Molec. Biol. Rep. Vol. 5, 3: 193-196, 1979
ELONGATION FACTOR Tu-INDUCED CONFORMATIONAL CHANGES OF RIBOSOMES DETECTED BY IODINATION
O. MARTINEZ & J. MODOLELL
Instituto de Bioquimica de Macromol~culas, Centro de Biologia Molecular, CSIC and (JAM, Canto Blanco, Madrid-34, Spain (Received April 1, 1979)
Abstract
The effect of elongation factor (EF) Tu, bound to the ribosome with the help of poly(uridylic) acid, Phe-tRNA and guanyl-5"-yl methylene diphosphonate, on the conformation and/or chemical environment of ribosomal proteins has been examined using, as a probe, protein iodination. Ribosomes complexed only with poly(uridylic acid) and Phe-tRNA have been used as a control. EF-Tu on the ribosome significantly increases the iodination of proteins $7, S10 and L3 and decreases that of $21 and L18.
Introduction
During polypeptide chain elongation, amirtoacyltRNA binds to the ribosome as a ternary complex with elongation factor (EF) Tu and GTP (1). After binding has occurred, GTP is hydrolyzed to GDP plus Pi and EF-Tu is released from the ribosome as a binary complex with GDP. Elongation factor Ts participates in the regeneration of the ternary complex from EF-Tu.GDP (1). In model systems, the nonhydrolyzable analogue of GTP guanyl-5'yl methylene diphosphonate (GppCH2 p) can replace GTP in the binding reaction, but, after binding is accomplished, EF-Tu and the analog remain bound to the ribosome (1). Moreover, under these conditions the aminoacyl-tRNA does not interact properly with the acceptor site of the peptidyl transferase centre, since it cannot accept the transfer of the nascent peptide chain (1) and it does not interfere with the reaction of donor site-bound peptidyltRNA with puromycin (2). In this paper we examine the effect of EF-Tu, bound to the ribosome with the help of Phe-tRNA
and GppCH2 p, on the conformation and/or chemical environment of ribosomal proteins. As a probe, we have used protein iodination catalyzed by the enzyme lactoperoxidase. The validity of this method for the study of structural modification of the ribosome has been previously established (3,4).
Materials and methods
The preparation of 1 M NI-I4Cl-washed E. coli MRE 600 ribosomes and purified EF-T (EF-TS + EF-Tu) has been described elsewhere (5, 6). Sources of bovine lactoperoxidase (E.C. 1.11.1.7), glucose oxidase (E.C. 1.1.3.4), Nal2SI (11-17 mCi/ug I), [14 C] phenylalanine (480 mCi/mmol), [3 H] phenylalanine (1 Ci/mmol), and GppCH2p have been previously reported (7, 8). P/'ior to use, ribosomes (100-145 A26o units/ml) were activated by incubating them at 30~ for 30 min in 70 mM NH4C1, 20 mM Mg(acetate)2, 10 mM Tris-HCl pH 7.8, and 0.1 mg/ml poly(U). Binding of labeled Phe-tRNA to ribosomes was carried out in mixtures (50-400/al) containing: 70 mM NH4CI, 10 mM Mg(acetate), 10 mM Tris-HCI, pH 7.8, 15-70 A26 o units/ml of ribosomes complexed with poly(U), 0.58-0.74 /aM [a H] Phe-tRNA (21 cpm/pmol) containing the remaining 19 deacylated tRNA's, 45-110 /ag/ml EF-T and 0.05-0.2 mM of either GTP or GppCH2p. In some experiments [all] Phe-tRNA was replaced by 0.64/aM purified [14 C] Phe-tRNAPh e (950 cpm[pmol), in which case, to decrease the background binding to the ribosomal donor site, 20 /ag/ml deacylated tRNA Phe was also present in the incubation mixture. After incubation at 30~ for 5-10 rain, portions of one tenth volume of the 193
mixture were taken out and analyzed for binding of labeled Phe-tRNA to ribosomes by the nitrocellulose filter technique (9). Incorporation of 12sI was effected by supplementing the remaining 45-360 /al (final volume 50-400/al) with 10/~g/ml lactoperoxidase, 0.1-0.2 mM Nal2SI (120-1200 cpm/pmol), 0.4 mM D-glucose and 7 /ag/ml glucose oxidase. The ionic conditions were identical to those for the binding of labeled Phe-tRNA. After inegbation at 30~ for 20 min to 1 h, the incorporation was stopped by addition of 50 mM 2-mercaptoethanol and incubation at 30~ for 10 min. To determine the amount of ~2sI incorporated, ribosomes in 5/A samples were separated from unreacted Na ~2 s I and iodinated factors and enzymes by passage through small Sepharose 6B columns (8) and their radioactivity was measured. Extraction of ribosomal proteins from the remainder of the incorporation mixture, separation of ribosomal proteins by twodimensional electrophoresis, and measurement of ~2sI incorporated into individual proteins were performed as previously described (7).
Results and discussion To determine the influence of ribosome-bound EFTu on the iodination of ribosomal proteins, we used Phe-tRNA.poly(U).ribosome complexes (prepared in the presence of GTP and EF-Tu) and
GppCH2 p.EF-Tu.Phe-tRNA.poly(U),ribosome complexes. First, we verified that these complexes were stable during the incorporation of iodine. Table 1 shows that, after one hour of iodination with unlabeled Nal, []4C]Phe-tRNA was not released from the first complex and only a small fraction of this ligand was lost from the second. Moreover, with [all] EF-Tu we confirmed that the factor was practically absent from the complex formed with GTP and was present, before and after iodination, in that formed with GppCH2p (Table 1, Expt. 2). (A ratio of [3H]EF-Tu to [14C]Phe-tRNA somewhat lower than 1 was consistently found, possibly due to uncertainties in the specific activity of the factor). We performed twelve 12s I-incorporation experiments using two different ribosomal preparations. On average, 8.4 and 7.6 pmol of labeled Phe-tRNA were bound per A26o unit of ribosomes in the presence of GTP and GppCH2p , respectively. For unknown reasons, incorporation varied from experiment to experiment between 13 and 28 atoms of ] 2sI per ribosome (except in one instance where approximately 5 atoms were incorporated per ribosome) despite our efforts to maintain it as constant as possible. (Variations in incorporation time (see Methods) were employed in attempts to compensate, at least partially, for the different incorporation rates). Nevertheless, in each experiment the incorporation was the same, within the experimental
Table 1. Binding of [~4C]Phe-tRNA and [3H]EF-Tu to ribosomes; effect of iodination. Expt. Guanosine nucleotide
Iodination [ 14C] Phe-tRNA bound (a) (pm~
[ 3H] EF-Tu bound
b/a
(b) o unit ribosomes)
GTP GTP GppCH2P GppCH~p
+ +
10.5 10.2 9.4 7.8
-
-
GTP GppCH2p GppCH:p
+
4.8 3.3 2.4
0.16 2.3 1.8
0.03 0.70 0.75
Binding of [14ClPhe-tRNA to ribosomes in the presence of either GTP or GppCH2p was performed essentially as described in Methods, except that in Expt. 2 [3H]EF-Tu (290 cpm/pmol; a gift from Dr. Pargmeggiani, Ecole Polytechnique, Palaiseau, France) was used. After binding was complete, a portion of the mixture was iodinated, when indicated, with unlabeled NaI for 1 h as described in the text. Bound [, 4C] Phe-tRNA and [SH] EF-Tu were determined in all mixtures by either the nitrocellulose filter technique (Expt. 1) or f'dtration through Sepharose 6B columns (Expt. 2). Values for bound [~H] EF-Tu were corrected by subtraction of blank values obtained in a parallel experiment without [s 4C] Phe-tRNA. 194
Table 2. Incorporation of 12 s I into proteins of r~osomes complexed with poly(U) and Phe-tRNA (abbreviated 70S) and of r~osomes complexed with poly(U), Phr EF-Tu and GppCHap (abbreviated 70S-EF-Tu). Protein
$7 SIO$21 L3 L18
Incorporation (% recovered radioactivRy) 70S
70S-EF-Tu
7.41 + 1.10 0.39 • 0.t5 1.51 • 0.25 0.49 9 0.09 0.92 • 0.18
8.07 • 1.24 0.44 ~O,~8 1.18 • 0;18 0.56 + 0.11 0.84 + 0.19
r
1.09 • t A1 • 0.80 • 1.15 • 0.91 •
p
0.07 0.1-1 0.10 0.14 0.08
0.975-0.99 0.95 -0.975 >0.995 0.95 -0.975 0.95 -0.975
The results, expressed as mean values • standard error of the mean, are average of 12 exp~iments performed as described in Methods. r. is the mean value of the ~atio of incorporation in the presence of bound EF-Tu to incorporation in its absenc~ for each protein in each of the 12 experiments. The prob2ibility of r being different from I is indicated by the value of p in Student's t significance test. Incorporation into proteins L4, L7, LS, L28, L31, L33 and L34 was very low or showed unacceptable scatter. Remaining proteins not shown in the table had r values between 0.91 and 1.09. For each of them p was
ELONGATION FACTOR Tu-INDUCED CONFORMATIONAL CHANGES OF RIBOSOMES DETECTED BY IODINATION
O. MARTINEZ & J. MODOLELL
Instituto de Bioquimica de Macromol~culas, Centro de Biologia Molecular, CSIC and (JAM, Canto Blanco, Madrid-34, Spain (Received April 1, 1979)
Abstract
The effect of elongation factor (EF) Tu, bound to the ribosome with the help of poly(uridylic) acid, Phe-tRNA and guanyl-5"-yl methylene diphosphonate, on the conformation and/or chemical environment of ribosomal proteins has been examined using, as a probe, protein iodination. Ribosomes complexed only with poly(uridylic acid) and Phe-tRNA have been used as a control. EF-Tu on the ribosome significantly increases the iodination of proteins $7, S10 and L3 and decreases that of $21 and L18.
Introduction
During polypeptide chain elongation, amirtoacyltRNA binds to the ribosome as a ternary complex with elongation factor (EF) Tu and GTP (1). After binding has occurred, GTP is hydrolyzed to GDP plus Pi and EF-Tu is released from the ribosome as a binary complex with GDP. Elongation factor Ts participates in the regeneration of the ternary complex from EF-Tu.GDP (1). In model systems, the nonhydrolyzable analogue of GTP guanyl-5'yl methylene diphosphonate (GppCH2 p) can replace GTP in the binding reaction, but, after binding is accomplished, EF-Tu and the analog remain bound to the ribosome (1). Moreover, under these conditions the aminoacyl-tRNA does not interact properly with the acceptor site of the peptidyl transferase centre, since it cannot accept the transfer of the nascent peptide chain (1) and it does not interfere with the reaction of donor site-bound peptidyltRNA with puromycin (2). In this paper we examine the effect of EF-Tu, bound to the ribosome with the help of Phe-tRNA
and GppCH2 p, on the conformation and/or chemical environment of ribosomal proteins. As a probe, we have used protein iodination catalyzed by the enzyme lactoperoxidase. The validity of this method for the study of structural modification of the ribosome has been previously established (3,4).
Materials and methods
The preparation of 1 M NI-I4Cl-washed E. coli MRE 600 ribosomes and purified EF-T (EF-TS + EF-Tu) has been described elsewhere (5, 6). Sources of bovine lactoperoxidase (E.C. 1.11.1.7), glucose oxidase (E.C. 1.1.3.4), Nal2SI (11-17 mCi/ug I), [14 C] phenylalanine (480 mCi/mmol), [3 H] phenylalanine (1 Ci/mmol), and GppCH2p have been previously reported (7, 8). P/'ior to use, ribosomes (100-145 A26o units/ml) were activated by incubating them at 30~ for 30 min in 70 mM NH4C1, 20 mM Mg(acetate)2, 10 mM Tris-HCl pH 7.8, and 0.1 mg/ml poly(U). Binding of labeled Phe-tRNA to ribosomes was carried out in mixtures (50-400/al) containing: 70 mM NH4CI, 10 mM Mg(acetate), 10 mM Tris-HCI, pH 7.8, 15-70 A26 o units/ml of ribosomes complexed with poly(U), 0.58-0.74 /aM [a H] Phe-tRNA (21 cpm/pmol) containing the remaining 19 deacylated tRNA's, 45-110 /ag/ml EF-T and 0.05-0.2 mM of either GTP or GppCH2p. In some experiments [all] Phe-tRNA was replaced by 0.64/aM purified [14 C] Phe-tRNAPh e (950 cpm[pmol), in which case, to decrease the background binding to the ribosomal donor site, 20 /ag/ml deacylated tRNA Phe was also present in the incubation mixture. After incubation at 30~ for 5-10 rain, portions of one tenth volume of the 193
mixture were taken out and analyzed for binding of labeled Phe-tRNA to ribosomes by the nitrocellulose filter technique (9). Incorporation of 12sI was effected by supplementing the remaining 45-360 /al (final volume 50-400/al) with 10/~g/ml lactoperoxidase, 0.1-0.2 mM Nal2SI (120-1200 cpm/pmol), 0.4 mM D-glucose and 7 /ag/ml glucose oxidase. The ionic conditions were identical to those for the binding of labeled Phe-tRNA. After inegbation at 30~ for 20 min to 1 h, the incorporation was stopped by addition of 50 mM 2-mercaptoethanol and incubation at 30~ for 10 min. To determine the amount of ~2sI incorporated, ribosomes in 5/A samples were separated from unreacted Na ~2 s I and iodinated factors and enzymes by passage through small Sepharose 6B columns (8) and their radioactivity was measured. Extraction of ribosomal proteins from the remainder of the incorporation mixture, separation of ribosomal proteins by twodimensional electrophoresis, and measurement of ~2sI incorporated into individual proteins were performed as previously described (7).
Results and discussion To determine the influence of ribosome-bound EFTu on the iodination of ribosomal proteins, we used Phe-tRNA.poly(U).ribosome complexes (prepared in the presence of GTP and EF-Tu) and
GppCH2 p.EF-Tu.Phe-tRNA.poly(U),ribosome complexes. First, we verified that these complexes were stable during the incorporation of iodine. Table 1 shows that, after one hour of iodination with unlabeled Nal, []4C]Phe-tRNA was not released from the first complex and only a small fraction of this ligand was lost from the second. Moreover, with [all] EF-Tu we confirmed that the factor was practically absent from the complex formed with GTP and was present, before and after iodination, in that formed with GppCH2p (Table 1, Expt. 2). (A ratio of [3H]EF-Tu to [14C]Phe-tRNA somewhat lower than 1 was consistently found, possibly due to uncertainties in the specific activity of the factor). We performed twelve 12s I-incorporation experiments using two different ribosomal preparations. On average, 8.4 and 7.6 pmol of labeled Phe-tRNA were bound per A26o unit of ribosomes in the presence of GTP and GppCH2p , respectively. For unknown reasons, incorporation varied from experiment to experiment between 13 and 28 atoms of ] 2sI per ribosome (except in one instance where approximately 5 atoms were incorporated per ribosome) despite our efforts to maintain it as constant as possible. (Variations in incorporation time (see Methods) were employed in attempts to compensate, at least partially, for the different incorporation rates). Nevertheless, in each experiment the incorporation was the same, within the experimental
Table 1. Binding of [~4C]Phe-tRNA and [3H]EF-Tu to ribosomes; effect of iodination. Expt. Guanosine nucleotide
Iodination [ 14C] Phe-tRNA bound (a) (pm~
[ 3H] EF-Tu bound
b/a
(b) o unit ribosomes)
GTP GTP GppCH2P GppCH~p
+ +
10.5 10.2 9.4 7.8
-
-
GTP GppCH2p GppCH:p
+
4.8 3.3 2.4
0.16 2.3 1.8
0.03 0.70 0.75
Binding of [14ClPhe-tRNA to ribosomes in the presence of either GTP or GppCH2p was performed essentially as described in Methods, except that in Expt. 2 [3H]EF-Tu (290 cpm/pmol; a gift from Dr. Pargmeggiani, Ecole Polytechnique, Palaiseau, France) was used. After binding was complete, a portion of the mixture was iodinated, when indicated, with unlabeled NaI for 1 h as described in the text. Bound [, 4C] Phe-tRNA and [SH] EF-Tu were determined in all mixtures by either the nitrocellulose filter technique (Expt. 1) or f'dtration through Sepharose 6B columns (Expt. 2). Values for bound [~H] EF-Tu were corrected by subtraction of blank values obtained in a parallel experiment without [s 4C] Phe-tRNA. 194
Table 2. Incorporation of 12 s I into proteins of r~osomes complexed with poly(U) and Phe-tRNA (abbreviated 70S) and of r~osomes complexed with poly(U), Phr EF-Tu and GppCHap (abbreviated 70S-EF-Tu). Protein
$7 SIO$21 L3 L18
Incorporation (% recovered radioactivRy) 70S
70S-EF-Tu
7.41 + 1.10 0.39 • 0.t5 1.51 • 0.25 0.49 9 0.09 0.92 • 0.18
8.07 • 1.24 0.44 ~O,~8 1.18 • 0;18 0.56 + 0.11 0.84 + 0.19
r
1.09 • t A1 • 0.80 • 1.15 • 0.91 •
p
0.07 0.1-1 0.10 0.14 0.08
0.975-0.99 0.95 -0.975 >0.995 0.95 -0.975 0.95 -0.975
The results, expressed as mean values • standard error of the mean, are average of 12 exp~iments performed as described in Methods. r. is the mean value of the ~atio of incorporation in the presence of bound EF-Tu to incorporation in its absenc~ for each protein in each of the 12 experiments. The prob2ibility of r being different from I is indicated by the value of p in Student's t significance test. Incorporation into proteins L4, L7, LS, L28, L31, L33 and L34 was very low or showed unacceptable scatter. Remaining proteins not shown in the table had r values between 0.91 and 1.09. For each of them p was
