Eur. J. Biochem. 82. 123-131 (1978)
Control of Globin Synthesis by the Haemin-Controlled Translational Repressor in a Fractionated Cell-Free System from Rabbit Reticulocytes Thomas SARRE and Kurt HILSE Institut fur Biologie 111, Universitat Freiburg (Received July 11, 1977)
The control of protein synthesis by haemin in rabbit reticulocyte lysates is mediated by a translational repressor protein. We have developed a fractionated globin-synthesizing system from rabbit reticulocyte lysates that is free of endogenous repressor and therefore well suited to study the action of purified translational repressor and its antagonism to haemin. The major components of this system are: (a) rabbit reticulocyte polysomes, (b) a mouse liver pH-5 enzyme as the source of all components for peptide chain elongation, and (c) a fraction pelleted from the rabbit reticulocyte postpolysomal supernatant by ultracentrifugation (named 200000 x g fraction). Crude rabbit reticulocyte lysates, when stimulated to extensive globin synthesis by haemin, can be inhibited by preparationes of purified translational repressor in its haemin-irreversible form. In contrast, globin synthesis directed by isolated polysomes is only slightly affected by the addition of purified repressor. Addition of the 200000 x g fraction to polysomes produces a strong stimulation of globin synthesis that is severely inhibited by the translational repressor. If the haemin-reversible form of the repressor is used, this inhibition can be prevented by the addition of haemin. Thus, the fractionated system shows the same effects observed with crude rabbit reticulocyte lysates and, moreover, supplies a convenient tool for the purification of the translational repressor in its haemin-reversible form. Addition of the 200000 x g fraction to a protein-synthesizing system from Ehrlich ascites tumour cells reveals the existence of a-globin mRNA within this fraction. As determined by sucrose gradient analysis, the 200000 x g fraction contains two major components sedimenting at 50 S and 80 S, respectively. In view of the results presented here, the nature of these two components and their relevance to the observed features of the 200000 x g fraction is discussed. Haemin deficiency in rabbit reticulocyte lysates causes the cessation of globin synthesis after several minutes [l-41 due to the accumulation of a translational inhibitor [4- 71. This so-called haemincontrolled repressor is a high-molecular-weight protein and can be purified from the rabbit reticulocyte postribosomal supernatant [6 - 91. It appears to exist in three distinct forms : prorepressor (inactive in the presence of haemin, forms active repressor in the absence of haemin), reversible repressor (active but its action reversed by haemin) and irreversible repressor (active and insensitive to haemin) Abbreviations. eIF-2, eIF-3, eucaryotic initiation factors, corresponding to Staehelin's IF-E2. IF-E3 [19] ; Hepes, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid. Definition. A260 unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm pathlength cell.
[S- 101. The translational repressor acts at the level of protein chain initiation [6,7,11,12], either by preventing the binding of Met-tRNAf to the 40-S ribosomal subunit [ l l - 131 or by blocking the formation of the 80-S initiation complex [14]. This is supported by the finding that crude rabbit reticulocyte initiation factors [7,13 - 151 or purified eIF-2 may overcome the inhibition of protein synthesis exerted by the translational repressor [13,15 - 171, eIF-2 being the initiation factor that promotes the association of the 40-Ssubunit . Met-tRNA, preinitiation complex [18,19]. From the finding that CAMP and GTP overcome repressor action in rabbit reticulocyte lysates [20, 211 it was suggested that the repressor may act as a protein kinase [21], which, in fact, copurifies with repressor preparations [22- 241. This CAMP-independent protein kinase phosphorylates eIF-2 [23- 291, some proteins from the 4 0 4 ribosomal subunit and/or other
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initiation factors [23,25,29]. However, the effect of phosphorylation on the activity of eIF-2 is not at present clear. Some results show a definite reduction of its Met-tRNA, . 40-S-subunit binding capacity [27,30], other authors report that phosphorylation does not result in any detectable loss of eIF-2 activity [17,24,28,31]. In order to elucidate the mechanism of repressor action further, we have developed a fractionated globin-synthesizing system from rabbit reticulocytes that is free of endogenous repressor activity, but extremely sensitive to the addition of purified repressor. Surprisingly, this sensibility of the system is not correlated to polyribosomes or initiation factors, but to a fraction that can be pelleted from the rabbit reticulocyte postpolysomal supernatant by lengthy centrifugation. The utilization of the repressor-sensitive fractionated system might be helpful in clarifying the contradictions about the regulatory mechanism mediated by the translational repressors. MATERIALS AND METHODS Materials Haemin was bought from Carl Roth OHG (Karlsruhe, F.R.G.) and DEAE-cellulose (DE-52) from Whatman Biochemical Ltd (Maidstone, U.K.). L-[U-'~C]Lysine (287 Ci/mol) was purchased from the Radiochemical Center (Amersham). Deproteinized globin mRNA and ribosomal subunits from rabbit reticulocytes were prepared by W. Buhl of our institute. Ehrlich ascites tumour cells were generously donated by Godecke AG (Freiburg, F.R.G.). Preparation of Rabbit Reticulocyte Lysates Reticulocytes were obtained from anaemic rabbits following the method of Borsook et al. [32]. After three-fold washing with an isotonic buffer [0.15 M NaCl, 0.005 M Mg(Ac),, 0.005 M KCl] the reticulocytes were sedimented by low-speed centrifugation at 2000 x g for 15 min and lysed for 10 min by dilution with a 1.5-fold volume of 0.003 M glutathione in H,O. After centrifugation of the lysate for 10 min at 27000 xg, the upper 70% of the postmitochondrial supernatant was withdrawn and stored in I-ml aliquots under liquid nitrogen. Preparation of Polyribosomes and the 200000 x g Fract ion All procedures were carried out at 4 "C. A freshly prepared solution of haemin (0.4 mM haemin, 0.05 M Tris-HC1 pH 7.5) was added to the lysate described above to give a haemin concentration of 0.05 mM. In order to sediment the polyribosomes this lysate was centrifuged for 30 min at 45000 rev./min in a Ti-50
Control of Globin Synthesis by Translational Repressor
rotor of a Spinco ultracentrifuge. The resulting pellet was rinsed twice with buffer A containing 0.01 M Tris-HC1 pH 7.6, 0.25 M sucrose, 0.005 M MgCl,, 0.001 M dithioerythritol, 0.25 mM EDTA) and then resuspended in the same buffer to a concentration of 100 A,,, units/ml. Polyribosomes were found to be more active in protein synthesis if isolated in the presence of haemin. The postpolysomal supernatant was recentrifuged for 90 rnin at 45000 rev./min in a Ti-50 rotor to pellet most of the ribosomal material (mainly 80-S ribosomes). The supernatant obtained was subsequently layered on a 40% sucrose cushion containing 0.01 M Tris-HC1 pH 7.6, 0.005 M MgCI,, 0.001 M dithioerythritol, 0.25 mM EDTA and centrifuged for 8 h at 48000 rev./min (200000 x g) in a Ti40 rotor to yield a small pellet containing postpolysomal 50-S and 80-S particles. After rinsing this pellet twice with buffer A, it was dissolved in the same buffer by stirring the solution with a glass rod for 20 min. Using this procedure, most of the 80-S particles form aggregates that are pelleted by a 5-min centrifugation at 15000 x g. The remaining supernatant (50 A2.50 units/ml), which we shall refer to as 200000 x g fraction, contains the 50-S and 80-S particles in a 1 : 1 ratio and was stored in 100-p1 aliquots under liquid nitrogen. Preparation of the Translational Repressor All procedures were carried out at 4 "C. Lysate without added haemin was centrifuged for 10 h (0.5 3.5 8 h) as described above. 4 ml of the resulting supernatant were immediately applied to a CM-Sephadex column (1 .O x 25 cm) equilibrated with a buffer containing 10 mM Hepes pH 7.5, 2 mM mercaptoethanol and 0.1 mM EDTA. Elution was carried out using the same buffer and 1-ml fractions were collected. The three fractions with the highest absorbance at 280 nm from the first protein peak were pooled and denoted as prorepressor. 1 ml of this material was incubated at 37 "C for 2 h and denoted as step-1 repressor. Both samples were stored at - 60 "C in 100-pl aliquots. For further purification 10 ml of prorepressor was subjected to chromatography on DEAE-cellulose as described by Beuzard and London [15]. The fractions eluted with 300 mM KC1 were dialysed against the buffer mentioned above and tested for inhibitory activity in the rabbit reticulocyte lysate following the method of Maxwell et al. [5] or in the fractionated system described below. The fraction showing repressor activity were pooled and stored at - 60 "C in 100-p1 aliquots denoted as step-2 repressor. After this purification step-2 repressor activity appeared to be completely haemin-irreversible. Protein concentration was 7.3 mg/ml as determined by the method of Lowry et al. [33], using bovine serum albumin as standard.
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T. Sarre and K . Hihe
Preparation of Crude Initiation Factors
The preparation of crude initiation factors from rabbit reticulocyte polysomes followed the method of Schreier and Staehelin [I81 with slight modifications. All procedures were carried out at 4 "C. Ribosomal material from rabbit reticulocyte lysates (see above) was pelleted by a 3-h centrifugation at 45000 rev./min in a Ti-50 rotor of a Spinco ultracentrifuge. After rinsing the pellet twice with buffer A, it was dissolved in buffer A up to a concentration of 300 A260 units/ml. This solution was brought to 0.5 M KCl by addition of 4.0 M KCl in H 2 0 and was gently stirred for 30 min. The salt-washed ribosomes were spun down by centrifugation in a Ti-50 rotor for 3 h at 45000 rev./min. The upper 80% of the postribosomal supernatant was withdrawn and protein was concentrated by addition of neutralized solid ammonium sulfate to 60% saturation. The resulting precipitate was pelleted by a 10-min centrifugation at l5OOOxg, dissolved in a buffer containing 0.02 M Tris-HC1 pH 7.6, 0.1 M KC1, 0.001 M dithioerythritol, 0.1 mM EDTA, 10% glycerol (IF buffer) and dialysed against the same buffer overnight. This solution was brought to 0.05 M KCl by addition of an equal volume of IF buffer (containing no KCI) and directly applied to a DEAE-cellulose column (1 x 5 cm) equilibrated with IF buffer (containing 0.05 M KCl). Non-initiationfactor proteins were washed from the column by the equilibration buffer. Initiation factors were eluted by IF buffer (containing 0.27 M KCl) and tested in the system described in the next section. The fractions containing stimulatory activity were pooled. Crude initiation factors contained 2.0 mg protein/ml estimated by the method of Lowry et al. [33] and were stored in 2 0 0 4 portions under liquid nitrogen. Protein Synthesis in the Fructionated System
The standard assay contained: 30 mM Hepes pH 7.6, 1 mM dithioerythritol, 1 mM ATP, 0.4 mM GTP, 20 mM phosphocreatine, 3.75 units/ml of creatine phosphokinase (Boehringer, Mannheim), 3 mM Mg(Ac), ,74mM KCl, 36 pM each of thecommon amino acids except lysine, 32 pM unlabelled lysine and 4 pM ['4C]lysine (specific activity 287 Ci/mol). The reaction mixture volume of 0.1 ml contained: 5 p1 of mouse liver pH-5 enzyme isolated as described elsewhere [34], 0.5 - 1.O A260 units of polyribosomes, 5- 10 p1 of crude initiation factors, 0.2-1.0 ,4260 units of the 200000 x g fraction and 5 - 20 p1 of translational repressor as specified in the appropriate figure legends. When assaying unfractionated rabbit reticulocyte lysate, polyribosomes and the mouse liver pH-5 enzyme were omitted and 10 pl of lysate (prepared as described above) were added per 0.1 ml of reaction
mixture. Incubation was carried out at 34 "C for 60 min and 20-p1 aliquots were taken after the times indicated in the appropriate figure. The synthesis of protein was measured by incorporation of [14C]lysine (60 counts min-' pmol-') in acid-insoluble material following the method of Mans and Novelli [35]. Protein Synthesis in the Ehrlich Ascites Cell-Free System
Preparation of the cell-free extract (30000 x g supernatant) and assay conditions were as described by Morrison and Lodish [36], except that the 30000 x g supernatant was prepared in the presence of haemin (0.05 mM). The reaction mixture volume was 0.1 ml and contained: 30 p1 of the 30000xg supernatant, 12 nmol each of unlabelled amino acids except lysine, 0.05 pCi of ['4C]lysine (specific activity 287 Ci/mol) and 5 p1 ofcrude initiation factors. The amount ofdeproteinized mRNA, 200000 x g fraction and purified repressor added to the system is specified in the legend to Fig. 5. Incubation was carried out at 37 "C for 120 min; 20-4 aliquots were withdrawn and incorporation of [14C]lysine (510 counts min-' pmol-' determined as described by Mans and Novelli [35]. Globin a and fi chains labelled with ['4C]lysine were analysed as described by Dintzis [37]. Analysis of the 200000 x g Fraction on Sucrose Density Gradients
3 A260 units of the 200000 x g fraction dissolved in 1 ml of buffer A were layered onto 11.5 ml of a linear l0-30% sucrose gradient, containing 10 mM TrisHCl pH 7.6, 50 mM KCI, 5 mM MgCl,, 1 mM dithioerythritol, 0.25 mM EDTA. Centrifugation was carried out in a Beckman SW-41 rotor at 40000 rev./min for 2.5 h. For estimation of sedimentation coefficients, parallel gradients with ribosomal subunits from rabbit reticulocytes were performed. After the run the gradients were displaced from the bottom of the tubes by a Mico puncturing apparatus (Molecular Instruments Corp., Evanston, Ill.) and the absorbance at 260 nm was monitored continously in a Perkin-Elmer recording spectrophotometer, using a Mico flow cell. RESULTS Efect of Partiully Purijied Translational Repressor on Protein Synthesis in the Fractionated Rabbit Reticulocyte System
When crude rabbit reticulocyte lysates are incubated in the presence of haemin, protein synthesis can be inhibited by addition of the translational re-
126
Control of Globin Synthesis by Translational Repressor
r
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Fig. 1. Efect of the translational repressor and crude initiation factors on protein synthesis in the rabbit reticulocyte lysate ( A , B ) and in the fractionated system ( C ) . Protein synthesis was assayed as described in Materials and Methods. All values represent the I4C in a 2 0 4 aliquot, which were removed from a 0.1-ml assay at the times indicated. Assays contained (A, B) 10 pl of crude rabbit reticulocytes lysate (equivalent to 0.7 A260 unit of ribosomal material) in the presence of haemin (0.012 mM) unless otherwise stated, (C) 1.0 A,,, unit of polyribos o m a and 5 pI of mouse liver pH-5 enzyme. Incubations were carried out under the following conditions: (A) (0-0) no addition; (0+) minus haemin; (mm) plus 10 p1 step-2 repressor; ( x x ) plus aurin tricarboxylic acid (0.06 mM); (B) (A-A) plus 5 pI initiation plus 5 pl initiation factors, plus 10 pl step-2 repressor; (m-m) no initiation factors, plus 10 pl factors, plus 5 p1 step-2 repressor; (A-A) step-2 repressor; (C) (0- -0) no addition; (m-m) plus 10 pl step-2 repressor; (0-0) plus 5 pl initiation factors; (0-0) plus 5 1 1 initiation factors, plus 10 pl step-2 repressor; (A-A) plus aurin tricarboxylic acid (0.06 mM); (A-A) plus 5 p1 initiation factors, ~
plus aurin tricarboxylic acid (0.06 mM). The incubations with the rabbit reticulocyte lysate were performed simultaneously but the points have been plotted on two graphs for convenience of display
pressor if its haemin-irreversible form is used [4- 151. The repressor reduces amino acid incorporation to the same level obtained with lysates unsupplemented with haemin (Fig. 1A). Since the translational repressor is reported to act at the level of protein chain initiation [6,7,11- 141, we have isolated polyribosomes and crude initiation factors from rabbit reticulocytes in order to investigate repressor action upon these components in a protein-synthesizing system that does not include the reticulocyte postribosomal supernatant, i.e. endogenous translational repressor. A mouse liver pH-5 enzyme preparation was used instead to maintain globin chain elongation [34]. In this fractionated system, polyribosomes direct protein synthesis with or without the addition of crude initiation factors (Fig. 1C ) . Even without additional initiation factors, polyribosomes are capable of promoting reinitiation of protein synthesis during incubation since the polyribosomal fraction itself provides a limited amount of initiation factors [18,19]. This is supported by the data obtained with aurin tricarboxylic acid, which is reported to inhibit protein chain initiation at a concentration of about 100 pM [38]. As shown in Fig.lC, aurin tricarboxylic acid reduces amino acid incorporation in the presence or absence of crude initiation factors to a level which is comparable to the level of protein synthesis in rabbit
reticulocyte lysates treated with the same amount of aurin tricarboxylic acid (Fig. 1A). Though all target components for the action of the translational repressor known from the rabbit reticulocyte lysate should be present in the described system, addition of purified translational repressor causes only a 20% inhibition of protein synthesis (Fig. lC), whereas the same amount of repressor leads to a 700/, inhibition in a comparable rabbit reticulocyte lysate (Fig. 1A). Crude initiation factors and purified translational repressor are reported to exhibit a somewhat antagonistic behaviour, depending on their relative concentrations when added to crude rabbit reticulocyte lysates [7,13]. An excess of initiation factors can fully overcome low concentrations of purified translational repressor, but not saturating amounts (Fig. 1B). In the fractionated system, however, reduction of added crude initiation factors or increase of added repressor did not render a better response of the system to repressor action; nor did the use of salt-washed, i.e. initiation-factor-free polyribosomes (see Materials and Methods) or polyribosomes purified by chromatography on Sepharose 6B (data not shown). From these results we realized that one or more essential components susceptible to repressor action were lost during the isolation of polyribosomes and/or
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40s 60s
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in)
Fig. 2. Time course of protein synthesis directed by the 200000 x g fraction in the fractionated system in the presence or absence oj translational repressor and crude initiation factors. Protein synthesis was assayed as described in Materials and Methods. All values represent in a 20-pI aliquot from a 0.1-ml assay containing 0.5 A,,, unit of polyribosomes and 0.5 A,,, unit of 200000xg fraction prepared in the absence of KCI (A) or in the presence of 0.1 M KCI (B). Incubations were carried out under the following conditions: ( 0 0 )no addition; (m- 4)plus 10 PI step-2 repressor; (o-) plus 3 p1 initiation factors; (04) plus 3 PI initiation factors, plus 10 pl step-2 repressor. Control incubations (plotted on A) contained 0.5 A,,, unit ofpolyribosomes and (A-A) no addition, (A-A) 10 pl of step-2 repressor
initiation factors. They were found in the pellet of a lengthy ultracentrifugation of the postpolysomal supernatant from rabbit reticulocyte lysates (named 200000 x g fraction). Addition of this fraction to polyribosomes yields a five-fold stimulation of protein synthesis per 0.5 A260 unit of 200000 x g fraction in the fractionated system (Fig. 2A), the stimulation per A260 unit of 200000 x g fraction being linear in the range of 0.2 - 1.O '4260 unit (data not shown). Additional initiation factors do not produce a further enhancement of protein synthesis, if it is already stimulated by the addition of the 200000xg fraction (Fig. 2A). Purified translational repressor causes a drastic inhibition of the protein synthesis promoted by the 200000 x g fraction (Fig. 2A), so that the fractionated system becomes very similar to the crude rabbit reticulocyte lysate (compare Fig. 1A and 2A). However, crude initiation factors cannot overcome repressor action in the fractionated system (Fig. 2A). Obviously, the mechanism of stimulation of globin synthesis as well as the mechanism of inhibition by the translational repressor has become completely independent of the addition of crude initiation factors.
Fig. 3 . Analysisof the 200000 x gfraction bysucrosedensitygradients. 3.0 A,,, units of 200000 x g fraction prepared either in the absence of KCl (A) or in the presence of 0.1 M KCI (B) were centrifuged as described in Materials and Methods. Sedimentation was from left to right. Arrows indicate the position of 40-S and 6 0 4 ribosomal subunits from rabbit reticulocytes determined in parallel gradients
Analysis of the 200000 x g Fraction by Sucrose Density Gradients
In order to elucidate the nature of the 200000 x g fraction, we have analysed it by sucrose gradient centrifugation (Fig. 3A). The arrows indicate the position of 40-S and 60-S ribosomal subunits from rabbit reticulocytes determined in parallel gradients. The 200000 x g fraction yields two major peaks, one in the 50-S and one in the 80-S region of the gradient, and the question arises as to which of the two components is responsible for the effects observed with the 200000 x g fraction. A modification of the isolation procedure for the 200000 x g fraction (see Materials and Methods) allowed a preliminary conclusion. When the 40% sucrose cushion and the solution buffer for the preparation of the 200000 x g fraction were supplemented with 0.1 M KCl, the aggregation of the 80-S particles is prevented so that the 200000xg fraction becomes enriched by the 80-S particles rather than by the 50-S component (Fig. 3B). When isolated in this way, the 200000 x g fraction enhances protein synthesis in the fractionated system only two-fold/0.5 ,4260 unit of 200000 x g fraction (Fig. 2B), its behaviour towards addition of translational repressor and/or crude initiation factors being similar to that of the 200000 x g fraction prepared without KCI (compare Fig. 2A and 2B). Stimulation and repressor response might be due
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to the small amount of the 50-S component that is still present in the 200000 x g fraction prepared in the presence ofO.1 M KC1 (Fig. 3B). Ifthe 80-Scomponent was the crucial material within the 200000 x g fraction one would expect an amplification of its features when it is prepared in the presence of 0.1 M KCl; this is not, however, observed (Fig. 2B). Though this result indicates that the 50-S component rather than the 80-S component is responsible for the observed features of the 200000 x g fraction, we cannot exclude the possibility that an interaction of the two, i.e. 50-S and 80-S components, leads to the effects of the 200000 x g fraction presented here. At this point it should be noted that the activities associated with the 200000 x g fraction appear to be extremely labile, not only towards rather low concentrations of salt, 0.1 M KCl partially destroying the 50-Scomponent (Fig. 3B), but also towardsdilution. For this reason, almost no activity can be retained from material that has been further purified by a method which includes a dilution of the starting material, such as preparative sucrose gradients or chromatography on Sepharose columns. Attempts to regain purified material of the 200000 x g fraction by precipitation with ethanol or ammonium sulfate or by pH-5 precipitation lead to complete loss of activity (data not shown). Antagonism of Haemin and Purified Translational Repressor in the Fractionated Rabbit Reticulocyte System Since the fractionated system shows the same response to repressor action as crude rabbit reticulocyte lysates (Fig.1A and 2A), one would expect that the antagonism of haemin and the translational repressor should also be demonstrable in the fractionated system. For this purpose it was necessary to prepare the translational repressor in its prorepressor or haeminreversible form (see Materials and Methods), and to examine the concentration of haemin that was optimal for protein synthesis in the fractionated system. It appeared to be much lower, i.e. 0.004 mM, for the fractionated system than for the crude rabbit reticulocyte lysate (0.012 mM). Addition of haemin to the fractionated system does not enhance the efficiency of protein synthesis promoted by the 200000 x g fraction (Fig.4A). As expected from the data obtained in the reticulocyte lysate [5 - 101 (and Fig. 1A), inhibition of protein synthesis by the irreversible repressor is not affected by haemin (Fig. 4B). Suppression of protein synthesis by the prorepressor preparation in the absence of haemin (Fig.4C) is incomplete as compared to the effect of the irreversible repressor (Fig. 4B). This can be explained by the fact that the prorepressor preparation represents a mixture of prorepressor and reversible
Control of Globin Synthesis by Translational Repressor
repressor; only the latter is capable of inhibiting protein synthesis immediately after the start of incubation. In contrast to the repression by the irreversible repressor, however, the inhibitory effect of the prorepressor preparation is completely prevented by the addition 'of haemin (Fig. 4C). These results clearly show that haemin acts by preventing or reversing the transition of prorepressor to active repressor [8 - lo] and not by protecting any globin synthesis component from inhibitory action 141. Furthermore, the fractionated system appears to offer a convenient assay for the purification of the translational repressor in its prorepressor or haeminreversible form. Analysis ofthe 200000 x g Fraction in the Ehrlich Ascites Cell-Free System For further characterization, we investigated for globin mRNA activity the 200000 x g fraction in the Ehrlich ascites cell-free system which is dependent on exogenous mRNA [36]. Limited amounts of deproteinized globin mRNA from rabbit reticulocytes are efficiently translated in this system and mRNAdirected globin synthesis can be enhanced by the addition of crude rabbit reticulocyte initiation factors (Fig. 5A). Addition of the 200000 x g fraction to the Ehrlich ascites cell-free system reveals the existence of an mRNA-containing component within the 200 000 x g fraction (Fig. 5B). Compared to the experiment without exogenous mRNA (Fig. 5A), stimulation is about three-fold/0.4 A260 unit of the 200000 x g fraction. Product analysis of the synthesized protein shows that a large excess of a-globin chain ( > 90%) is translated from the mRNA of the 200000xg fraction (Fig. 6). In contrast to the corresponding experiment in the reticulocyte fractionated system (Fig. 2A), globin synthesis promoted by the 200000 x g fraction is not affected by the addition of purified translational repressor (Fig. 5B). If, however, globin synthesis directed by the 200000 x g fraction is enhanced by the addition of crude rabbit reticulocyte initiation factors, this stimulation can be reversed by the translational repressor (Fig. 5B). This result can be explained if one assumes a difference in the pattern of factors between ribosomes from rabbit reticulocytes and from Ehrlich ascites cells. Ehrlich ascites ribosomes are capable of translating the globin mRNA due to the inherent initiation factors, but must be reconstituted with rabbit reticulocyte initiation factors to exhibit the observed response to the translational repressor. Furthermore, a comparison of Fig. 5A and B clearly indicates a difference between the mRNA associated with the 200000 x g fraction and deproteinized globin mRNA. The latter shows no change of function in the presence of the translational repressor, whether or not additional rabbit reticulocyte initiation factors are present (Fig. 5A).
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10 2 0
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Fig 4 Effect of huemin und trunslutionulrepressor on globin tbnthesis directed bj 200000 x g fraction in the fructionritedsvstem Protein synthesis was assayed as described in Materials and Methods All values represent I4C in 2 0 4 aliquot from a 0 I-ml assay containing 0 5 AZ6,, unit minus haemin (A) No addition, of polyribosomes and 0 5 A,,, unit of 200000 x g fraction, ( 0 4 ) plus haemin (0 004 mM), (0-0) (B) plus 20 pl step-1 repressor, (C) plus 20 PI prorepressor
04
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.; .... ..~ , O.... ...,. ....... .,....,..
0 0
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Fig 5 Time course of globin sbnthesrs directed bv deproteinr-ed globin m R N A ( A ) or bv the 200000 x g fraction ( B ) in the Ehrlirh uscites cell-free srstem. Protein synthesis was assayed as described in Materials and Methods All values represent I4C in a 20-p1 aliquot from a 0 1-ml assay containing (A) 0 02 A,,, unit of deproteinized globin m R N A (except control incubations, see below) and (B) 0 4 A,,, unit of 200000 x g fraction. under the following conditions ( 0 - 0 ) n o addition, (0-4)plus 3 p1 initiation plus 10 p1 step-2 repressor. (0-0) plus 3 pl factors, (w+) initiation factors. plus 10 pI step-2 repressor Control incubations (plottedon A)containednomRNAor200000 x gfraction (A- A) A ) plus 3 pI initiation factors no addition. ( A
23
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,
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, . ......., 60 70
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Fig 6 Product unaljsis o f protein svnthesis directed bi the 200000 x g fraction in (he Ehrhch usciter cell-free rvstcm Protein synthesis was assayed as described in Materials and Methods Incubation of a 0 1 ml assay containing 0 4 AZ6()unit of 200000 x g fraction was stopped after 120 min by addition of 2 0 ml cold water Isolation of complete chains followed the method of Dintzis [37], including chromatography on CM-cellulose Elution of the a and 1 chains was monitored by an Isco recording spectrophotometer, measuring the absorbance dt 280 nni () Fractions of 5 0 ml meie collected and Incorporation of ['4C]lysine into acid-insoluble material was medsured by precipitation with triLhlorodcetic dcid ( -- )
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DISCUSSION Initiation of protein synthesis in rabbit reticulocytes is reported to occur in three steps [19]: (a) association of the 40-S ribosomal subunit with Met-tRNA, promoted by G T P and eIF-2; (b) binding of mRNA to the 40-S-subunit . Met-tRNA, . eIF-2 preinitiation complex mediated by eIF-3; (c) joining of the 60-S ribosomal subunit to the 40-S-subunit . Met-tRNA, . mRNA complex yielding the 80-S-ribosome . MettRNA, . mRNA complex ready for peptide chain elongation. Several groups have reported that the translational repressor from rabbit reticulocytes acts by impairing the formation of the 40-S-subunit . Met-tRNA, preinitiation complex [ l l - 13,27,30], i.e. step (a) of the initiation process. This is in good agreement with a previous finding indicating that the purified Mett RNA, binding factor, eIF-2, can reactivate protein synthesis in inhibited rabbit reticulocyte lysates [13, 15-17]. On the assumption that this mechanism represents the regulation mediated by the translational repressor, we have investigated the effect of partially purified repressor on a globin-synthesizing system from rabbit reticulocytes that was free of endogenous repressor activity. In this system, however, globin synthesis directed by isolated polyribosomes is affected by the translational repressor only to a limited extent (Fig. 1C), although the polyribosomes appear to be capable of reinitiating globin synthesis, especially in the presence of crude rabbit reticulocyte initiation factors (Fig. 1C). This finding is not consistent with the proposed mechanism of repressor action mentioned above, which implies that the initiation process should be sensitive to the translational repressor. The response to repressor action in the fractionated system could be reconstituted by a fraction pelleted from the rabbit reticulocyte postpolysomal supernatant by ultracentrifugation at 200000 x g for a long time (200000 x g fraction). Addition of this fraction to polyribosomes in the fractionated system results in a dramatic stimulation of globin synthesis as well as in a strong response to the translational repressor (Fig. 2A). Moreover, inhibition of globin synthesis by the repressor in its haemin-reversible form can be completely prevented by the addition of haemin (Fig. 4C). Thus, the control of globin synthesis by the haemincontrolled repressor can be fully demonstrated in the fractionated system described in this paper. Analysis of the 200000 x g fraction, which appears to be the crucial component of the fractionated system, shows the following attributes: (a) the 200000 x g fraction contains postpolysomal material that is large enough to be pelletable by ultracentrifugation (see Materials and Methods); (b) it contains globin mRNA activity (Fig. 5B) which is, with respect to repressor
Control of Globin Synthesis by Translational Repressor
action, different from the template activity of deproteinized globin mRNA (Fig.5A); (c) the mRNA associated with the 200000 x g fraction codes mainly for the c1 chain of globin (Fig. 6); (d) 200000 x g fraction contains a 50-S and 80-S component (Fig. 3A). From findings (a) and (b), one must assume that the observed mRNA activity is not due to protein-free 9-S globin mRNA, which cannot be pelleted from the postpolysomal supernatant by mere ultracentrifugation, but to an mRNA-containing particle that is of a rather large size. Thus, the mRNA must be present in a form associated either with a ribosomal subunit (e.g. with the 40-S subunit) or with high-molecularweight proteins as a messenger ribonucleoprotein (mRNA . protein) particle. As a matter of fact, several authors [39 -421 have described such a cytoplasmic (or postpolysomal) messenger ribonucleoprotein from rabbit reticulocytes, which contains exclusively CIglobin mRNA [39-421. This is in good agreement with our findings (a - c). However, this messenger ribonucleoprotein is reported to sediment at 20-S [39 -421, whereas the 200000 x g fraction contains 50-S and 80-S particles (d). This finding suggests the possibility that under the conditions described in this paper the cytoplasmic 20-S messenger ribonucleoprotein is associated with the 40-S-subunit . Met-tRNA, preinitiation complex to produce a particle sedimenting at 50-S. We are aware that the results presented here can offer only presumptive evidence for the existence of such a 40-S-subunit . Met-tRNA, . mRNA . protein complex and that this complex corresponds to the 50-S component within the 200000 x g fraction. If this were so, the observed high sensitivity ofthe fractionated system towards the translational repressor could be explained by the fact that the translational repressor might act by dissociating the 40-S-subunit . MettRNA, ' mRNA . protein complex and/or by impairing the binding of the 20-S mRNA . protein to the 40-S preinitiation complex. This hypothesis implies that step (b) of the initiation process may also be involved in the highly complex mode of regulation of globin synthesis. We thank Mrs Gabriele Rietdorf for excellent technical assistance and Drs T. Staehelin (Institute for Immunology, Bask) and R. Hertel for valuable discussions. This investigation was supported by the Deutsche Forscliungsgemeinschcrfi within the SFB46.
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T. S a m and K. Hike, lnstitut fur Biologie 111 der Albert-Ludwigs-Universitat Freiburg, SchanzlestraBe 1, D-7800 Freiburg I Br , Federal Republic of Germany