Eur. J . Biochem. 58, 501 -510 (1975)
The Importance of Escherichia coli Ribosomal Proteins L1, L11 and L16 for the Association of Ribosomal Subunits and the Formation of the 70-S Initiation Complex Mirabotalib KAZEMIE Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmaiin, Berlin-Dahlcm (Received March 20 July 10, 1975)
50-S subunits were washed with LiCl solutions of different concentrations. After washing with 1 M LiCl solution the particles lost their ability to attach either to 30-S subunits or to the AUG . 30-S subunit . fMet-tRNAfMe'complex or to a poly(U) . 30-S subunit . Phe-tRNAPhecomplex. Those proteins which were removed by LiCl were fractionated on a Sephadex G-100 column. Of the fractionated proteins only the combinations L1 and L11 or L1 and L16 were essential for the association of 50-S 1.O cores (particles prepared by washing 50-S subunits in 1 .O M LiCl) with 30-S subunits. These three proteins were also required for the formation of a stable complex between 50-S 1 .O cores, mRNA, 3 0 3 subunits and aminoacyl-tRNA.
During the process of protein synthesis in bacteria the 50-S subunits become associated with 30-S subunits which are already charged with mRNA (for review see [1,2]). This attachment of 50-S subunits to 30-S subunits must take place in a very specific manner, in order to guarantee that the recognized aminoacyl-tRNA species are bound to their proper sites on the 50-S subunit. Thus, the formation of a 70-S initiation complex requires the interaction of a 50-S subunit with both the aminoacyl-tRNA moiety and the 30-S subunit moiety of the 30-S initiation complex. The purpose of this paper is the identification of those 50-S ribosomal proteins which are essential for the association of subunits and the formation of a 7 0 4 initiation complex.
with buffers containing LiCl(10mM Tris-HC1,pH 7.5; 10 mM magnesium acetate; varying concentrations of LiCl: 0.5 M, 1.0 M, 2.0 M, 3.0 M) to 15 A260 unitsjml and incubated for 5 h at O'C, with gentle stirring. The suspensions were centrifuged at 250000 x g for 5 h in a Spinco 60 Ti rotor at 2°C. The supernatants (split proteins) were collected, dialyzed overnight against core buffer (20 mM Tris-HC1, pH 7.5; 20 mM magnesium acetate; 1 mM EDTA; 200 mM NH,Cl; 2 mM 2-mercaptoethanol) with two changes. Thereafter, the samples were lyophilized, taken up in 4 ml bidistilled water, dialyzed against protein buffer (the same as core buffer, except that it contained 400 mM NH,Cl), lyophilized, taken up in 1 ml water and dialyzed once more against protein buffer. The samples were clarified by centrifugation at 10000 rev./min for 10 min, frozen in liquid nitrogen and kept at - 80 "C for use as split proteins.
MATERIALS AND METHODS
Preparation and Analysis of LiCl Cores and Split Proteins from 50-S Ribosomal Subunits
The pellets obtained after the 250000xg centrifugation (cores) were suspended in core buffer at a concentration of 500 A260 units/ml, dialyzed overnight against core buffer with two changes, clarified as above and kept at - 80°C for use (as 0.5 cores, 1.0 cores, 2.0 cores, and 3.0 cores respectively, according to the concentrations of LiCl which were used for the extraction).
'Cores' and 'split proteins' were prepared as described by Nierhaus and Montejo [4].1000 A260 units of 503 subunits were incubated for 20 min at 37°C in 2ml buffer (10mM Tris-HC1, pH 7.5; 10 mM magnesium acetate; 60mM NH,Cl; 6mM 2-mercaptoethanol). Subsequently the suspensions were diluted
The protein compositions of cores and split proteins were determined by the two-dimensional gel electrophoresis technique of Kaltschmidt and Wittmann [5]. The fractionation of split proteins was carried out on a Sephadex G-100 column as described by Nierhaus and Montejo [4].
Preparation of Ribosomal Subunits
Ribosomal subunits were prepared according to the procedure reported by Schreiner and Nierhaus [3].
502
Functions of Ribosomal Proteins L1, L11 and L16
-Sedimentation
direction
Fig. 1. The effect of LiCl washing of 50-S subunits on their ability to ussociate with 3 0 3 subunits. Cores from 50-S subunits were prepared as described under Materials and Methods. 50 pmol of each core was incubated with or without 50 pmol of 30-S subunits for 10 min a t 22°C in 5O-pl reaction mixtures containing 20 mM Tris-HCI, pH 7 . 5 ; 30 mM magnesium acetate; 150 mM NH,CI and 2 mM 2-mercaptoethanol. At thc cnd of the incubation the samples were layered on to 10-30% sucrose gradients. The gradients had the same salt composition as the samples. Centrifugation was performed at 30000 rev./min in a Spinco SW 40 rotor for 5 h at 3°C. The monitoring of the gradients at 260 nm is described under Materials and Methods. (A) 5 0 4 subunits, (B) 0.5 cores, (C) 1.0 cores, (D) 2.0 cores, (E) 3.0 cores. 30-S subunits were added to (F) 50-S subunits, (G) 0.5 corcs, (H) 1.0 cores, (I) 2.0 cores, (J) 3.0 cores
Partial Reconstitution of 50-S Subunits
Reconstitution experiments were performed by incubating 50-S cores with split proteins in reaction mixtures containing 20 mM Tris-HC1, pH 7.5 ; 20 mM magnesium acetate; 1 mM EDTA; 350 mM NH,C1 and 2 mM 2-mercaptoethanol for 90 min at 50 "C [6].
Binding of Aminoacyl-tRNAs to Subunits The procedure for binding aminoacyl-tRNA has been described elsewhere [7]. The reaction products were assayed either by the Millipore technique of Nirenberg and Leder [8] or on 13 ml10- 30 % sucrose gradients, which were prepared in 14-ml tubes of a Spinco SW 40 rotor. The centrifugations were performed at 30000 rev./min for 5 h at 3 "C. The gradients were pumped out from the bottom of the tubes at a constant rate. The effluents were monitored with a flow-through cell (l-cm light path) at 260 nm. For measuring the radioactivity 20-drop fractions were collected; 5-pl aliquots of a serum albumin solution (10 mg/ml) were added to the fractions, which were subsequently made 10% with respect to trichloroacetic acid at 0 "C. The precipitates were collected on
Millipore filters and the radioactivity was measured in a liquid-scintillation spectrometer (Packard, model 3380). Counting efficiency for 3H was 38 %.
RESULTS The Eflect of LiCl Washing on the Ability of 5 0 3 Subunits to Associate with 30-S Subunits
At distinct concentrations of LiC1, definite groups of proteins can be extracted from the ribosomal particles [9]. In order to determine which groups contain the protein required for the association of 50-S subunits with 30-S subunits, one has to find the concentration of LiCl at which this function is impaired. For this purpose 50-S subunits were washed with increasing concentrations of LiCl(0.5 M, 1.0 M, 2.0 M, and 3.0 M) as described under Materials and Methods. The 0.5, 1.0, 2.0, and 3.0 cores were incubated with 30-S subunits and the reaction products were analysed on sucrose gradients (Fig. 1). The upper part of the figure shows the sedimentation pattern of the 5 0 4 core particles in the absence of 30-S subunits.
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Fig.2. The effect of' LiCl washing (u' SO-S subunits on their ability to .forin comjdexes with 30-S subunits rind [3H]Phe-tRNAPh'. Cores from 50-S subunits were prepared as described under Materials and Methods. 50pmol of 30-S subunits was incubated at 22'C for 10 min with 50 pmol of (A) 50-S subunits, (B) 0.5 cores, (C) 1.0 cores, (D) 2.0 cores and (E) 3.0 cores, or (F) alone in SO-pI reaction mixtures containing 20 m M Tris-HC1, pH 7.5; 30 mM magnesium acetate; 150 mM NH,CI; 2 mM 2-mercaptoethanol; 15 pg poly(U), 40 pmol ['HH]Phe-tRNAPhe(spec. act. 1152 mCi/mmol, 52 charged). The monitoring of the gradients at 260 nm, fractionation and Absorbance at 260 nm, preparation of the fractions for measuring- radioactivity are described under Materials and Methods. (-) ( ~radioactivity b)
The lower part of the figure shows the result of mixing these different core particles with 30-S subunits. Whereas 50-S subunits and (to a lesser extent) 0.5 cores could form 70-S ribosomes with 30-S subunits, 1.0 cores and cores prepared with higher concentrations of LiCl were unable to do so. It should be noted that the appearance of a peak heavier than 70-S in the gradients of 2.0 and 3.0 cores is possibly due to aggregation of these core particles (compare the curves F -J of Fig. 1 with the corresponding curves A - E). The EjJect of LiCl Washing on the Ability of 50-S Subunits to Form an Initiation Complex with Poly(U), 30-S Subunits and Phe-tRNA
50-S subunit cores (0.5, 1.0, 2.0, 3.0 cores) were incubated with 30-S subunits in the presence of poly(U) and [3H]Phe-tRNA. Subsequently the reaction products were analysed on sucrose gradients (Fig. 2). The distribution of the radioactivity on the gradients shows that the ability of 5 0 4 subunits to form 70-S or heavier complexes in the presence of the
other reactants was lost almost entirely after washing with 1.0 M or higher concentrations of LiC1. The results obtained from this and the previous experiment indicate that some components of the 50-S subunit, which are important for subunit association and binding of aminoacyl-tRNA, were removed by washing with 1.0 M LiCl. As a first step in the identification of these components the RNA moieties from 1.0 cores and 50-S subunits were compared electrophoretically. No difference in their electrophoretic pattern was observed. Further, the 1.0 split fraction contained only faint traces of 23-S RNA. Next, the protein compositions of 1.0 cores and 1.0 split proteins were analysed by two-dimensional gel electrophoresis, and the distribution of proteins found is shown in Fig. 3. Identijkation of'50-S Ribosomal Proteins Needed for the Association of 50-S Subunits with 30-S Subunits
In order to determine whether the 1.O split proteins could restore the ability of 1.0 core particles to bind to 30-S subunits 1.0 cores were incubated with 1.0
504
Functions of Ribosomal Proteins L1, L11 and L16
split proteins under reconstitution conditions. Subsequently the particles were assayed for their activities, and it was found that the ability to associate was indeed restored. In order to find out which protein(s) in the 1.0 split protein fraction is (are) responsible for this effect, 1.0 split proteins were fractionated on a Sephadex G-100 column (2 x 200 cm) as described by Nierhaus and Montejo [4]. L1, L2, L6, L27 and L26/28 could be reasonably well separated; L30 and L33 were cross-contaminated with each other to the extent of about 40%; L11 was contaminated by 30% with L6, and L16 was contaminated by 20% with L11 and by 5-20% with L10, L8/9, L7/12 and L15. 1.O core particles were incubated with the various isolated protein fractions under reconstitution conditions. After subsequent incubation with 3 0 3 subunits the reaction products were analysed on sucrose gradients (Fig. 4A, B). When the core particles were incubated with each protein separately, no 7 0 3 couple formation was observed. The patterns of the gradients in these cases were similar to the pattern obtained by incubating 1.0 cores with 30-S subunits in the absence of added proteins (Fig. 4Ae) and are thercfore not shown. However, by using various combinations of the proteins a conspicuous effect was observed with L1, L11 and L16: If L1 was omitted there was a noticeable reduction in the formation of 70-S couples (Fig. 4A b). This was further reduced when, besides L1, either L11 or L16 was omitted (Fig.4Ac,d). Absence of L l l and L16 together also led to the disappearance of 7 0 4 couples (Fig.4Af). Fig.4B shows that the combined effect of L1, L11 and L16 (Fig.4Bd) on the restoration of the activity of 1.0 cores is almost as great as the effect of all nine protein fractions together (Fig. 4B e). The combination of L1 with either L11 and L16 enables the 1.0 cores to associate with 30-S subunits to a large extent. The Influence of I .O Split Proteins on the Reactivation oJ’50-s 1.0 Cores to Stimulate the Binding of Aminoacyl-tRNA
Table 1 demonstrates the effect of the 1.0 split proteins on the ability of 1.0 cores to stimulate the binding of Phe-tRNA. The presence of complete 50-S subunits in the reaction mixture caused more than a two-fold stimulation of binding. On the other hand if 1.O core particles were added to the reaction mixture in place of untreated 50-S subunits no stimulation of binding was observed. 0.5 cores could bring about only 113 of the stimulation effect observed with intact 50-S subunits. However, when the cores were incubated with their corresponding split protein fractions the particles were able to regain their original activity. This apparent stimulation of binding by 50-S subunits or by reactivated core particles relies on the stabilization of the binding of aminoacyl-tRNA to
Fig. 3. Two-dimensional gel eleccrophoresis pattern oj the proreins .from 50-S subunits, 1.0 cores and 1.0 split proteins
3 0 3 subunits in the presence of 50-S subunits [lo- 121. The rather loose binding of aminoacyl-tRNA to the 30-S subunits alone can be shown by the following experiment.
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Fig.4. Injluence of 1.0 split proteins on the cissociation of' 1.0 cores with 30-S subunits. 50 pmol of 1.0 cores were preincubated with or without the corresponding proteins (4-6 molar equivalents) in 40-pI reaction mixtures containing 1 volume of core buffer and 3 volume of protein buffer for 90 min at 50°C. Subsequently the volume of the reaction medium was raised to 100 pI and the salt concentrations were adjusted to 20 m M Tris-HCI, p H 7.5; 30 mM magnesium acetate, 150 m M NH,CI; 2 mM 2-mercaptoethanol. 50 pmol of 30-S subunits was added and incubation was carried out for a further 10 min at 22 T. At the end of the incubation the samples were layered on to sucrose gradients as described under Materials and Methods. (A) Additions during reconstitution were: (a) all nine protein fractions, (b) all minus L1, (c) all minus L1 and L11, (d) all minus L1 and L16, (e) no protein, (0 all minus L11 and L16. (B) Additions during reconstitution were: (a) no protein, (b) L1 and L11, (c) L1 and L16, (d) L1, L11 and L16, (e) all proteins, (0 all proteins minus L1, L11 and L16
30-S subunits were incubated either alone or together with 50-S subunits, 0.5 cores or 1.0 cores for 10 min at 22°C in the standard reaction mixture for assaying the poly(U)-dependent binding of Phe-tRNA. At the end of the incubation the reaction mixtures were diluted 50-fold and incubated further at 40°C for different periods of time. The curves in Fig.5 show clearly that 30-S subunits alone and the mixture of 30-S subunits with 1.0 cores have, within 30 min,
lost up to 70% of the Phe-tRNA which was retained on filter-sheets at the beginning of the incubation at 40°C. Within the same period of time there was no loss of bound aminoacyl-tRNA from the mixture of 30-S subunits with complete 50-S subunits. The loss of activity from the mixture of 30-S subunits with 0.5 cores was much less than the corresponding loss observed in the case of the mixture of 30-S subunits with 1.O cores.
506
Functions of Ribosomal Proteins L1, L11 and L16
Table 1. Influence of split proteins on binding of ['HH]Phe-tRNAPh' 10 50-S subunit cores in the presence of 30-S subunits Preparations of core particles and split proteins are described under Materials and Methods. 50 pmol of the cores was preincubatcd with or without thc split protcins (4-6 molar equivalents) in 40-pl reaction mixtures, containing 1 volume of core buffer and 3 volumes of protein buffer, for 90min at 50°C. Subsequently thc samples were placed on ice and the volume of the reaction mediums was raised to 100 pl. The salt concentration was adjusted to 20 mM Tris-HCI, pH 7.5; 30 mM magnesium acetate; 150 mM NH,Cl and 2 mM 2-mercaptoethanol. The following components were present in each reaction mixture: 50 pmol 30-S subunits, 15 pg poly(U), 40 pmol ["H]Phe-tRNAPhe (spec. act. 1152 mCi/mmol, 52'j: charged). The mixtures were incubated for 10 min at 22°C. (0-0.5 sp): Split proteins prepared by washing 5 0 3 subunits in 0.5 M LiCI; (0.5- 1.0 sp): split proteins prepared by washing 0.5 cores in 1.0 M LiCI; (0- 1.0 sp): split proteins preparcd by washing 5 0 3 subunits in 1.O M LiCl [3H]Phe-tRNA"he bound
Additions
Table 2. Comparison of the ejyect of total 1.0 split protein fiaciion and u mixture of ,fractionated 1.0 split proleins on the reactivation o j 1.0 cores with respect to ihe binding oj/3H]Phe-tRNAPh' Preparation of 1.0 cores and 1.0 split protein is dcscribed under Materials and Methods. 1.0 split proteins were fractionated on a G-100 column according to Nierhaus and Montejo [4]. The reaction conditions were similar to those described in the legend to Table 1. In the case of fractionated proteins (Ll, L2, L6, L t l , L16, L27, L26/28, L30, L33) 4-6 molar equivalents of each protein were incubated with 1.O core particles Additions
r3H lPhe-t R N APhr bound countsimin
+ 50-S + 1.0 core 30-S + 1.0 core + 0-1.0 sp 30-S + 1.0 core + a mixture of
304 30-S
L1, L2, L6, L11, L16, L27, L26/28 L30, L33
12015 4233 12813
10235
counts/min 30-S 30-S + 303 + 30-S + 30-S + 30-S +
50-S 0.5 core 0.5 corc + 0-0.5 sp 1.O core 1.0 core + 0-0.5 sp 30-S + 1.0core + 0 . 5 - 1 . 0 s ~ 30-S + 1.0corc + 0-0.5 sp + 0 . 5 - 1 . 0 ~ ~ 30-S + 1.0core + 0 - 1 . 0 s ~
6491 14385 9381 14521 6432 11 383 11 802 13 644 13250
Time (rnin)
Fig. 5. Stability of 30-S und 70-S intiution complexes. Ribosomal particles were incubated with poly(U) and rH]Phe-tRNAPh' for 10 min at 22"C, under conditions similar to those in Fig.2. At the cnd of the incubation the samples were diluted 50-fold with wash buffcr (20 mM Tris-HC1, pH 7.5; 30 mM magnesium acetate; 150mM NH,CI) and incubated further for different pcriods of time at 40 'C. (0)30-S subunits; (0)30-S subunits plus 50-S subunits; (A) subunits plus 0.5 cores; (0)30-S subunits plus 1.0 corcs. The amount of aminoacyl-tRNA retained on the filter at zero time of incubation at 40°C is designated as loo%, which was = 17000 countslmin for 30-S + 50-S subunits, 11100 countslmin for 3 0 3 subunits + 0.5 cores, 9600 counts/min for 30-S subunits + 1.0 cores and 10400 counts/min for 30-S subunits
Identificution of the 503 Ribosomal Proteins which are Required for the Formation of a 70-S Initiation Complex Reconstitution of active 50-S subunits from 1.0 core particles and the 1.0 split protein fraction has been shown above. Next, an attempt was made to identify those proteins in the 1.0 split fraction which are responsible for this reactivation. As shown in Table 2, a mixture of proteins Ll, L2, L6, L11, L16, L27, L26/L28, L30, and L33 (4- 6 molar equivalents of each) was almost as effective as total 1.O split fraction in the restoration of the binding activity of 1.0 cores. Since this was the case, each of the nine protein fractions was tested separately or in various combinations (Tables 3 and 4). In these experiments the binding of both M e t tRNA and Phe-tRNA werc studied in parallel. When thc proteins were omitted one at a timc from the mixture, a strong reduction in the binding of aminoacyl-tRNAs was observed only in the case of omission of L1 (Table 3). However, when each protein was added alone, L1 (like all the other protcins) showed no effect (Table 4). Using different combinations of the proteins it was found that L1 plus L11 and L1 plus L16 were the combinations with the minimal number of proteins which could cause 45- 60 % stimulation of aminoacyl-tRNA binding. The binding of both Phe-tRNA and met-tRNA were dependent on these combinations of proteins. The effect of these two groups could be enhanced further by the addition of the other proteins. In the presence of L1 plus L11 the degree of enhancement of met-tRNA binding was in the following order: L26/28 > L6 > L27 > L16 = L30 = L33 > L2. When L1 plus L16 were used as the essential proteins, only L6 and L11 further enhanced their effect on binding of Met-tRNA. The
M. Kazemie
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Table 3. t R N A binding activity of 1.0 cores. Effect of the omission qf the individual split proteins f r o m the reconstitution medium 1.0 cores and the proteins were prepared as described under Materials and Methods and in the legend to Table 2. 50 pmol of 1 .O cores were preincubated with the proteins (4- 6 molar equivalents) in 40-pl reaction mixtures under the reconstitution conditions described in the legends to Tables 3 and 2. Subsequently the samples were placed on ice and the volume of the reaction mcdium was raised to 100 PI. The salt concentration was adjusted to 20 mM Tris-HCI, pH 7.5; 30 mM magnesium acetate; 150 mM NH,CI; 2 mM 2-mercaptoethanol. 50 pmol 30-S subunits was added. To the poly(U)-dependent system, 15 pg of poly(U) and 40 pmol [3H]Phe-tRNAPhe (spec. act. 1152 mCi/mmol, 52% charged) were added. To the AUG-dependent system 0.25 A260 unit of the AUG and 60 pmol f[3H]Met-tRNA'Me' (spec. act. 1720 mCi/mmol, 40% charged) were added. The mixtures were further incubated for 10 min at 22°C. Binding of the aminoacyltRNAs to the mixture of 30-S subunits plus 1.0 cores is taken as the background value, and the stimulation of binding over this value caused by addition of all nine proteins is designated as 100%. I n the poly(U) system: 3 0 4 + 1.0 cores = 4233 counts/min; 30-S 1.0 cores + all proteins = 10235 counts/min; 100% stimulation = 6000 counts/min. In the AUG system: 30-S + 1.0 cores = 3320 counts/min; 30-S 1.0 cores + all proteins = 9403 counts/min; 100".; stimulation = 6083 countslmin '
+
+
Omission
Stimulation of aminoacyl-tRNA binding
0'
/o
None L1 L2 L6 L11 L16 L27 L26/28 L30 L33
100 37 79 82 92 78 63 88 78 71
100 50 90 81 87 73 77 84 90 81
binding of Phe-tRNA was slightly enhanced when L1 and L16 were supplemented by L6 or L11 or L27. The difference between the stimulatory effect of L1 plus L11 and L1 plus L16, which has been observed before and also after the addition of some of the proteins, for example L6, L27 and L26/28 (Table 4), is important in order to argue that the effect of L16 is independent of that of L11, which contaminates L16 fraction by 20% (see above). Moreover, the data given in Table 3 also confirm this assumption: the omission of L16 from the reaction mixture causes more reduction of aminoacyl-tRNA binding than the omission of L11 does. The results displayed in Fig.6 show the absolute requirement for L1 plus L11 and L1 plus L16 for reactivation of 1.0 cores with respect to the binding of aminoacyl-tRNAs. Omission of only L11 or only L16 caused no significant reduction in binding. How-
Table 4. The effect of different combinations of the individual split proteins on the binding activity of 1.0 cores Incubations were performed as for Table 3. Percentage stimulation was calculated as in the legend to Table 3 Addition
Stimulation of aminoacyl-tRNA binding
L1 L2 L6 L11 L16 L27 L26128 L30 L33
no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation
no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation
L1, L2 L1, L6 L l , L11 L1, L16 L3, L27 L1, L26/28 L1, L30 L1, L33
9 6 49 58 11 7 13 15
14 20 45 54 18 12 15 18
56
L1, L11, L2 L1, L11, L6 L1, L11, L16 L1, L11, L27 L1, L11, L26/28 L1, L11, L30 L1, L11, L33
63 78 84 64 64
58 65 64 74 65 68 65
L1, L16, L2 L1, L16, L6 L1, L16, L11 L1, L16, L27 L1, L16, L26/28 L1, L16, L30 L1, L16, L33
48 66 63 49 45 42 37
54 64 64 67 54 50 60
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Fig.6. The ejjkct oj proteins L I , LII, mid L16 on binding of ( A ) I3HJPhe-tRNAPheund (R)f[3H]Met-tRNA*M"'to 30-S suhunits plus 1.O cores. Reconstitution, binding and calculations were donc as in Table 3. (a) All proteins added; (b) minus L11; (c) minus L16; (d) minus L1; (e) minus L11 and L16; (1) minus L1,LIl andL16
Functions of Ribosomal Proteins L1, L11 and L16
508
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Fractions
Fig. I . 1nJuenc.e o j 1.0 split proteins on the formation ojihe poly( UJ-dependent iniliation complex. Sucrose gradient analysis of the reaction Reconstitution and binding were performed as described products obtained from 1.O cores, 30-S subunits, poly(U) and [3H]Phe-~RNAPhe. in the legend of Table 3. 1.0 cores were incubated with (A) L1, (B) L l l , (C) L16, (D) L1 and L11, (E) L1 and L16, (F) no protcin, ( G ) all nine protein fractions, (H) 1.0 split, (I) all nine protein fractions minus L1, L11 and L16. (-) Absorbance at 260nm; (+0) radioactivity
ever, if L11 and L16 were both omitted, a strong reduction of binding was observed. When all three proteins L1, L11 and L16 were omitted, the binding of aminoacyl-tRNAs was reduced to the same level as was observed when all nine proteins were absent. The data described above were consolidated further by sucrose gradient analysis of the reaction products (Fig.7 and 8). The results of these analyses can be summerized as follows. (a) The core particles obtained after washing 5 0 4 subunits with 1.0 M LiCl have almost no stimulatory effect on the binding of aminoacyl-tRNAs (Fig. 7F, 8 E). (b) This activity could be restored when 1.0 cores were reincubated with the 1.O split protein fraction (Fig. 7 G, H ;8 A). (c) This effect is attributed to proteins L1, L11 and L16. In the absence of these proteins the remaining proteins of the 1.0 split fraction are ineffective (Fig.71; 8D). (d) The reactivation in the presence of L1 together with either L11 or L16 is a co-operative effect (Fig.7D,E; SB,C), since each of these proteins by itself had no effect (Fig.7A-C). In the case of the poly(U)-dependent Phe-tRNAbinding system, the appearance of an absorbance peak in the 70-S region of some gradients (Fig.7A-
C, F) is an artefact which requires explanation. Besides preventing formation of 70-S initiation complexs the absence of L1, L11 and L16 also prevents formation of 7 0 4 ribosome couples (see Fig. 1; 4A,B). The reason for the appearance of '7043' peaks in Fig.7 A-C,F was found to be that more than one 30-S subunit was bound to each poly(U) chain. Addition of poly(U) to a mixture of 1.O cores and 30-S subunits (used in excess) shifted the 30-S peak to the 70-S position, whereas the peak corresponding to 1.0 cores remained unchanged (data not shown). Puromycin Reaction
According to current thinking, binding of PhetRNA occurs at the A-site and that of Met-tRNA at the P-site (or a site very similar to it). Therefore it is reasonable to assume that proteins which are involved in binding of the former aminoacyl-tRNA should differ from the proteins which are involved in binding of the latter. It was found (Table 4), however, that the same proteins (Ll, L11 and L16) effect the binding of both tRNAs. An explanation of this somewhat surprising result could be that, under the nonenzymic conditions used,
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Fig. 8. InJIuenc,e o j ' 1.0 split proreins o t i rlir ,formation of' A U G - t l ~ ~ ~ , l I c / ~initiariorz .llr conip1e.x. Sucrose gradient analysis of the reactions products obtained from 1.0 cores, 30.4 subunits, A U G and f['H]Met-tRNAfM". The conditions for reconstitution and binding were similar to those of Table 4 and Fig.6. 1.0 cores were incubated with (A) all nine fractions, (B) L1 and L11, (C) L1 and L16, (D) all protein fractions minus L1, L11 and L16, (E) no protein; (F) instead of 1.0cores 5 0 3 subunits were used. (-----) Absorbance at 260nm; (0-
0 )radioactivity
both tRNAs may bind to the same site. In order to examine this possibility the reactivities of the bound aminoacyl-tRNAs towards puromycin were tested according to the procedure described by Leder and Bursztyn [13]. Table 5 shows the puromycin reactivity of Net-tRNA and Phe-tRNA, which were bound to mixtures of 1.0 cores with 30-S subunits. The cores were preincubated with either L1 plus L11 or L1 plus L16. The two types of aminoacyl-tRNA reacted differently, in that all of the Met-tRNA and only a small fraction of Phe-tRNA reacted with puromycin. This is an indication that the Met-tRNA was bound to the peptidyl site or/and to a prepeptidyl site [14] and Phe-tRNA was bound to a different site. The amount of Met-puromycin formed was much higher than the amount of Met-tRNA bound to the subunits. This is possibly caused by the displacement of the tRNA binding equilibrium by puromycin, which favours the binding reaction [14]. These data show that the involvement of proteins L1, L11 and L16 in the binding of both types of tRNA can not be explained by the assumption that the tRNAs bind to an identical site. Assembly of Radioactive Labelled L I , LlI and L16 with 1 .O Cores
The mutual interdependence observed between L1 and L11 or L1 and L16 could be due to the specific
Table 5. Binding of aminoacj>l-tRNAsto rihosomul subunits and thc fivmarion of aminoacyl-puromycin complex Reconstitution and binding were performed as described in the legends to Fig. 2 and Table 1. Two parallel samples of each reaction mixture were incubated similarly as far as to the end of the binding reaction, at which point the reaction of one of the two samples was stopped. IO-pI aliquots of a puromycin solution ( 5 mglml) were added to the other tubes and the mixtures were further incubated for 30 min at 37°C. Subsequently 1.0 ml 0.1 M sodium acetate (pH 5 . 5 in the case of m e t - t R N A ; pH 8.0 in the case of Phe-tRNA) and 1.5 ml ethyl acetate were added. The mixtures were stirred vigorously for 3 min. The radioactivity in the ethyl acetate phase was measured in Bray's solution System
Aminoacylt R N A bound in 10 min a t 22 "C
Aminoacylpuromycin formed in 30 min at 37 "C
t3H]Phe- ft3H]Met- t3H]PhetRNAPhe tRNArMe' puromycin
f[3H]Metpuromvcin
pmol
+ 1 .O cores + L1 + L11 30-S + 1.O cores + L1 + L16 30-S
5.1
5.0
1.46
11.4
5.6
3.8
1.2x
12.0
assembly sequence of these proteins with 1.0 cores. To examine this possibility, each of the three proteins were labelled with 14C by reductive methylation according to the procedure of Rice and Means [15].
510
M. Kazemie: Functions of Ribosomal Proteins L1, L11 and L16
The labelled proteins were incubated under reconstitution conditions with 1.0 cores, either separately or together with the other (unlabelled) protein of the pairs L1 and L11 or L1 and L16. The reaction products were subsequently analysed on sucrose gradients. It was found that each of the three proteins L1, L11 and L16 could independently assemble with 1 .O cores. Supplementation of each labelled protein with the unlabelled complementary one during reconstitution had no influence on the radioactivity profiles of the sucrose gradients (data not shown).
ing binding sites on 30-S subunits. Proteins L1, L11 and L16 seem to fulfill this requirement, and the aminoacyl-tRNA molecules carried by the 30-S subunits can then react with their proper sites on the 50-S subunits. Thus, the three proteins seem to be of central importance for the binding of aminoacyl-tRNAs to the 70-S ribosomes. Recent studies on affinity labelling with aminoacyl-tRNA and peptidyl-tRNA analogues [18-201 also show the importance of L11 and L16 in aminoacyl-tRNA binding.
DISCUSSION
I wish to thank Dr H. G. Wittmann for support and many discussions, Mr H. Hampl and D r K. Nierhaus for providing me with some ribosomal proteins and Drs R. Brimacombe, N. Kcnnedy and S. Hall for critical reading of the manuscript.
In this work the involvement of 50-S proteins in subunit association and in binding of aminoacyltRNAs to subunits has been investigated. It is shown here that L1 plus L11 or L1 plus L16 are important for the association of 50-S 1.0cores with 30-S subunits. The same proteins are also required for the formation of a mRNA . 30-S . 50-S 1.0-core . aminoacyl-tRNA complex. The dependence of the subunit association on these proteins can be explained in two ways: (a) the proteins may induce a conformational change in 50-S 1 .O core particles which then makes the association of these particles with 30-S subunits possible; or (b) they are involved directly in this process possibly at the subunit interface. The latter possibility is supported by the finding that L1 is an ‘interface’ protein [16,17]. Even if L11 and L16 are not located directly at the interface they may influence the conformation of L1 in such a way that the latter can interact with 30-S subunits directly and thereby enable the two subunits to associate. With regard to the influence of L1, L11 and L16 on the binding of Met-tRNA and Phe-tRNA to the ribosomes, the puromycin test shows that these proteins promote the proper binding of both aminoacyltRNA molecules, namely Met-tRNA to the peptidyl site and Phe-tRNA to the aminoacyl site. A possible explanation of how these proteins, which are important for the association of the subunits, also effect the binding of aminoacyl-tRNA is the following. During the formation of the 70-S initiation complex the binding sites on 5 0 4 subunits must be brought into a proper conformation with respect to the correspond-
REFERENCES 1. Lucas-Lenard, I. & Lipmann, F. (1971) Annu. Rev. Biochem.
40,409 - 448. 2. Kurland, C. G. (1972) Annu. Rev. Biochem. 41, 377-408. 3. Schreiner, G. & Nierhaus, K. H. (1973) J . Mol. Biol. 81, 71 - 82. 4. Nierhaus, K. H. & Montejo, V. (1973) Prric. Natl Acud. Sci. U.S.A. 70,1931 - 1935. 5. Kaltschmidt, E. & Wittmann, H. G. (1970) Proc. Natl Acud. Sci. U.S.A. 67, 1276- 1282. 6. Staehelin, T., Maglott, D. & Monro, R. E. (1969) Cold Spring Harbor Symp. Quunt. Biol. 34, 39-48. 7. Kazemie, M. (1974) Mol. Gen. Genet. 128, 171 -182. 8. Nirenberg, M. & Leder, P. (1964) Science (Wash. 0 . C . ) 145, 1399-1407. 9. Homdnn, H. E. & Nierhaus, K. H. (1971) Eur. J . Biochem. 20,249 -257. 10. Suzuka, I., Kaji, H. & Kaji, A. (1965) Biochem. Biophys. Rex Commun. 21,187- 193. 11. Pestka, S. (1967) J . Biol. Chem. 242,4939-4947. 12. Grunberg-Manago, M., Clark, B. F. C., Revel, M., Rudland, P. S. & Dondon, J. (1969) J . Mol. B i d . 40, 33-44. 13. Leder, P. & Bursztyn, H. (1966) Biochern. Biophys. Res. Commun. 25,233-238. 14. Zagorska, L., Dondon, J., Lelong, J. C., Gros, F. & Grunberg-Manago, M. (1971) Biochirnie (Paris) 53, 63-70. 15. Rice, R. H. & Means, G. E. (1971) J . B i d . Chem. 246, 831832. 16. Morrison, C. R., Garrett, R. R., Zeichhardt, H. & Stoffler, G. (1973) Mol. Gen. Genet. 127,359-368. 17. Hsiung, N. & Cantor, C. R. (1973) Arch. Biochem. Biophys. 157, 125- 132. 18. Czernilofsky, A. P., Collatz, E. E., Stoffler, G. & Kiichler, E. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 230-234. 19. Hsiung, N., Reines, S. A. & Cantor, C. R. (1974) J . Mol. B i d . 88, 841 -855. 20. Pongs, O., Nierhaus, K. H., Erdmann, V. A. & Wittmann, H. G. (1974) FEBSLett. 40, 28-37.
M. Kazemie, Max-Planck-lnstitut fur Molekulare Genetik, Abteilung Wittmann, D-1000 Berlin (West)-Dahlem 33, IhnestraSc 63/73
The Importance of Escherichia coli Ribosomal Proteins L1, L11 and L16 for the Association of Ribosomal Subunits and the Formation of the 70-S Initiation Complex Mirabotalib KAZEMIE Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmaiin, Berlin-Dahlcm (Received March 20 July 10, 1975)
50-S subunits were washed with LiCl solutions of different concentrations. After washing with 1 M LiCl solution the particles lost their ability to attach either to 30-S subunits or to the AUG . 30-S subunit . fMet-tRNAfMe'complex or to a poly(U) . 30-S subunit . Phe-tRNAPhecomplex. Those proteins which were removed by LiCl were fractionated on a Sephadex G-100 column. Of the fractionated proteins only the combinations L1 and L11 or L1 and L16 were essential for the association of 50-S 1.O cores (particles prepared by washing 50-S subunits in 1 .O M LiCl) with 30-S subunits. These three proteins were also required for the formation of a stable complex between 50-S 1 .O cores, mRNA, 3 0 3 subunits and aminoacyl-tRNA.
During the process of protein synthesis in bacteria the 50-S subunits become associated with 30-S subunits which are already charged with mRNA (for review see [1,2]). This attachment of 50-S subunits to 30-S subunits must take place in a very specific manner, in order to guarantee that the recognized aminoacyl-tRNA species are bound to their proper sites on the 50-S subunit. Thus, the formation of a 70-S initiation complex requires the interaction of a 50-S subunit with both the aminoacyl-tRNA moiety and the 30-S subunit moiety of the 30-S initiation complex. The purpose of this paper is the identification of those 50-S ribosomal proteins which are essential for the association of subunits and the formation of a 7 0 4 initiation complex.
with buffers containing LiCl(10mM Tris-HC1,pH 7.5; 10 mM magnesium acetate; varying concentrations of LiCl: 0.5 M, 1.0 M, 2.0 M, 3.0 M) to 15 A260 unitsjml and incubated for 5 h at O'C, with gentle stirring. The suspensions were centrifuged at 250000 x g for 5 h in a Spinco 60 Ti rotor at 2°C. The supernatants (split proteins) were collected, dialyzed overnight against core buffer (20 mM Tris-HC1, pH 7.5; 20 mM magnesium acetate; 1 mM EDTA; 200 mM NH,Cl; 2 mM 2-mercaptoethanol) with two changes. Thereafter, the samples were lyophilized, taken up in 4 ml bidistilled water, dialyzed against protein buffer (the same as core buffer, except that it contained 400 mM NH,Cl), lyophilized, taken up in 1 ml water and dialyzed once more against protein buffer. The samples were clarified by centrifugation at 10000 rev./min for 10 min, frozen in liquid nitrogen and kept at - 80 "C for use as split proteins.
MATERIALS AND METHODS
Preparation and Analysis of LiCl Cores and Split Proteins from 50-S Ribosomal Subunits
The pellets obtained after the 250000xg centrifugation (cores) were suspended in core buffer at a concentration of 500 A260 units/ml, dialyzed overnight against core buffer with two changes, clarified as above and kept at - 80°C for use (as 0.5 cores, 1.0 cores, 2.0 cores, and 3.0 cores respectively, according to the concentrations of LiCl which were used for the extraction).
'Cores' and 'split proteins' were prepared as described by Nierhaus and Montejo [4].1000 A260 units of 503 subunits were incubated for 20 min at 37°C in 2ml buffer (10mM Tris-HC1, pH 7.5; 10 mM magnesium acetate; 60mM NH,Cl; 6mM 2-mercaptoethanol). Subsequently the suspensions were diluted
The protein compositions of cores and split proteins were determined by the two-dimensional gel electrophoresis technique of Kaltschmidt and Wittmann [5]. The fractionation of split proteins was carried out on a Sephadex G-100 column as described by Nierhaus and Montejo [4].
Preparation of Ribosomal Subunits
Ribosomal subunits were prepared according to the procedure reported by Schreiner and Nierhaus [3].
502
Functions of Ribosomal Proteins L1, L11 and L16
-Sedimentation
direction
Fig. 1. The effect of LiCl washing of 50-S subunits on their ability to ussociate with 3 0 3 subunits. Cores from 50-S subunits were prepared as described under Materials and Methods. 50 pmol of each core was incubated with or without 50 pmol of 30-S subunits for 10 min a t 22°C in 5O-pl reaction mixtures containing 20 mM Tris-HCI, pH 7 . 5 ; 30 mM magnesium acetate; 150 mM NH,CI and 2 mM 2-mercaptoethanol. At thc cnd of the incubation the samples were layered on to 10-30% sucrose gradients. The gradients had the same salt composition as the samples. Centrifugation was performed at 30000 rev./min in a Spinco SW 40 rotor for 5 h at 3°C. The monitoring of the gradients at 260 nm is described under Materials and Methods. (A) 5 0 4 subunits, (B) 0.5 cores, (C) 1.0 cores, (D) 2.0 cores, (E) 3.0 cores. 30-S subunits were added to (F) 50-S subunits, (G) 0.5 corcs, (H) 1.0 cores, (I) 2.0 cores, (J) 3.0 cores
Partial Reconstitution of 50-S Subunits
Reconstitution experiments were performed by incubating 50-S cores with split proteins in reaction mixtures containing 20 mM Tris-HC1, pH 7.5 ; 20 mM magnesium acetate; 1 mM EDTA; 350 mM NH,C1 and 2 mM 2-mercaptoethanol for 90 min at 50 "C [6].
Binding of Aminoacyl-tRNAs to Subunits The procedure for binding aminoacyl-tRNA has been described elsewhere [7]. The reaction products were assayed either by the Millipore technique of Nirenberg and Leder [8] or on 13 ml10- 30 % sucrose gradients, which were prepared in 14-ml tubes of a Spinco SW 40 rotor. The centrifugations were performed at 30000 rev./min for 5 h at 3 "C. The gradients were pumped out from the bottom of the tubes at a constant rate. The effluents were monitored with a flow-through cell (l-cm light path) at 260 nm. For measuring the radioactivity 20-drop fractions were collected; 5-pl aliquots of a serum albumin solution (10 mg/ml) were added to the fractions, which were subsequently made 10% with respect to trichloroacetic acid at 0 "C. The precipitates were collected on
Millipore filters and the radioactivity was measured in a liquid-scintillation spectrometer (Packard, model 3380). Counting efficiency for 3H was 38 %.
RESULTS The Eflect of LiCl Washing on the Ability of 5 0 3 Subunits to Associate with 30-S Subunits
At distinct concentrations of LiC1, definite groups of proteins can be extracted from the ribosomal particles [9]. In order to determine which groups contain the protein required for the association of 50-S subunits with 30-S subunits, one has to find the concentration of LiCl at which this function is impaired. For this purpose 50-S subunits were washed with increasing concentrations of LiCl(0.5 M, 1.0 M, 2.0 M, and 3.0 M) as described under Materials and Methods. The 0.5, 1.0, 2.0, and 3.0 cores were incubated with 30-S subunits and the reaction products were analysed on sucrose gradients (Fig. 1). The upper part of the figure shows the sedimentation pattern of the 5 0 4 core particles in the absence of 30-S subunits.
M. Kazemie
503
0.4
0.3
0.2
0.1
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m
0 : m
0.4
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0
5
10
15
0
5
10 15 Fractions
0
5
10
2
4"
15
Fig.2. The effect of' LiCl washing (u' SO-S subunits on their ability to .forin comjdexes with 30-S subunits rind [3H]Phe-tRNAPh'. Cores from 50-S subunits were prepared as described under Materials and Methods. 50pmol of 30-S subunits was incubated at 22'C for 10 min with 50 pmol of (A) 50-S subunits, (B) 0.5 cores, (C) 1.0 cores, (D) 2.0 cores and (E) 3.0 cores, or (F) alone in SO-pI reaction mixtures containing 20 m M Tris-HC1, pH 7.5; 30 mM magnesium acetate; 150 mM NH,CI; 2 mM 2-mercaptoethanol; 15 pg poly(U), 40 pmol ['HH]Phe-tRNAPhe(spec. act. 1152 mCi/mmol, 52 charged). The monitoring of the gradients at 260 nm, fractionation and Absorbance at 260 nm, preparation of the fractions for measuring- radioactivity are described under Materials and Methods. (-) ( ~radioactivity b)
The lower part of the figure shows the result of mixing these different core particles with 30-S subunits. Whereas 50-S subunits and (to a lesser extent) 0.5 cores could form 70-S ribosomes with 30-S subunits, 1.0 cores and cores prepared with higher concentrations of LiCl were unable to do so. It should be noted that the appearance of a peak heavier than 70-S in the gradients of 2.0 and 3.0 cores is possibly due to aggregation of these core particles (compare the curves F -J of Fig. 1 with the corresponding curves A - E). The EjJect of LiCl Washing on the Ability of 50-S Subunits to Form an Initiation Complex with Poly(U), 30-S Subunits and Phe-tRNA
50-S subunit cores (0.5, 1.0, 2.0, 3.0 cores) were incubated with 30-S subunits in the presence of poly(U) and [3H]Phe-tRNA. Subsequently the reaction products were analysed on sucrose gradients (Fig. 2). The distribution of the radioactivity on the gradients shows that the ability of 5 0 4 subunits to form 70-S or heavier complexes in the presence of the
other reactants was lost almost entirely after washing with 1.0 M or higher concentrations of LiC1. The results obtained from this and the previous experiment indicate that some components of the 50-S subunit, which are important for subunit association and binding of aminoacyl-tRNA, were removed by washing with 1.0 M LiCl. As a first step in the identification of these components the RNA moieties from 1.0 cores and 50-S subunits were compared electrophoretically. No difference in their electrophoretic pattern was observed. Further, the 1.0 split fraction contained only faint traces of 23-S RNA. Next, the protein compositions of 1.0 cores and 1.0 split proteins were analysed by two-dimensional gel electrophoresis, and the distribution of proteins found is shown in Fig. 3. Identijkation of'50-S Ribosomal Proteins Needed for the Association of 50-S Subunits with 30-S Subunits
In order to determine whether the 1.O split proteins could restore the ability of 1.0 core particles to bind to 30-S subunits 1.0 cores were incubated with 1.0
504
Functions of Ribosomal Proteins L1, L11 and L16
split proteins under reconstitution conditions. Subsequently the particles were assayed for their activities, and it was found that the ability to associate was indeed restored. In order to find out which protein(s) in the 1.0 split protein fraction is (are) responsible for this effect, 1.0 split proteins were fractionated on a Sephadex G-100 column (2 x 200 cm) as described by Nierhaus and Montejo [4]. L1, L2, L6, L27 and L26/28 could be reasonably well separated; L30 and L33 were cross-contaminated with each other to the extent of about 40%; L11 was contaminated by 30% with L6, and L16 was contaminated by 20% with L11 and by 5-20% with L10, L8/9, L7/12 and L15. 1.O core particles were incubated with the various isolated protein fractions under reconstitution conditions. After subsequent incubation with 3 0 3 subunits the reaction products were analysed on sucrose gradients (Fig. 4A, B). When the core particles were incubated with each protein separately, no 7 0 3 couple formation was observed. The patterns of the gradients in these cases were similar to the pattern obtained by incubating 1.0 cores with 30-S subunits in the absence of added proteins (Fig. 4Ae) and are thercfore not shown. However, by using various combinations of the proteins a conspicuous effect was observed with L1, L11 and L16: If L1 was omitted there was a noticeable reduction in the formation of 70-S couples (Fig. 4A b). This was further reduced when, besides L1, either L11 or L16 was omitted (Fig.4Ac,d). Absence of L l l and L16 together also led to the disappearance of 7 0 4 couples (Fig.4Af). Fig.4B shows that the combined effect of L1, L11 and L16 (Fig.4Bd) on the restoration of the activity of 1.0 cores is almost as great as the effect of all nine protein fractions together (Fig. 4B e). The combination of L1 with either L11 and L16 enables the 1.0 cores to associate with 30-S subunits to a large extent. The Influence of I .O Split Proteins on the Reactivation oJ’50-s 1.0 Cores to Stimulate the Binding of Aminoacyl-tRNA
Table 1 demonstrates the effect of the 1.0 split proteins on the ability of 1.0 cores to stimulate the binding of Phe-tRNA. The presence of complete 50-S subunits in the reaction mixture caused more than a two-fold stimulation of binding. On the other hand if 1.O core particles were added to the reaction mixture in place of untreated 50-S subunits no stimulation of binding was observed. 0.5 cores could bring about only 113 of the stimulation effect observed with intact 50-S subunits. However, when the cores were incubated with their corresponding split protein fractions the particles were able to regain their original activity. This apparent stimulation of binding by 50-S subunits or by reactivated core particles relies on the stabilization of the binding of aminoacyl-tRNA to
Fig. 3. Two-dimensional gel eleccrophoresis pattern oj the proreins .from 50-S subunits, 1.0 cores and 1.0 split proteins
3 0 3 subunits in the presence of 50-S subunits [lo- 121. The rather loose binding of aminoacyl-tRNA to the 30-S subunits alone can be shown by the following experiment.
M . Kazemie
505 7 0 5 305
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Fig.4. Injluence of 1.0 split proteins on the cissociation of' 1.0 cores with 30-S subunits. 50 pmol of 1.0 cores were preincubated with or without the corresponding proteins (4-6 molar equivalents) in 40-pI reaction mixtures containing 1 volume of core buffer and 3 volume of protein buffer for 90 min at 50°C. Subsequently the volume of the reaction medium was raised to 100 pI and the salt concentrations were adjusted to 20 m M Tris-HCI, p H 7.5; 30 mM magnesium acetate, 150 m M NH,CI; 2 mM 2-mercaptoethanol. 50 pmol of 30-S subunits was added and incubation was carried out for a further 10 min at 22 T. At the end of the incubation the samples were layered on to sucrose gradients as described under Materials and Methods. (A) Additions during reconstitution were: (a) all nine protein fractions, (b) all minus L1, (c) all minus L1 and L11, (d) all minus L1 and L16, (e) no protein, (0 all minus L11 and L16. (B) Additions during reconstitution were: (a) no protein, (b) L1 and L11, (c) L1 and L16, (d) L1, L11 and L16, (e) all proteins, (0 all proteins minus L1, L11 and L16
30-S subunits were incubated either alone or together with 50-S subunits, 0.5 cores or 1.0 cores for 10 min at 22°C in the standard reaction mixture for assaying the poly(U)-dependent binding of Phe-tRNA. At the end of the incubation the reaction mixtures were diluted 50-fold and incubated further at 40°C for different periods of time. The curves in Fig.5 show clearly that 30-S subunits alone and the mixture of 30-S subunits with 1.0 cores have, within 30 min,
lost up to 70% of the Phe-tRNA which was retained on filter-sheets at the beginning of the incubation at 40°C. Within the same period of time there was no loss of bound aminoacyl-tRNA from the mixture of 30-S subunits with complete 50-S subunits. The loss of activity from the mixture of 30-S subunits with 0.5 cores was much less than the corresponding loss observed in the case of the mixture of 30-S subunits with 1.O cores.
506
Functions of Ribosomal Proteins L1, L11 and L16
Table 1. Influence of split proteins on binding of ['HH]Phe-tRNAPh' 10 50-S subunit cores in the presence of 30-S subunits Preparations of core particles and split proteins are described under Materials and Methods. 50 pmol of the cores was preincubatcd with or without thc split protcins (4-6 molar equivalents) in 40-pl reaction mixtures, containing 1 volume of core buffer and 3 volumes of protein buffer, for 90min at 50°C. Subsequently thc samples were placed on ice and the volume of the reaction mediums was raised to 100 pl. The salt concentration was adjusted to 20 mM Tris-HCI, pH 7.5; 30 mM magnesium acetate; 150 mM NH,Cl and 2 mM 2-mercaptoethanol. The following components were present in each reaction mixture: 50 pmol 30-S subunits, 15 pg poly(U), 40 pmol ["H]Phe-tRNAPhe (spec. act. 1152 mCi/mmol, 52'j: charged). The mixtures were incubated for 10 min at 22°C. (0-0.5 sp): Split proteins prepared by washing 5 0 3 subunits in 0.5 M LiCI; (0.5- 1.0 sp): split proteins prepared by washing 0.5 cores in 1.0 M LiCI; (0- 1.0 sp): split proteins preparcd by washing 5 0 3 subunits in 1.O M LiCl [3H]Phe-tRNA"he bound
Additions
Table 2. Comparison of the ejyect of total 1.0 split protein fiaciion and u mixture of ,fractionated 1.0 split proleins on the reactivation o j 1.0 cores with respect to ihe binding oj/3H]Phe-tRNAPh' Preparation of 1.0 cores and 1.0 split protein is dcscribed under Materials and Methods. 1.0 split proteins were fractionated on a G-100 column according to Nierhaus and Montejo [4]. The reaction conditions were similar to those described in the legend to Table 1. In the case of fractionated proteins (Ll, L2, L6, L t l , L16, L27, L26/28, L30, L33) 4-6 molar equivalents of each protein were incubated with 1.O core particles Additions
r3H lPhe-t R N APhr bound countsimin
+ 50-S + 1.0 core 30-S + 1.0 core + 0-1.0 sp 30-S + 1.0 core + a mixture of
304 30-S
L1, L2, L6, L11, L16, L27, L26/28 L30, L33
12015 4233 12813
10235
counts/min 30-S 30-S + 303 + 30-S + 30-S + 30-S +
50-S 0.5 core 0.5 corc + 0-0.5 sp 1.O core 1.0 core + 0-0.5 sp 30-S + 1.0core + 0 . 5 - 1 . 0 s ~ 30-S + 1.0corc + 0-0.5 sp + 0 . 5 - 1 . 0 ~ ~ 30-S + 1.0core + 0 - 1 . 0 s ~
6491 14385 9381 14521 6432 11 383 11 802 13 644 13250
Time (rnin)
Fig. 5. Stability of 30-S und 70-S intiution complexes. Ribosomal particles were incubated with poly(U) and rH]Phe-tRNAPh' for 10 min at 22"C, under conditions similar to those in Fig.2. At the cnd of the incubation the samples were diluted 50-fold with wash buffcr (20 mM Tris-HC1, pH 7.5; 30 mM magnesium acetate; 150mM NH,CI) and incubated further for different pcriods of time at 40 'C. (0)30-S subunits; (0)30-S subunits plus 50-S subunits; (A) subunits plus 0.5 cores; (0)30-S subunits plus 1.0 corcs. The amount of aminoacyl-tRNA retained on the filter at zero time of incubation at 40°C is designated as loo%, which was = 17000 countslmin for 30-S + 50-S subunits, 11100 countslmin for 3 0 3 subunits + 0.5 cores, 9600 counts/min for 30-S subunits + 1.0 cores and 10400 counts/min for 30-S subunits
Identificution of the 503 Ribosomal Proteins which are Required for the Formation of a 70-S Initiation Complex Reconstitution of active 50-S subunits from 1.0 core particles and the 1.0 split protein fraction has been shown above. Next, an attempt was made to identify those proteins in the 1.0 split fraction which are responsible for this reactivation. As shown in Table 2, a mixture of proteins Ll, L2, L6, L11, L16, L27, L26/L28, L30, and L33 (4- 6 molar equivalents of each) was almost as effective as total 1.O split fraction in the restoration of the binding activity of 1.0 cores. Since this was the case, each of the nine protein fractions was tested separately or in various combinations (Tables 3 and 4). In these experiments the binding of both M e t tRNA and Phe-tRNA werc studied in parallel. When thc proteins were omitted one at a timc from the mixture, a strong reduction in the binding of aminoacyl-tRNAs was observed only in the case of omission of L1 (Table 3). However, when each protein was added alone, L1 (like all the other protcins) showed no effect (Table 4). Using different combinations of the proteins it was found that L1 plus L11 and L1 plus L16 were the combinations with the minimal number of proteins which could cause 45- 60 % stimulation of aminoacyl-tRNA binding. The binding of both Phe-tRNA and met-tRNA were dependent on these combinations of proteins. The effect of these two groups could be enhanced further by the addition of the other proteins. In the presence of L1 plus L11 the degree of enhancement of met-tRNA binding was in the following order: L26/28 > L6 > L27 > L16 = L30 = L33 > L2. When L1 plus L16 were used as the essential proteins, only L6 and L11 further enhanced their effect on binding of Met-tRNA. The
M. Kazemie
507
Table 3. t R N A binding activity of 1.0 cores. Effect of the omission qf the individual split proteins f r o m the reconstitution medium 1.0 cores and the proteins were prepared as described under Materials and Methods and in the legend to Table 2. 50 pmol of 1 .O cores were preincubated with the proteins (4- 6 molar equivalents) in 40-pl reaction mixtures under the reconstitution conditions described in the legends to Tables 3 and 2. Subsequently the samples were placed on ice and the volume of the reaction mcdium was raised to 100 PI. The salt concentration was adjusted to 20 mM Tris-HCI, pH 7.5; 30 mM magnesium acetate; 150 mM NH,CI; 2 mM 2-mercaptoethanol. 50 pmol 30-S subunits was added. To the poly(U)-dependent system, 15 pg of poly(U) and 40 pmol [3H]Phe-tRNAPhe (spec. act. 1152 mCi/mmol, 52% charged) were added. To the AUG-dependent system 0.25 A260 unit of the AUG and 60 pmol f[3H]Met-tRNA'Me' (spec. act. 1720 mCi/mmol, 40% charged) were added. The mixtures were further incubated for 10 min at 22°C. Binding of the aminoacyltRNAs to the mixture of 30-S subunits plus 1.0 cores is taken as the background value, and the stimulation of binding over this value caused by addition of all nine proteins is designated as 100%. I n the poly(U) system: 3 0 4 + 1.0 cores = 4233 counts/min; 30-S 1.0 cores + all proteins = 10235 counts/min; 100% stimulation = 6000 counts/min. In the AUG system: 30-S + 1.0 cores = 3320 counts/min; 30-S 1.0 cores + all proteins = 9403 counts/min; 100".; stimulation = 6083 countslmin '
+
+
Omission
Stimulation of aminoacyl-tRNA binding
0'
/o
None L1 L2 L6 L11 L16 L27 L26/28 L30 L33
100 37 79 82 92 78 63 88 78 71
100 50 90 81 87 73 77 84 90 81
binding of Phe-tRNA was slightly enhanced when L1 and L16 were supplemented by L6 or L11 or L27. The difference between the stimulatory effect of L1 plus L11 and L1 plus L16, which has been observed before and also after the addition of some of the proteins, for example L6, L27 and L26/28 (Table 4), is important in order to argue that the effect of L16 is independent of that of L11, which contaminates L16 fraction by 20% (see above). Moreover, the data given in Table 3 also confirm this assumption: the omission of L16 from the reaction mixture causes more reduction of aminoacyl-tRNA binding than the omission of L11 does. The results displayed in Fig.6 show the absolute requirement for L1 plus L11 and L1 plus L16 for reactivation of 1.0 cores with respect to the binding of aminoacyl-tRNAs. Omission of only L11 or only L16 caused no significant reduction in binding. How-
Table 4. The effect of different combinations of the individual split proteins on the binding activity of 1.0 cores Incubations were performed as for Table 3. Percentage stimulation was calculated as in the legend to Table 3 Addition
Stimulation of aminoacyl-tRNA binding
L1 L2 L6 L11 L16 L27 L26128 L30 L33
no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation
no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation no stimulation
L1, L2 L1, L6 L l , L11 L1, L16 L3, L27 L1, L26/28 L1, L30 L1, L33
9 6 49 58 11 7 13 15
14 20 45 54 18 12 15 18
56
L1, L11, L2 L1, L11, L6 L1, L11, L16 L1, L11, L27 L1, L11, L26/28 L1, L11, L30 L1, L11, L33
63 78 84 64 64
58 65 64 74 65 68 65
L1, L16, L2 L1, L16, L6 L1, L16, L11 L1, L16, L27 L1, L16, L26/28 L1, L16, L30 L1, L16, L33
48 66 63 49 45 42 37
54 64 64 67 54 50 60
80
100 I
P
a
L1
f
a c
-2 I
2 L
-
a b c d e f
0
a b c d e f
Fig.6. The ejjkct oj proteins L I , LII, mid L16 on binding of ( A ) I3HJPhe-tRNAPheund (R)f[3H]Met-tRNA*M"'to 30-S suhunits plus 1.O cores. Reconstitution, binding and calculations were donc as in Table 3. (a) All proteins added; (b) minus L11; (c) minus L16; (d) minus L1; (e) minus L11 and L16; (1) minus L1,LIl andL16
Functions of Ribosomal Proteins L1, L11 and L16
508
0
5
10
15 0
15 0
10
5
5
10
15
Fractions
Fig. I . 1nJuenc.e o j 1.0 split proteins on the formation ojihe poly( UJ-dependent iniliation complex. Sucrose gradient analysis of the reaction Reconstitution and binding were performed as described products obtained from 1.O cores, 30-S subunits, poly(U) and [3H]Phe-~RNAPhe. in the legend of Table 3. 1.0 cores were incubated with (A) L1, (B) L l l , (C) L16, (D) L1 and L11, (E) L1 and L16, (F) no protcin, ( G ) all nine protein fractions, (H) 1.0 split, (I) all nine protein fractions minus L1, L11 and L16. (-) Absorbance at 260nm; (+0) radioactivity
ever, if L11 and L16 were both omitted, a strong reduction of binding was observed. When all three proteins L1, L11 and L16 were omitted, the binding of aminoacyl-tRNAs was reduced to the same level as was observed when all nine proteins were absent. The data described above were consolidated further by sucrose gradient analysis of the reaction products (Fig.7 and 8). The results of these analyses can be summerized as follows. (a) The core particles obtained after washing 5 0 4 subunits with 1.0 M LiCl have almost no stimulatory effect on the binding of aminoacyl-tRNAs (Fig. 7F, 8 E). (b) This activity could be restored when 1.0 cores were reincubated with the 1.O split protein fraction (Fig. 7 G, H ;8 A). (c) This effect is attributed to proteins L1, L11 and L16. In the absence of these proteins the remaining proteins of the 1.0 split fraction are ineffective (Fig.71; 8D). (d) The reactivation in the presence of L1 together with either L11 or L16 is a co-operative effect (Fig.7D,E; SB,C), since each of these proteins by itself had no effect (Fig.7A-C). In the case of the poly(U)-dependent Phe-tRNAbinding system, the appearance of an absorbance peak in the 70-S region of some gradients (Fig.7A-
C, F) is an artefact which requires explanation. Besides preventing formation of 70-S initiation complexs the absence of L1, L11 and L16 also prevents formation of 7 0 4 ribosome couples (see Fig. 1; 4A,B). The reason for the appearance of '7043' peaks in Fig.7 A-C,F was found to be that more than one 30-S subunit was bound to each poly(U) chain. Addition of poly(U) to a mixture of 1.O cores and 30-S subunits (used in excess) shifted the 30-S peak to the 70-S position, whereas the peak corresponding to 1.0 cores remained unchanged (data not shown). Puromycin Reaction
According to current thinking, binding of PhetRNA occurs at the A-site and that of Met-tRNA at the P-site (or a site very similar to it). Therefore it is reasonable to assume that proteins which are involved in binding of the former aminoacyl-tRNA should differ from the proteins which are involved in binding of the latter. It was found (Table 4), however, that the same proteins (Ll, L11 and L16) effect the binding of both tRNAs. An explanation of this somewhat surprising result could be that, under the nonenzymic conditions used,
M. Kazemie
7OS3oS
A
509 705 305
70S30S
B
T
C
D
0.8
,I
1
1 A
0.6
is
E N 4,
c
m
0.4 8 c
e z:
\
9
( 8
0.2
%/
d 10
20
10
20
4, 10
x
n
20
10
Fractions
Fig. 8. InJIuenc,e o j ' 1.0 split proreins o t i rlir ,formation of' A U G - t l ~ ~ ~ , l I c / ~initiariorz .llr conip1e.x. Sucrose gradient analysis of the reactions products obtained from 1.0 cores, 30.4 subunits, A U G and f['H]Met-tRNAfM". The conditions for reconstitution and binding were similar to those of Table 4 and Fig.6. 1.0 cores were incubated with (A) all nine fractions, (B) L1 and L11, (C) L1 and L16, (D) all protein fractions minus L1, L11 and L16, (E) no protein; (F) instead of 1.0cores 5 0 3 subunits were used. (-----) Absorbance at 260nm; (0-
0 )radioactivity
both tRNAs may bind to the same site. In order to examine this possibility the reactivities of the bound aminoacyl-tRNAs towards puromycin were tested according to the procedure described by Leder and Bursztyn [13]. Table 5 shows the puromycin reactivity of Net-tRNA and Phe-tRNA, which were bound to mixtures of 1.0 cores with 30-S subunits. The cores were preincubated with either L1 plus L11 or L1 plus L16. The two types of aminoacyl-tRNA reacted differently, in that all of the Met-tRNA and only a small fraction of Phe-tRNA reacted with puromycin. This is an indication that the Met-tRNA was bound to the peptidyl site or/and to a prepeptidyl site [14] and Phe-tRNA was bound to a different site. The amount of Met-puromycin formed was much higher than the amount of Met-tRNA bound to the subunits. This is possibly caused by the displacement of the tRNA binding equilibrium by puromycin, which favours the binding reaction [14]. These data show that the involvement of proteins L1, L11 and L16 in the binding of both types of tRNA can not be explained by the assumption that the tRNAs bind to an identical site. Assembly of Radioactive Labelled L I , LlI and L16 with 1 .O Cores
The mutual interdependence observed between L1 and L11 or L1 and L16 could be due to the specific
Table 5. Binding of aminoacj>l-tRNAsto rihosomul subunits and thc fivmarion of aminoacyl-puromycin complex Reconstitution and binding were performed as described in the legends to Fig. 2 and Table 1. Two parallel samples of each reaction mixture were incubated similarly as far as to the end of the binding reaction, at which point the reaction of one of the two samples was stopped. IO-pI aliquots of a puromycin solution ( 5 mglml) were added to the other tubes and the mixtures were further incubated for 30 min at 37°C. Subsequently 1.0 ml 0.1 M sodium acetate (pH 5 . 5 in the case of m e t - t R N A ; pH 8.0 in the case of Phe-tRNA) and 1.5 ml ethyl acetate were added. The mixtures were stirred vigorously for 3 min. The radioactivity in the ethyl acetate phase was measured in Bray's solution System
Aminoacylt R N A bound in 10 min a t 22 "C
Aminoacylpuromycin formed in 30 min at 37 "C
t3H]Phe- ft3H]Met- t3H]PhetRNAPhe tRNArMe' puromycin
f[3H]Metpuromvcin
pmol
+ 1 .O cores + L1 + L11 30-S + 1.O cores + L1 + L16 30-S
5.1
5.0
1.46
11.4
5.6
3.8
1.2x
12.0
assembly sequence of these proteins with 1.0 cores. To examine this possibility, each of the three proteins were labelled with 14C by reductive methylation according to the procedure of Rice and Means [15].
510
M. Kazemie: Functions of Ribosomal Proteins L1, L11 and L16
The labelled proteins were incubated under reconstitution conditions with 1.0 cores, either separately or together with the other (unlabelled) protein of the pairs L1 and L11 or L1 and L16. The reaction products were subsequently analysed on sucrose gradients. It was found that each of the three proteins L1, L11 and L16 could independently assemble with 1 .O cores. Supplementation of each labelled protein with the unlabelled complementary one during reconstitution had no influence on the radioactivity profiles of the sucrose gradients (data not shown).
ing binding sites on 30-S subunits. Proteins L1, L11 and L16 seem to fulfill this requirement, and the aminoacyl-tRNA molecules carried by the 30-S subunits can then react with their proper sites on the 50-S subunits. Thus, the three proteins seem to be of central importance for the binding of aminoacyl-tRNAs to the 70-S ribosomes. Recent studies on affinity labelling with aminoacyl-tRNA and peptidyl-tRNA analogues [18-201 also show the importance of L11 and L16 in aminoacyl-tRNA binding.
DISCUSSION
I wish to thank Dr H. G. Wittmann for support and many discussions, Mr H. Hampl and D r K. Nierhaus for providing me with some ribosomal proteins and Drs R. Brimacombe, N. Kcnnedy and S. Hall for critical reading of the manuscript.
In this work the involvement of 50-S proteins in subunit association and in binding of aminoacyltRNAs to subunits has been investigated. It is shown here that L1 plus L11 or L1 plus L16 are important for the association of 50-S 1.0cores with 30-S subunits. The same proteins are also required for the formation of a mRNA . 30-S . 50-S 1.0-core . aminoacyl-tRNA complex. The dependence of the subunit association on these proteins can be explained in two ways: (a) the proteins may induce a conformational change in 50-S 1 .O core particles which then makes the association of these particles with 30-S subunits possible; or (b) they are involved directly in this process possibly at the subunit interface. The latter possibility is supported by the finding that L1 is an ‘interface’ protein [16,17]. Even if L11 and L16 are not located directly at the interface they may influence the conformation of L1 in such a way that the latter can interact with 30-S subunits directly and thereby enable the two subunits to associate. With regard to the influence of L1, L11 and L16 on the binding of Met-tRNA and Phe-tRNA to the ribosomes, the puromycin test shows that these proteins promote the proper binding of both aminoacyltRNA molecules, namely Met-tRNA to the peptidyl site and Phe-tRNA to the aminoacyl site. A possible explanation of how these proteins, which are important for the association of the subunits, also effect the binding of aminoacyl-tRNA is the following. During the formation of the 70-S initiation complex the binding sites on 5 0 4 subunits must be brought into a proper conformation with respect to the correspond-
REFERENCES 1. Lucas-Lenard, I. & Lipmann, F. (1971) Annu. Rev. Biochem.
40,409 - 448. 2. Kurland, C. G. (1972) Annu. Rev. Biochem. 41, 377-408. 3. Schreiner, G. & Nierhaus, K. H. (1973) J . Mol. Biol. 81, 71 - 82. 4. Nierhaus, K. H. & Montejo, V. (1973) Prric. Natl Acud. Sci. U.S.A. 70,1931 - 1935. 5. Kaltschmidt, E. & Wittmann, H. G. (1970) Proc. Natl Acud. Sci. U.S.A. 67, 1276- 1282. 6. Staehelin, T., Maglott, D. & Monro, R. E. (1969) Cold Spring Harbor Symp. Quunt. Biol. 34, 39-48. 7. Kazemie, M. (1974) Mol. Gen. Genet. 128, 171 -182. 8. Nirenberg, M. & Leder, P. (1964) Science (Wash. 0 . C . ) 145, 1399-1407. 9. Homdnn, H. E. & Nierhaus, K. H. (1971) Eur. J . Biochem. 20,249 -257. 10. Suzuka, I., Kaji, H. & Kaji, A. (1965) Biochem. Biophys. Rex Commun. 21,187- 193. 11. Pestka, S. (1967) J . Biol. Chem. 242,4939-4947. 12. Grunberg-Manago, M., Clark, B. F. C., Revel, M., Rudland, P. S. & Dondon, J. (1969) J . Mol. B i d . 40, 33-44. 13. Leder, P. & Bursztyn, H. (1966) Biochern. Biophys. Res. Commun. 25,233-238. 14. Zagorska, L., Dondon, J., Lelong, J. C., Gros, F. & Grunberg-Manago, M. (1971) Biochirnie (Paris) 53, 63-70. 15. Rice, R. H. & Means, G. E. (1971) J . B i d . Chem. 246, 831832. 16. Morrison, C. R., Garrett, R. R., Zeichhardt, H. & Stoffler, G. (1973) Mol. Gen. Genet. 127,359-368. 17. Hsiung, N. & Cantor, C. R. (1973) Arch. Biochem. Biophys. 157, 125- 132. 18. Czernilofsky, A. P., Collatz, E. E., Stoffler, G. & Kiichler, E. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 230-234. 19. Hsiung, N., Reines, S. A. & Cantor, C. R. (1974) J . Mol. B i d . 88, 841 -855. 20. Pongs, O., Nierhaus, K. H., Erdmann, V. A. & Wittmann, H. G. (1974) FEBSLett. 40, 28-37.
M. Kazemie, Max-Planck-lnstitut fur Molekulare Genetik, Abteilung Wittmann, D-1000 Berlin (West)-Dahlem 33, IhnestraSc 63/73
