandez, A. Lbpez-Rivas, J. A. Pintor-Toro, D. Vizquez, and E. Palaciin 139
I I I 5000 Do00
[Butanedione]/[ribosornal particles] (rnolhol)
Fig. 2. Efyects of modifi'cution by hutunrdionr qf 70-S ribosomes and 30-S subui1it.r on the binding of'poly(U). 70-S ribosomes (0) and 30-S subunits (0) were treated with butanedione at the indicated molar ratios of reagent to ribosomal particle. Binding of poly(U) was determined in aliquots of the preparations, and expressed as percentages of poly(U) bound by the Corresponding control sub- jected to the same treatments as the modified preparations but in the absence of reagent. The untreated control of 7 0 3 ribosomes bound 25 604 counts/min and that of 30-S subunits 17 926 counts/ min
I 1 ... ..Ap- 2 m 200
[Reagent] (rnM)
Fig. 3. l . / /c , i I ( I / modific,utron (?f the split ~~ro~cir i . s fruc./ior, on llie ~ i f i ~ / ; i / , ~ ( ~ ~ / ~ ( ~ I i , f 0). Split proteins, obtained as indicated in Materials and hlc1licids, were treated with phenylglyoxal (0) and butanedione (A) a t the indicated concentrations of reagent. Binding of poly(U) was determined in aliquots of the preparations, and expressed as pcrcentages of poly(U) bound to the control subjected to the same treatments a5 the modified split proteins but in the absence of reagent. The untreated split proteins bound 10 999 countsjmin
Table 2. Polyphenylalanine synthesis and poly( U ) binding of ribosomul partirles containing components f rom treated und untreuled 3 0 3 subunits 303 subunits were modified with phenylglyoxal at molar ratios of reagent to subunit equal to 1800 (expt 1) and 2200 (expt 2). In Expt 1 total protein (TP and phenylglyoxal/TP) and 16-S RNA (RNA and phenylglyoxal/RNA) were prepared from untreated (TP and RNA) and modified subunits (phenylglyoxal/TP and phenylglyoxal/RNA). 30-S subunits were reconstituted from the components indicated in the table (the reconstitution mixtures contained 58 pmol 16-S RNA in 280 pl, and twice the corresponding amount of proteins). Polyphenyl- alanine synthesis was determined in the presence of 5 0 3 subunits (ratio of 30-S to 50-S subunits equal to 1.2). The control 303 subunits containing untreated components incorporated in 30 min 0.72 pmol phenylalanine/pmol 16-S RNA. Prior to determination of poly(U) binding, the ribosomal particles present in aliquots of the reconstitution mixtures were precipitated with one volume of cold ethanol, collected by centrifugation, and the supernatant discarded, to eliminate the excess of proteins. The control 30-S subunits, containing untreated components, bound 1002 counts min pmol30-S subunits-'. In Expt 2, untreated and modified 303 subunits were dialyzed at low ionic strength to separate the split proteins (SP and phenylglyoxal/SP) from the corresponding 'cores' (30-S ~ SP and phenylglyoxal/30-S - SP). 30-S subunits were reconstituted by incubation (40'C and 10 min) of the indicated components in 10 m M Tris-HC1 (pH 7.4), 300 mM KC1, and 10 mM MgCI2 (10 pmol 'cores' and the corresponding amount of split proteins in 20 p1 reconstitution mixture). Polyphenylalanine synthesis was determined in the presence of 50-S subunits (ratio of 30-S to 5 0 4 subunits equal to 1.2). The control containing 30-S -SP plus SP incorporated in 30 min 3.3 pmol phenylalanine/pmol 30-S subunits, and bound 1354 counts min-' pmol 304 subunits-'
effective at concentrations about 100 times lower than butanedione. Amino acid analysis of the prep- arations treated with phenylglyoxal showed a de- crease in the amount of arginine residues. The sample treated with 20 niM phenylglyoxal had only To see whether the inactivation of 30-S subunits 45% of the arginine present in the untreated prep- is produced by modification of only ribosomal aration. proteins, ribosomal particles were reconstituted from
Reconstituted Ribosomal Particles Containing Componentsfrom Phenylglyoxal-Treated and Untreated 30-S Subunits
140 Implication of Arginyl Residues in mRNA Binding to Ribosomes
16-S RNA and total proteins obtained from phenyl- glyoxal-treated and untreated 30-S subunits. Table 2 (expt 1) shows that when either the 16-S RNA or the proteins were obtained from phenylglyoxal-treated subunits, the reconstituted particles had very little activity in polyphenylalanine synthesis. On the other hand, the modification of 16-S RNA affects very little the capacity of the reconstituted subunits to bind poly( U), while modification of the ribosomal proteins is accompanied by a substantial inhibition. These results indicate that both RNA and protein com- ponents are modified by phenylglyoxal, and the modification of either of them prevents the reconsti- tution of active 30-S subunits.
Modification of only a small group of proteins is accompanied by inactivation of polyphenylalanine synthesis and inhibition of poly(U) binding, as shown in Table 2 (expt 2). While addition of the split proteins fraction obtained by dialysis to the corresponding ‘cores’ reconstitutes subunits active in polyphenyl- alanine synthesis and poly(U) binding, when the ribosomal particles were reconstituted with split proteins from modified subunits very little poly- phenylalanine synthesis was obtained, as well as decreased poly(U)-binding. The split proteins isolated from modified 30-S subunits have, like those modified directly (Fig. 3), a decreased poly(U)-binding capacity.
DISCUSSION
The loss of the capacity to synthesize polyphenyl- alanine that takes place upon modification of 70-S ribosomes with the arginine reagents phenylglyoxal and butanedione is the result of modification of the 30-S subunit. This subunit is responsible for the binding of mRNA during the initiation of polypeptide synthesis. The inactivation of poly(U) binding to 70-S ribosomes and to 30-S subunits that accompanies modification indicates that mRNA binding to the 30-S subunit might be the step responsible for the inactivation of protein synthesis.
The reconstitution experiments indicate that both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and that modification of either of them prevents reconstitution of active ribosomes. However, while modification of ribosomal proteins is accompanied by a decrease in poly(U)- binding capacity, that of RNA is not, indicating that although in both cases there is loss of polyphenyl- alanine synthesis the inhibited step seems to be different. Since modification of 16-S RNA with the related reagent kethoxal produces inhibition of the binding of transfer RNA without affecting that of messenger RNA [8], the tRNA binding might also be the inhibited step in 30-S subunits reconstituted with 16-S RNA modified with phenylglyoxal.
Inactivation of polypeptide synthesis produced by modification of the protein moiety of 30-S subunits appears to be the result of the modification of a group of proteins, which contains mainly protein S1, released from 30-S subunits by prolonged dialysis at low ionic strength [3] . This group of proteins 1s able to bind poly(U) in the absence of other components of the 3 0 3 subunit. Modification of these split proteins eliminates their poly(U)-binding capacity. The split proteins obtained from modified 30-S subunits show a similar lack of poly(U)-binding capacity. When this protein fraction is added to complementary ribosomal ‘cores’ obtained from untreated 30-S subunits, there is no recovery of polyphenylalanine-synthesizing ac- tivity, in contrast with the results obtained with split proteins from untreated subunils. A separate ex- periment showed that modified split proteins bind to the ‘cores’ as well as those untreated (unpublished).
Chang and Craven [9] modified the 30-S ribosomal subunit with a large variety of chemical reagents, and determined the effects of modification on the capacity of the ribosomal particles to bind mRNA. They concluded that lysyl residues play an important role in mRNA binding to 30-S subunits. These authors did not find any inactivating effect of the reagents specific for arginine, and concluded that arginyl residues do not appear to be implicated in the binding. On the other hand, reductive methylation of lysyl residues of protein S1 causes the loss of the capacity of this protein to form stable complexes with poly(U) [lo]. In addition to lysyl residues, histidinyl residues have been implicated in the binding of mRNA to ribosomes (M. Tal, personal com- munication).
The results presented in this paper indicate that arginyl residues of ribosomal proteins from the 30-S subunit are also implicated in the binding of mRNA to ribosomes, being in agreement with the role played by arginyl residues in many enzymes as positive binding sites for anionic substrates and cofactors [l]. It is interesting to note that the three residues that have been implicated in mRNA binding (lysine, histidine and arginine) are those that carry a positive charge, being able to interact with the negatively charged phosphate moieties of mRNA. Our results agree with the proposal of Lange et al. [ l l ] considering arginyl residues essential for the binding of substrates containing phosphate moieties, and with the model proposed by Helene [12], according to which Arg-Glu-Lys sequences are involved in the specific interactions between proteins and nucleic acids. It has recently been found that arginine inhibits peptidyl transferase [13]. Apparently two arginine molecules bind to the A-site, probably by interacting with a nucleotide sequence of 2 3 3 RNA. Since D-arginine has the same inhibitory effect as L-arginine, the interaction might be mainly dependent
F. Hernandez, A. Lopez-Rivas, J. A. Pintor-Toro, D. Vazquez, and E. Palacian 141
on the guanidiiiium group. These results point to the generality of interaction between polynucleotides and the guanidinium groups of arginine in ribosomes.
In conclusion, the arginyl residues of a few ribosomal proteins from the 30-S subunit, which in- clude protein S1, seem to play an important role in mRNA binding to 30-S subunits through interaction of their positively charged guanidinium groups with the negatively charged phosphate moieties of mRNA.
We thank Dr Magdalena Ugarte and Miss Paloma Sanz for per- forming the amino acid analysis and Miss Asuncion Martin for technical assistance. This work was supported in par1 by a n institutional grant to the Centrode Biologiu Moleculur from Comisicin del Descuento Complementario.
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F. Hernandez, A. Lopez-Rivas, J . A. Pintor-Toro, D. Vazquez, and E. Palaciin, Instituto de Bioquimica de Macromolt-culas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid, Facultad de Ciencias, Universidad AUthomd de Madrid, Canto Blanco, Madrid-34, Spain
