A New Concept of the Function of Elongation Factor 1 in Peptide Chain Elongation

Transcription

1 Eur. J. Biochem. 71, (1976) A New Concept of the Function of Elongation Factor 1 in Peptide Chain Elongation Hans GRASMUK, Robert D. NOLAN, and Jiirgen DREWS Sandoz Forschungsinstitut, Wien (Received August 6/0ctober 15, 1976) An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments, in which mixtures of rh]ef-1, ribosomes from Krebs I1 ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a trna in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound. Elongation factor 1 (EF-l), the enzyme which attaches aminoacyl-trna to the ribosomal A-site in eukaryotic cells, has been shown to occur in multiple forms [l -41. All of these appear to be composed of aggregates containing different numbers of a single polypeptide chain with a molecular weight ranging from to 60000, depending on the type of tissue from which the factor is prepared [l, The light form of the enzyme (EF-~L) can be isolated in good yield from ascites tumor cells [7] and other tissues using a modification of the purification procedure of Kaziro et al. [S]. In aminoacyl-trna binding, EF-~L functions stoichiometrically. Upon addition of elongation factor 2 (EF-2), however, EF-1 functions catalytically [2,3,9]. In order to gain further insight into this change from stoichiometric to catalytic function of EF-1 we examined the binding of tritiated elongation factors to 80-S ribosomes from Krebs I1 ascites cells in the various phases of the peptide elongation process. In our earlier studies [7,10,11] we used sucrose gradient centrifugation for the analysis of complex formation between ribosomes and elongation factors. This method was time consuming and had the disadvantage of subjecting complexes between ribosomes, aminoacyltrna and elongation factors to the disruptive influences of hydrodynamic shearing forces. It was replaced, therefore, in the present study by the more rapid and gentle filtration of ribosomal complexes through small Sepharose 6B columns. Now intermedi- Abbreviations. EF-I, elongation factor 1 ; EF-2, elongation factor 2; GDP, guanosine diphosphate; GTP, guanosine triphosphate; GMP-P(NH)P, guanylyl-imido-diphosphate; GMP-P(CH2)- P, 5 -guanylyl-methylene diphosphonate ; HO-tRNAPh, deacylated phenylalanine-specific trna. ary complexes, not detectable by the earlier method, became visible and could be isolated and studied with respect to their function in the peptide chain elongation cycle. Some new properties essential for defining the role of EF-~L in peptide elongation became evident, as follows. a) EF-IL binds to empty ribosomes alone and more tightly to ribosomes programmed with poly(u) carrying an HO-tRNAPhe molecule in their P-site. b) The binding to ribosomes occurs independently of GTP or aminoacyl-trna. c) The resulting complexes comprising pro- grammed ribosomes and EF-~L are able to accept aminoacyl-trna and GTP. d) Subsequent cleavage of GTP to GDP prepares the aminoacyl-trna for translocation by EF-2. e) Bound EF-1, however, is not released from its ribosomal attachment by GTP cleavage or by EF-2 binding and subsequent translocation. We conclude from these results that EF-~L, once associated with ribosomes, which have completed their initiation arrangement, remains bound until termination has occurred. MATERIALS AND METHODS Chemicals The materials came from the same sources as stated previously [7]. Sepharose 6B was a product of Pharmacia, Fine Chemicals A B (Uppsala, Sweden). [3H]GMP-P(NH)P came from the Radiochemical Centre Amersham, England and was diluted with

2 272 A Novel EF-I Cycle unlabeled material to a specific activity of 0.77 Ci/ mmol. Buffer Solutions Column elution buffer, buffer 1 : 25 (v/v) glycerol, 20 mm KCl, 20 mm Tris-HC1 (ph 7.2 at 20 "C), 10 mm 2-mercaptoethanol and 7 mm Mg (CH3C00)2, buffer 2: 160 mm KCI, 30 mm Tris-HC1 (ph 7.2 at 20 "C) and 6 mm Mg (CH3COO)z. Preparations The labeled and unlabeled biological compounds used in these experiments were prepared in exactly the same manner ; therefore, they could easily be interchanged with one another depending on which compounds' binding to the ribosome one wanted to observe. The ascites EF-1 used in these experiments corresponds to the final product described previously [7]. The monomers (EF-lL), which comprised 88 % of the preparation were not separated from the aggregated form of the factor (EF-1"). The specific activity of the [3H]EF-1 was 44 dis. x min-' x -' (1 = 50 ng). EF-2 was prepared as described previously [7]. The specific activity of 3H-labeled EF-2 was 173 dis. xmin-l x -' (1 = 107 ng). The charging of crude E. coli trna with ['4C]phenylalanine (specific activity 1099 dis. x min-' x -') followed published procedures [12]. Ascites 'run off ribosomes were prepared and freed of elongation factors as described previously [7]. Methods Phe-tRNA binding to ribosomes and poly(pheny1- alanine) synthesis were assayed as described earlier [3]. Formation of ['4C]phenylalanyl-puromycin followed the technique given elsewhere [13]. To estimate ribosomal binding of the different components directly, the reaction mixtures were chromatographed on a 1 x 26-cm Pharmacia column of Sepharose 6B, preequilibrated with buffer 1 in the presence of sodium azide. Immediately before use, the columns were washed free from sodium azide, and a large excess of ribosomes (some nanomoles) and 10mg of bovine serum albumin were applied and eluted in order to reduce the considerable adsorption of EF-1 to the column. After application of the reaction mixture, the column was developed at room temperature with buffer 1, using a flow rate of 18 ml/h. Fractions (0.74 ml, 13 drops) were collected after a passage through an LKB Uvicord ultraviolet scanner and recording system. Depending on the experiment involved the fractions were counted in toto or a small aliquot was taken for radioactivity estimation. RESULTS Fig.1 shows the calibration of Sepharose 6B columns used in this study, with three main constituents participating in the elongation process. As can be seen [3H]EF-1 and [3H]EF-2 were found resolved from the column's void volume, in which the ribosomes appeared. The elution profile of EF-lL shows that this protein has an affinity for and is retained on Sepharose 6B and its complete elution requires much larger volumes than one would expect on the basis of its known molecular weight. For a better understanding of the following we refer the reader to the reaction scheme depicted in Fig. 5 and will make use of the abbreviations contained therein. Binding of ('HIEF-1 to Empty Ribosomes or to Programmed Ribosomes Independent of GTP and Phe-tRNA When empty ribosomes (RSo) are incubated with [3H]EF-1 for 20 min at 37 "C and the reaction mixture is then filtered on a Sepharose 6B column, a small amount of [3H]EF-1 elutes with the ribosomal peak (Fig.2A). A much more substantial attachment of [3H]EF-1 is found when the same experiment is carried out with ribosomes programmed with poly(u) and carrying a deacylated trna (RS2) in their P-site (Fig. 2B). v, 6 c. 3c5 Fr,jcliorl rijiibw Fig. 1. Elution pattern oj ribosomes, [ 3H]EF-l und [ H]EF-2 after.filtration through a typicul Sephurose 6B column. (-----) 400 of washed ribosomes, absorbance at 280 nm (relative scale); (0) 640 of [3H]EF-1;(0) 75 of [3H]EF-2. All components were applied to the column in 150 p1 of elution buffer. The scale for molecular weights was introduced after calibration with defined marker proteins

3 ~ ~ H. Grasmuk, R. D. Nolan, and J. Drews 273 l A In order to demonstrate the specificity of [3H]- EF-1 binding to poly(u)-programmed ribosomes, control experiments were carried out to estimate the amount of the enzyme which was nonspecifically bound to poly(u) in the absence of ribosomes. Correction was made for this low amount (which was less than 10% of the specific binding seen in Fig. 2B) in all the corresponding experiments with the exception of results shown in Table 2, where this value is given. In neither case, did the addition of GTP to the reaction mixture lead to an increase in the amount of [3H]EF-1 bound (results not shown). Fraction number Fig.2. 13H]EF-I binding to empty (A) or poly(u)-programmed ribosomes (S). (A) 0.14-ml reaction mixtures containing 40 mm sucrose, 2.5 "/, (v/v) glycerol, 140 mm KCI, 7 mm creatine phosphate, 6 mm Mg(CH3COO)Z, 4 mm dithioerythritol, 400 washed ribosomes and 640 [3H]EF-1 were incubated for 20 min at 37 "C. (B) 400 of ribosomes were first incubated in 0.17-ml reaction volumes for 10 min at 37 "C with 160 pg poly(u) and 500 deacylated trnaphe. After addition of 640 [3H]EF-1 incubation was continued for 20 min. Salt concentrations were as indicated above. In a control experiment, ribosomes were omitted and the results are presented in (B) (0). After incubation the reaction mixtures were applied to the Sepharose 6B columns without cooling and chromatographed as described in Methods. 5O-pl aliquots of the fractions appearing in the ribosomal peak area and 0.74 ml of the remaining fractions were assayed for radioactivity. The remaining portions of the ribosomal peak fractions (9 and 10) were stored at 0 "C for further experiments depicted in Table 1. (-----)Absorbance at 280nm, relative scale, (0)[3H]EF-1 in the presence of ribosomes; (0)[3H]EF-1 in the absence of ribosomes Prebinding of EF-1 to Empty or Programmed Ribosomes Is Functional Small aliquots of the ribosomal peak shown in Fig. 2A containing the ribosome.ef-1 complex were supplemented stepwise with poly(u), GTP, [14C]PhetRNA and EF-2. The results obtained after filtration of the corresponding reaction mixtures through nitrocellulose filters are shown in Table 1 (A). It is obvious that prebinding of EF-1 to empty ribosomes is functional. Binding of [I4C]Phe-tRNA to this complex is dependent on the addition of poly(u) and stimulated significantly by GTP. Upon addition of EF-2, bound Phe-tRNA is converted to trichloroacetic-acid-precipitable poly( [14C]phenylalanine). When the same experiment was carried out with aliquots of fraction 9 (Fig. 2 B), which contained most of the ribosome. poly(u). trnaphe. EF-1 complex Table 1. EF-1 bound to ribosomes in the absence of GTP and Phe-tRNA is functionally active (A) 100-p1 aliquots of fraction 10 obtained after gel filtration of the ribosome. [3H]EF-1 complex (Fig. 2A) were supplemented stepwise as indicated in the table with 62 [14C]Phe-tRNA, 80 pg poly(u), 20 pm GTP, 2.5 EF-2, 30 pg creatine kinase and salts as indicated in the legend to Fig.2. After incubation for 30 min at 37 "C the reactions were stopped by addition of 2 ml of ice-cold buffer 2 and filtered through nitrocellulose filters as described previously 131. The filters were dried and counted for I4C radioactivity in a liquid scintillation counter. (B) Experiments carried out with complexes comprising ribosomes, [3H]EF-1, poly(u) and trnaphe (fraction 9 from ribosomal complex depicted in Fig. 2B). Otherwise, conditions were as described under (A). The control for nonenzymic Phe-tRNA binding was performed analogously with a ribosomal complex containing poly(u) and trnaphe but lacking [3H]EF-1 Preformed and isolated complex Additions [14C]Phe-tRNA bound Acid-insoluble [14C]Phe [14C]Phe-tRNA poly(u) GTP EF-2 -~ A RS.EF B RS trnaphe poly(u) EF ~ RS trnaphe poly(u) _

4 274 A Novel EF-1 Cycle f i r I trac::;~ riwiti.r Fig. 3. Binding of f3h]gmp-p(nh)p (A) and ['HIGTP (B) to ribosomes. 400 of washed ribosomes, 80 pg of poly(u), approximately 280 of unlabeled Phe-tRNA, 1 nmol of unlabeled EF-1 and either 20 pm ['HIGMP-P(NH)P or [3H]GTP plus 50 pg creatine kinase were incubated in 0.25-ml volumes containing salts as indicated under Fig. 2 for 30 min at 37 "C. After incubation samples were applied to Sepharose 6B columns, chromatographed and analyzed by liquid scintillation counting (-----) Absorbance at 280 nm, relative scale; (H) distribution of [3H]GMP-P(NH)P (A) and [3H]GTP(B) Table 2. Presence of [3H]EF-1 on ribosomes programmed with poly(u) after interaction with Phe-tRNA, GTP or GMP-P(NH)P and EF ml reaction volumes contained in addition to 320 [3H]- EF-1, 200 unlabeled Phe-tRNA, 80 pg poly(u) and salts as described in Fig. 2: 30 pm GTP and 30 pg creatine kinase (Expt l), or 200 washed ribosomes, 30pM GTP and 30pg creatine kinase (Expt 2), or 200 washed ribosomes, 170 pm GMP-P- (NH)P (Expt 3), or 200 washed ribosomes, 30 pm GTP and 30 pg creatine kinase plus 10 of EF-2 (Expt 4). Incubations lasted for 20 min at 37 "C. Filtration was carried out on Sepharose 6B columns as described under Methods. 3H radioactivity appearing in the ribosomal peak fractions (7 to 12) was counted and expressed as picomoles EF-1 Expt Additions to ['HIEF-1 ['HIEF-1 eluting with the ribosomal region 1 Phe-tRNA + poly(u) + GTP 3 2 Ribosomes + Phe-tRNA + POlY(U) 66 3 Ribosomes + Phe-tRNA + POIY(U) + GMP-P(NH)P 62 4 Ribosomes + Phe-tRNA + poly(u) + GTP + EF-2 53 (RS3), [14C]Phe-tRNAPhe binding was now much enhanced and appeared less dependent on the addition of GTP. The introduction of additional poly(u) into the incubation mixtures had no influence on PhetRNA binding. A control experiment in which a ribosome. trnaphe. poly(u) complex, containing no EF-1 was incubated with [14C]Phe-tRNA resulted in very little binding of Phe-tRNA to the ribosomal Table 3. ['H]EF-2 binding to empty or poly( U)-programmed ribosomes Each 0.1-ml reaction volume contained: salts as indicated in the legend of Fig. 2, 200 of washed ribosomes, 88 ['H]EF-2 and 28 pm GTP or 50 pm GMP-P(NH)P as indicated. The reaction mixtures were analyzed by gel filtration on Sepharose 6B columns and the amounts of [3H]EF-2 eluting with the ribosomal peak were calculated. Expt 4 was performed in two steps: 200 of washed ribosomes were first incubated with 80 pg poly(u) and 250 deacylated trnaphe for 10 min, at 37 "C. 88 ['HI- EF-2 and 50 pm GMP-P(NH)P were then added and incubations continued for 20 min at 37 "C. ['H]EF-2 bound to the ribosomal peak fractions (fractions 7-10) were analyzed by gel filtration Expt Ribosomes Guanine [3H]EF-2 bound nucleotide to ribosomes 1 Empty GMP-P(NH)P GTP Poly(U)- programmed GMP-P(NH)P complex, confirming the absolute requirement for EF-lL in this process (Table 1, B). GDP Remains Bound to the Ribosomal Complex following y-phosphate Cleavage of GTP The cleavage of GTP to GDP and phosphate after completion of EF-1 -catalyzed binding of PhetRNA to the ribosomal A-site was demonstrated in a previous publication [3]. In order to determine if the

5 H. Grasmuk, R. D. Nolan, and J. Drews 275 GDP moiety is subsequently released from the ribosomal complex, binding reactions between RS3 complexes and Phe-tRNAPhe were carried out with [3H]- GTP and in parallel with the analogue t3h]gmp-p- (NH)P from which the y-phosphate cannot be split. As shown in Fig.3, the elution profiles of both reaction mixtures were almost identical. It is clear from Fig. 3 B that GDP remains bound after y-phosphate splitting. The fact that the radioactivity peak eluting with the ribosomes in this figure (GTP/GDP binding) is less symmetric than the peak representing [3H]- GMP-P(NH)P bound to the ribosomal complex (Fig. 3A) may indicate that the interaction between GDP in complex RS5 is not very stable. Evidence for the Presence of r3h]ef-1 on Complex RS5 Ribosomes, poly(u), trnaphe, [3H]EF-1 and PhetRNA were incubated with GTP at a concentration which had previously been found to be saturating with respect to the stimulation of Phe-tRNA binding. In addition, a GTP-regenerating system (creatine phosphate/creatine kinase) was introduced into the reaction mixture. After incubation of this mixture and filtration on a Sepharose 6B column the amount of [3H]EF-1 appearing in the ribosomal peak was comparable to that measured in a parallel experiment in which GMP-P(NH)P had been used instead of GTP (Table 2, Expts 2 and 3). Experiment 2 was repeated in the absence of ribosomes to estimate the low nonspecific binding of t3h]ef-1 to poly(u), which appeared in the ribosomal peak region of the columns (Table 2, Expt 1). Interaction of r3h]ef-2 with Empty or Poly( U) -Programmed Ribosomes and the Influence of GTP Substantial amounts of [jh]ef-2 are bound to empty ribosomes in the absence of guanine nucleotide when assayed by the column method (Table 3, Expt 1). Binding was significantly stimulated in the presence of GMP-P(NH)P (Table 3, Expt 2), but reduced to the amount observed in the nucleotide-free system when GTP was present (Table 3, Expt 3). The extent to which GMP-P(NH)P stimulated the binding of [3H]- EF-2 was not changed when ribosomes programmed with poly(u) and trnaphe, instead of empty ribosomes, were used (Table 3, Expt 4). These experiments suggest that EF-2, in contrast to EF-1, may be released from the ribosome upon cleavage of GTP. In order to confirm this suggestion, we compared the binding of [3H]EF-2 to an isolated A-site ribosomal complex (RS5). RS5 complexes were formed with unlabeled components, isolated by gel filtration, incubated with Table 4. Activity of isolated A-site complexes (RS5) in poly(plie) synthesis and binding of [3H]EF-2 to such complexes Complex RS5 was formed by incubation of 400 of washed ribosomes, 80 pg poly(u), 200 of unlabeled Phe-tRNA, 24 pm GTP, 50 pg of creatine kinase, 1 nmol of EF-1 and salts as described in the legend of Fig. 2. After incubation for 40 min at 37 "C the complexes were isolated by gel filtration. Peak fractions (8 and 9) containing the ribosomal complex were pooled. A 260-p1 aliquot of the pooled complex was then incubated with 96 [14C]PhetRNA, 30 pm GTP, 80 pg creatine kinase and a salt mixture to give final salt concentrations as indicated under Fig.2. After incubation for 30 min at 37 "C the mixture was filtered through nitrocellulose and assayed for the binding of [14C]Phe-tRNA (Expt 1). The second 260-p1 aliquot was supplemented as described above but received 88 of [3H]EF-2 in addition (Expt 2). Two additional aliquots ofthe pooled RS5 fractions were supplemented with 88 [3H]EF-2 and with either 25 pm GTP (Expt 3) or 33 pm GMP-P- (NH)P (Expt 4). The mixtures were incubated for 30 min at 37 "C and subjected to gel filtration. 'H radioactivity associated with the ribosomal peak (fractions 7-10) is expressed as picomoles of ['H]EF-2 bound to ribosomes (complex RSs) Expt Additions [14C]Phe-tRNA bound to isolated RS5 complex 1 GTP + [14C]Phe-tRNA GTP + [14C]Phe-tRNA + [3H]EF Nucleotide [3H]EF-2 bound to ribosomal complexes RS5 3 GTP GMP-P(NH)P 7.85 [3H]EF-2 in the presence of GTP or GMP-P(NH)P, and rechromatographed to analyze the amount of bound [3H]EF-2. When the RS5 complex is incubated with [3H]EF-2 and GTP, 2.6 of t3h]ef-2 elute with the ribosomal peak (Table 4, Expt 3). In contrast, almost 8 of [3H]EF-2 were eluted with the ribosomal peak when the same experiment was carried out with GMP-P(NH)P instead of GTP. These findings lend further support to the proposal that EF-2 is released from its ribosomal binding site after cleavage of GTP. In the experiments just described it was important to show that both elongation factors were functioning. It is shown in Table'4 that complex RS5, upon incubation with GTP and [14C]Phe-tRNA, binds relatively little additional [14C]Phe-tRNA. This is due to the fact that the A-sites of these complexes are already occupied (Expt 1). After the addition of ['4C]PhetRNA and EF-2 however, substantial amounts of ['4C]phenylalanine are incorporated into polypeptide chains (Expt 2). These two experiments clearly show that both the prebound EF-1 and the added EF-2 functioned catalytically.

6 ~~ 276 A Novel EF-1 Cycle Interaction of Ribosomal Complex RS5 with EF-2 Does Not Lead to the Release of r3h]ef-1 In Table2, Expt 4 shows the result of column analysis of an incubation mixture containing ribosomes, Phe-tRNA, poly(u), [3H]EF-1, GTP and EF-2. Obviously, the amount of ['HIEF-l found in the ribosomal region is not significantly reduced, com- Table 5. Interaction of EF-2 with ribosomal complex RS5 does not lead to the release o~[~h]ef-i RS5 complexes were formed by incubating 400 of washed ribosomes, 80 pg poly(u), 280 unlabeled Phe-tRNA, 1280 [3H]EF-1, 25 pm GTP, 60 pg creatine kinase and salts as described in Fig.2 in a 0.3-ml volume for 40 min at 37 "C. After filtration of the reaction mixture through a Sepharose 6B column, 400-pl aliquots of the pooled ribosomal peak fractions (9 and 10) eachcontaining 25 of [3H]EF-1 bound to 45 of ribosomes, were incubated with the following additions: 25 pm GTP (Expt l), 25 pm GTP with 10 of EF-2 (Expt 2), and 25 pm GTP, 10 of EF-2 and 530 of unlabeled EF-1 (Expt 3) for 10 min at 37 "C. Subsequently, the reaction mixtures were again subjected to gel filtration. 3H radioactivity appearing in the ribosomal peak fractions (7-12) was counted and expressed as picomoles of EF-1 bound to ribosomes Expt Additions to the [3H]EF-1 remaining isolated A-site complex bound to ribosomes GTP EF-2 EF pared to the values obtained in the Expts 2 and 3, in which EF-2 had not been present. Thus the interaction of EF-2 with the ribosomal complexes and several rounds of translocation which are likely to have occurred in these experiments did not diminish the number of EF-1 molecules attached to the ribosomes. In order to eliminate the possibility that [3H]EF-1 had been replaced by EF-2 during translocation and subsequently reattached to the ribosomes, we divided the isolated RS5 pool into three portions (each containing 45 ribosomes, 25 of which carried [3H]- EF-1). These were subsequently incubated either with GTP alone, or GTP and 10 of EF-2 or with the latter two components in combination with a 20-fold excess of unlabeled EF-1 which would dilute any labeled EF-1 released from the ribosome. It is obvious from Table 5 that the amount of [3H]EF-1 remaining attached to the ribosomal complex RSs was approximately the same as found on RS5. This result excludes the possibility of a recyling of [3H]EF-1 in the process of translocation. Evidence that the Interaction of EF-2 with Ribosome Complex RS5 Leads to Translocation The conclusion drawn in the last paragraph would be invalid without the demonstration that the interaction of ribosomal complex RS5 with EF-2 had indeed triggered translocation of the peptidyl-phe-trna moiety on the ribosomal decoding site into the peptidyl site. RS5 complexes were therefore formed as indicated Fraction numbei Fig.4. Isolation of ribosomal complexes loaded with ['4C]Plie-tRNA and EF-I in the absence (A) or presence (B) of GTP ml reaction volumes containing salts as indicated under Fig.2, 400 of washed ribosomes, 80 pg of poly(u) and 290 of [I4C]Phe-tRNA were preincubated for 5 min at 37 "C. After addition of 1 nmol of unlabeled EF-1 (A) or 1 nmol of EF-1 plus 25 pm of GTP (B) incubations were continued for another 30 min. The reaction mixtures were analyzed on Sepharose 6B columns. 50-pI aliquots of fractions appearing in the ribosomal area and 740 p1 ofthe remaining fractions were assayed for radioactivity. The larger part of the ribosomal peak fractions were stored in ice for experiments shown in Table 6. (-----) Absorbance at 280 nm, relative scale; (A) 14C radioactivity

7 ~ ~~~ ~~ ~ ~- ~~~~ H. Grasmuk, R. D. Nolan, and J. Drews 277 Table 6. The transfer of [14C]phenylalanine to puromycin after addition of EF-2 to isolated A-site complexes (RS5) 150-pl aliquots of fractions 9 and 10 containing RS5 complexes formed and isolated in the presence of [I4C]Phe-tRNA as described in Fig.4 were supplemented with 26 pm GTP, 40 pg creatine kinase and 5 EF-2. GTP was replaced by either 26 or 260 pm GMP- P(NH)P (+ +) as indicated in the table. Salt concentrations were adjusted to be the same as indicated in the legend to Fig.2. Incubations were carried out for 20 min at 37 "C. Subsequently the puromycin reaction was carried out with 1 mm puromycin as described previously [13] Expt Additions to A-site complex ['4C]Phenylalanineformed in the absence of GTP puromycin 1 GTP GMP-P(NH)P EF ~ _ 6 _ ~ ~ - Additions to A-site complex formed in the presence of GTP I above in the presence of ['4C]Phe-tRNA in the absence or in the presence of GTP and isolated by gel filtration as shown in Fig.4A and B. The passage through Sepharose columns results in some loss of ribosomebound [14C]Phe-tRNA, which has not been stabilised by GTP. In contrast the filtration of the complexes through nitrocellulose filters (Table 1) is very rapid and permits little distinction between the binding of [14C]Phe-tRNA with and without GTP. Aliquots from these complexes were then incubated with EF-2 in the absence or in the presence of GTP or GMP-P- (NH)P. As shown in Table 6, the addition of EF-2 and GTP leads to a definite increase in the amount of the ['4C]phenylalanine reacting with puromycin. GMP-P(NH)P, if introduced into the reaction mixtures in the same concentration as GTP does not replace the natural nucleotide. Upon increasing the GMP-P(NH)P concentrations by a factor of 10, however, translocation clearly occurs. Two conclusions can be derived from this observation. In the first place, interaction of ribosomal complex RS5 with EF-2 and GTP leads to translocation. Secondly, cleavage of GTP does not seem to be a prerequisite for translocation itself but rather for the release of EF-2 from the ribosome after translocation has occurred. Our observation that high concentrations of GMP-P- (NH)P can replace GTP in triggering translocation by EF-2 agrees with results communicated earlier by Lee et al. [14]. DISCUSSION By the combined use of purified tritiated elongation factors and a rapid and gentle method for the analysis and isolation of ribosomal complexes representing critical intermediary products of the peptide chain elongation cycle, it has, for the first time, been possible to delineate the function of EF-1,. during peptide chain elongation in greater detail. In contrast to conclusions put forward previously by others [ as well as by ourselves [lo], we have shown here that EF-1 L binds to the ribosome independently of aminoacyl-trna and GTP and that it remains bound during all phases of peptide chain elongation. While empty ribosomes interact only weakly with EF-1, substantial amounts of the factor become attached to ribosomes which are programmed with a polynucleotide chain and carry a trna in their P- site. Most likely, therefore, EF-1,. enters the ribosomal A-site after completion of the initiation process. In an earlier study [lo] we had been unable to detect the stable interaction between EF-1 and ribosomes in the presence of GTP or GDP. After replacement of GTP by the analogue GMP-P(CHz)P, however, EF-1 remained bound to the ribosomes and could be detected by sucrose gradient centrifugation. A significant displacement of [3H]EF-1 together with the aminoacyl-trna on the ribosomal peak to lighter parts of the gradients was regularly observed and interpreted as indicating sensitivity of the ribosome. EF-1. aminoacyl-trna complexes to the hydrodynamic shearing forces inherent in sucrose gradient analysis. The gel filtration method employed in the present study allows the isolation of ribosome. [3H]EF-1 complexes formed independently of any guanine nucleotide. Using gel filtration, the elution profiles representing the binding of radioactive elongation factor 1 to ribosomes programmed with poly(u) and carrying an HO-tRNAPhe in their P-site, are sharp and symmetric and show no smearing effects. The complexes formed between programmed ribosomes and EF-1 were shown to be functional: they readily accept Phe-tRNA and GTP and are capable of carrying out peptide chain elongation after the addition of EF-2. GTP does not seem to be an absolute requirement for the binding of aminoacyl-trna by ribosomal complexes carrying EF-1. [3H]EF-1 and aminoacyl-trna when bound to ribosomes in the presence of GMP-P(NH)P were always found to be displaced together from the ribosomal peak by hydrodynamic shearing or addition of EF-2 [lo] while aminoacyl-trna attached to ribosomes by EF-1 in the presence of GTP remained firmly bound under the same conditions.

8 278 A Novel EF-1 Cycle OH- trna GTP u-u-u-u-u-u T u-u-u-u-u-u T EF-II T RSo RSZ RS3 RSL R55 RS6 pep-trna GTP empty ribosomes programmed ribosomes, occupied in the P-site initiation or post - translocat ion complex, ready for A-site occupation A-site complex before accomodation A-site complex after peptidyl transfer, reaay for translocation transient post- translocatlon complex,only detectable in the absence of GTP splitting peptidyl- trna quanosine triphosphate GMP-P(NH)F guanj.l,lyllmido-diphosphate GDP Phe-tRNA? OH- trna I GTP GT P Fig.5. Proposed reaction scheme of EF-1L and EF-2 during the elongation cycle. Details are described in the Discussion In view of these observations we propose that GTP is necessary for the proper attachment of aminoacyltrna to ribosomes carrying EF-1 and that this attachment involves the - CCA - amino acid region of the molecule [21]. We also propose that upon hydrolysis of GTP to GDP and phosphate the association between EF-1 and the aminoacyl-trna is modified such that the aminoacyl-trna becomes available for transpeptidation and translocation. Obviously, the GDP moiety resulting from the cleavage of GTP after aminoacyl-trna binding remains attached to the ribosomal complex. Preliminary data, not presented in this paper, suggest that GDP is released from its binding to EF-1 as a result of the interaction of the RS5 complex with EF-2. The sequence of events reported here for the binding of EF-1 to programmed ribosomes and the subsequent addition of aminoacyl-trna and GTP to such complexes seems to resolve the controversial question of the formation of a ternary complex comprising aminoacyl-trna, GTP and EF-1. The formation of such complexes had previously been inferred from results obtained with EF-1 preparations from calf brain [5], sheep brain, calf liver [18], pig liver [19], rabbit reticulocytes [20,21], yeast [22], wheat embryo [23], and wheat germ [6]. Ternary complexes comprising EF-2 preparations from Krebs I1 ascites cells could never be isolated [24]. Moreover, preincubation of aminoacyl-trna with EF-1 and GTP did not enhance the rate of the aminoacyl-trna s subsequent binding to ribosomes. In view of the instability of this and other ternary complexes formed with eukarotic EF-1 preparations and considering the avidity with which ribosome. poly(u). trna complexes bind EF-1, the postulated physiological role of the ternary complex can now be abandoned. The weak interactions observed in the absence of ribosomes may be interpreted as residual recognition between the three components. If EF-1 remains attached to the ribosome during the elongation process, it should not interfere with the binding of EF-2. However, previous studies from other laboratories [25,26] as well as from our own [lo] have indicated that the two elongation factors apparently occupy partly overlapping binding sites on the ribosome. Thus, Richter first showed that ribosomes complexed with GMP-P(CH2)P and EF-2 can no longer serve as substrates for the attachment of aminoacyl-trna catalyzed by EF-1 [17]. This finding was confirmed by Nombela and Ochoa [15] as well as by ourselves [lo]. These apparent contradictions may, however, be interpreted in terms of interference between EF-2 and aminoacyl-trna bound in the presence of GMP-P(CHz)P, rather than between EF-2 and EF-1. We have recently reported that the plant toxins abrin and ricin and pokeweed antiviral protein (unpublished) inhibit the ribosomal binding of EF-2 without in any way affecting the interaction between ribosome and EF-1 [ll]. In addition recent unpublish-

9 H. Grasmuk, R. D. Nolan, and J. Drews 219 ed experiments have clearly demonstrated that the two factor binding sites are completely separate. The results presented in this paper are summarized graphically in Fig. 5, which illustrates the novel suggestion that, at least during translation, EF-lL becomes essentially a ribosomal protein. The experiments discussed in this paper have been concerned with the metabolism of the monomeric form of EF-1 (EF-1L). Recent experiments have shown that the aggregate form (EF-1H) also binds to ribosomes in a similar way. Details for the mechanism of disaggregation following this binding will be published shortly. The competent technical assistance of Miss E. Bauer and Mr K. Bednarik is gratefully acknowledged. We are grateful to Mrs G. Spiegelhofer for typing the manuscript. REFERENCES 1. McKeehan, W. L. & Hardesty, B. (1969) J. Bid. Chem. 244, Collins, J. F., Moon, H. M. & Maxwell, E. (1972) Biochemistry, 11, Drews, J., Bednarik, K. & Grasmuk, H. (1974) Eur. J. Biochem. 41, Bollini, R., Soffientini, A. N., Bertani, A. & Lanzani, G. A. (1974) Biochemistry, 13, Moon, H. M., Redfield, B., Millard, S., Vane, F. & Weissbach, H. (1973) Proc. Nut/ Acad. Sci. U.S.A. 70, Golinska, B. & Legocki, A. B. (1973) Biochim. Biophys. Actu, 324, Grasmuk, H., Nolan, R. D. & Drews, J. (1976) Eur. J. Biochem. 67, Iwasaki, K., Wagata, S., Mizumoto, K. & Kaziro, Y. (1974) J. Bid. Chem. 249, Lin, S. Y., McKeehan, W. L., Culp, W. & Hardesty, B. (1969) J. Biol. Chem. 244, Nolan, R. D., Grasmuk, H. &Drews, J. (1975) Eur. J. Biochem. 50, Nolan, R. D., Grasmuk, H. & Drews, J. (1976) Eur. J. Biochem. 64, Turnowsky, F. & Hogenauer, G. (1973) Biochem. Biophys. Res. Commun. 55, Leder, P. & Bursztyn, H. (1966) Proc. Nut1 Acud. Sci. U.S.A. 56, Lee, T., Tsai, P. & Heintz, R. (1973) Arch. Biochem. Biophys. 156, Nombela, C. & Ochoa, S. (1973) Proc. Nut/ Acud. Sci. U.S.A. 70, Modolell, J., Cabrer, B. & Vazquez, D. (1973) Proc. Nut1 Acud. Sci. U.S.A. 70, Richter, D. (1973) J. Biol. Chem. 248, Weissbach, H., Redfield, B. & Moon, H. M. (1973) Arch Biochem. Biophys. 156, Nagata, S., Iwasaki, K. & Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, Ravel, J. M., Dawkins, R. C., Lax, S., Odom, 0. W. & Hardesty, B. (1973) Arch. Biochem. Biophys. 155, Nombela, C., Redfield, B., Ochoa, S. & Weissbach, H. (1976) Eur. J. Biochem. 65, Richter, D. (1970) Biochem. Biophys. Res. Commun. 38, Lanzani, G. A,, Bollini, R. & Soffientini, A. N. (1974) Biochim. Biophys. Acta, 335, Nolan, R. D., Grasmuk, H., Hogenauer, G. & Drews, J. (1974) Eur. J. Biochem. 45, Okata, T. & Kaji, A. (1973) Eur. J. Biochem. 38, Okura, A,, Kinoshita, T. & Tanaka, N. (1970) Biochem. Bio- phys. Res. Commun. 41, H. Grasmuk, R. D. Nolan, and J. Drews, Sandoz Forschungsinstitut, GmbH, BrunnerstraRe 59, A-1235 Wien, Austria