Cell-free Hemoglobin Synthesis CHARACTERISTICS OF THE TRANSFER RIBONUCLEIC ACID-DEPENDENT ASSAY Sk-STEM*

A cell-free protein-synthesizing system derived from rabbit reticulocytes is described which is dependent on the addition of transfer RNA for the translation of endogenous hemoglobin messenger RNA. Product analysis indicates that the system is active in the initiation of new chains. When hemoglobin is synthesized in the presence of a limiting amount of tRNA, there is a 50% decrease in (Y chain production relative to /3 chain production.

From the Xection on Human Biochemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY A cell-free protein-synthesizing system derived from rabbit reticulocytes is described which is dependent on the addition of transfer RNA for the translation of endogenous hemoglobin messenger RNA.
Product analysis indicates that the system is active in the initiation of new chains. When hemoglobin is synthesized in the presence of a limiting amount of tRNA, there is a 50% decrease in (Y chain production relative to /3 chain production.
Deacylated rabbit liver tRNA can, but deacylated Escherichia coli tRNA cannot, be substituted for rabbit reticulocyte tRNA in the synthesis of hemoglobin.
Although many of the steps involved in Escherichia coli protein synthesis have been determined (I), gaps remain in understanding the mechanism and regulation of protein synthesis in higher organisms. The rabbit reticulocyte cell-free hemoglobin-synthesizing system appears to be well suited for studying mammalian protein synthesis.
Reticulocytes, obtainable in large quantity from phenylhydrazine-treated rabbits (2), have lost their nucleus and appear to contain only the apparatus necessary for translation of previously synthesized messenger RNA (3). In addition, 85 to 95% of the protein synthesized is one product, hemoglobin (4). Procedures for characterizing hemoglobin are well established (5).
Allen a,nd Schweet (6) described a cell-free system capable of completing the synthesis of nascent hemoglobin chains on rabbit reticulocyte polysomes.
Miller and Schweet (7) reported a similar system which was capable of initiating new hemoglobin chains and which depended for its activity on the addition of a ribosomal salt wash fraction.
Our work further extends these reports. The present paper describes a cell-free system derived from rabbit reticulocytes which is dependent on added transfer RNA for translating endogenous mRNA.
This system, which maintains the capacity to initiate new chains, is being utilized to study the functions of tRNA in hemoglobin biosynthesis (8,9) and to analyze the biological activity of individual tRNA species.' Isolation of active factors from the ribosomal wash (enzyme fraction) is being reported separately (10). tRNA-de-* The first paper in this series was Reference 8. 1 Unpublished experiments. pendent cell-free systems derived from E. coli a,nd rabbit reticulocytes which translate artificial mRNA templates have been described (11,12).

EXPERIMENTAL PROCEDURE
MatiriaZs-Uniformly labeled L-[i4C]-Valine, uniformly labeled n-[r4C]-leucine, and L-[4, 5-3II]-leucine were obtained from Amersham-Searle and L-[2,3-3H]-valine from Schwarz BioResearch. The isotopes were either used directly or diluted with 12C-L-amino acid; the specific activity used in each experiment is listed in the appropriate legend. Isotopes for t,he data in Table  IV  Preparation of Rabbit Reticulocyte Lysate-Lysate made from rabbit blood having a high reticulocyte count was used as the starting material for the preparation of polysomes, enzyme fraction, and tRNA.
New Zealand white rabbits less than 8 weeks of age and weighing less than 2.5 kg were injected subcutaneously with a 2.5% (w/v) phenylhydrazine solution at a dosage of 0.25 ml per kg per day for 6 consecutive days. On the 1st day of injections each rabbit was given also an intramuscular injection of a vitamin Bn (cyanocobalamin)folic acid solution containing 10 mg of cyanocobalamin and 100 mg of folic acid in 100 ml of 0.9% NaCl.
No phenylhydrazine injection was given on the 7th day, and the rabbits were bled via direct cardiac puncture on the 8th day with a heparinized needle and syringe. All subsequent procedures were carried out at O-4". The blood was centrifuged at 10,000 x q for 10 min, and the plasma was removed and discarded.
After washing the cells twice with 0.14 M NaCl, 0.05 M KCl, and 0.005 M MgC12 and removing the buffy coat, the packed cells were lysed by the addition of 4 volumes of 2 mM MgC12, 1 ml1 dithiothreitol, and 0.1 m&l EDT.1 (neutralized to pH 7 with NaOH).
The suspension was centrifuged at 15,000 X q for 20 min, and the supernatant (lysate) n-a.s used for subsequent preparations. a Brtivity is expressed as micromicromoles of 1%.valine incorporated into protein in 30 min.

Preparation of tRNA-free Polysomes (High-Low
Polysomes)-The lysate preparation was centrifuged at 78,000 x g for 90 min. The supernatant fraction was removed and used for preparation of tRNA (see below).
The upper surface of the polysome pellets was washed with standard sucrose (0.25 M sucrose, 1 mM dithiothreitol, and 0.1 rnnf EDTA, neutralized to pH 7 with NaOH) in order to remove amorphous material.
The pellets were then suspended in sufficient standard sucrose to give an absorbance at 260 rnp of between 275 and 300 units per ml (measured in water). The polysome suspension was treated with high salt as described by Miller and Schweet (7). KC1 (4.0 M) was added slowly over a 2-min period with stirring to the polysomes until a final KC1 concentration of 0.5 M was reached (0.1 ml of 4.0 M KC1 for each 0.7 ml of polysomes).
After I 5 min the solution was centrifuged at 10,000 x g for 10 min, and the pellet was discarded.
After centrifugation at 105,000 x g for 2 hours to obtain the polysome pellet, the upper four-fifths of the high salt wash supernatant was removed and used for the preparation of the enzyme fraction (see below).
The surface of the polysome pellet and the inside of the centrifuge tube were rinsed three times with 0.5 ml of standard sucrose in order to remove adherent tRNA.
The pellet was then suspended in standard sucrose to a concentration of 200 At60 units per ml. Three volumes of standard sucrose were added with mixing, and the suspension was centrifuged at 10,000 X g for 10 min. The pellet was discarded, and the supernatant was centrifuged at 105,000 x g for 2 hours. The final supernatant was discarded, and the polysome pellet was suspended in standard sucrose. After a final centrifugation at 10,000 X g for 10 min, the resulting polysome concentration was between 150 and 200 &,, units per ml. Both the high salt (0.5 M KC1 in standard sucrose) and the low salt (standard sucrose only) washes are necessary to obtain 30.fold tRNA dependence.
Polysomes prepared in this manner are referred to as high-low polysomes.
Aliquots, stored in liquid nitrogen, are stable for at least 6 months.  The yield is approximately 2 A260 units of high-low polysomes per ml of lysate.
Preparation of tRNA-free Enzyme Fraction-The tRNA-free enzyme fraction was prepared from the high salt polysome wash by treatment with DEAE-cellulose.
Cellex D anion exchange cellulose (0.91 meq per g of exchange capacity) was cycled according to the procedure of Peterson and Sober (13), equilibrated with 0.02 M Tris-HCl (pH 7), and stored at 2" in the same buffer. Sufficient DEAE-cellulose was centrifuged at top speed in an International clinical centrifuge for 3 min in order to obtain 3.5 ml of packed material.
The packed cellulose was washed three times with buffer (0.02 M Tris HCl (pH 7), 0.3 M KCI) by dispersal and centrifugation.
The polysome high salt wash (in 0.5 M KCI, see above) was diluted with standard sucrose to give a final KC1 concentration of 0.30 M. Six milliliters of the diluted solution were mixed with 3.5 ml of washed packed DEAE-cellulose. The cellulose was dispersed repeatedly over a IO-min period and then sedimented by centrifugation.
The supernatant was recentrifuged to remove any remaining DEAE-cellulose and then stored in liquid nitrogen.
The enzyme fraction is completely stable for 2 weeks and loses approximately 10% activity after 1 month.
Preparation of Unfractionated Rabbit Reticulocyte tRiVA-An equal volume of water-saturated redistilled phenol (but no potassium acetate) was added to the high speed (105,000 x g) supernatant from the lysate centrifugation (see above), and the mixture was shaken vigorously for 5 min at room temperature. The phases were separated by centrifugation at 10,000 X g for 10 min, and the aqueous phase was removed.
The phenol phase was re-extracted with 0.5 volume of 2% potassium acetate (pH 5.5)-l mM P-mercaptoethanol.
The aqueous phases were pooled, and the nucleic acid was precipitated by the addition of 2 volumes of -20" 95% ethanol.
The mixture was allowed to remain at -20" for 12 to 16 hours, and the RNA was collected by centrifugation at 10,000 X g for 15 minutes at -20".
The 37" and the reactions were stopped by the addition of 2 ml of 10% trichloracetic acid. The mixtures were heated at 90-95" for 10 min and then cooled in an ice bath for 10 min.
Each filter was washed with cold 5% trichloracetic acid, dried, and counted in 10 ml of Liquifluor in toluene in a liquid scintillation counter at an efficiency of 82% for 14C. Chromatography of Globin Chains on Carboxymethyl Cellulose-Separation of the a! and fi chains was carried out by the method of Dintzis (4).
Preparation of Uniformly Labeled Hemoglobin-Preparation of rabbit hemoglobin uniformly labeled with radioactive L-valine or L-leucine for use as standards was carried out by the method of Borsook as described by Schapira et al. (16).
Two-Dimensional Chromatography of Tryptic Peptides (Fingerprinting)-Globin was prepared from the salt-free hemoglobin sample by an acid-acetone precipitation and was subjected to tryptic digestion (16) and p chains obtained from carboxymethyl cellulose chromatography was performed in order to verify the origin of each peptide.

Peptides
were eluted from the chromatography paper with 5% acetic acid and counted in Bray's solution in a liquid scintillation counter at single label efficiencies of 74% for 14C and 40% for 3H, and double label efficiencies of 56% for 14C and 24% for 3H. NRz-Terminal Valine BnalysisNH%-Terminal analysis of hemoglobin was carried out as described by Bishop, Leahy,and Schweet (17). Derivatives of the NHz-terminal amino acid were made with 1-fluoro-2,4-dinitrobenzene.
The derivative is extracted into the ether phase after acid hydrolysis while unsubstituted amino acids remain in the aqueous phase. Aliquots of each phase were counted in Triton X-100 (1 liter of Triton X-100, 2 liters of toluene, 165 ml of Liquifluor) with an efficiency of 79 % for 14C and 44% for 3H.

RESULTS
Optimization of System-The optimum concentrations of components are listed under "Experimental Procedure." As can be seen in Table I, the system is dependent on Mg*, ATP, an ATP-generating system, amino acids, enzyme fraction, polysomes, and tRNA.
The nondependence on dithiothreitol is probably because an adequate amount of this component is already present in the enzyme and polysome fractions.
Since t,he enzyme frac- attempted. An optimum for KC1 was found at 80 to 90 mM. Optimum concentrations for Mg ++, ATP, phosphoenolpyruvate, phosphoenolpyruvate kinase, and amino acids were determined from the data showninFigs.
1 to 4. Although the use of creatine phosphate and creatine phosphokinase has been found to result in greater activity in the lysate system (18)) we have found these reagents to be less effective than phosphoenolpyruvate and phosphoenolpyruvate kinase in this fractionated system. Hemin has no effect in this system, and the addition of DPN+ results in a small, but not reproducible, increase in the extent of protein synthesis.
For maximum activity it is essential that a correct ratio of polysomes to enzyme fraction be used. The enzyme fraction contains, in addition to Tl, T2, and aminoacyl-tRNA synthetases, other factors required for protein synthesis de novo (7, 10). The concentration curve for this multifactor component is sigmoidal, not linear, as can be seen in Fig. 5. If a lesser amount of polysomes is used the enzyme fraction curve is shifted to the left, resulting in a more obvious saturation plateau; if a greater amount of polysomes is used the curve is shifted to the right, resulting in an inability to saturate the system for this particular enzyme fraction preparation within a KC1 concentration of 90 mM. In the presence of saturating concentrations of enzyme fraction and tRNA, the polysomes are rate-limiting as shown in Fig. 6. Under these conditions, incorporation of amino acids into hemoglobin is linear for 45 min, as shown in Fig. 7. The system has a 25-to 30-fold dependence on added tRNA as shown in Fig. 8 and Table I. Characterization of Products-To confirm that the products of the tRNA-dependent assay system are complete new chains of hemoglobin the following studies were performed.
(a) The '*C- given in Table II, show uniform labeling of the 14C peptides; i.e. as much label was found in the NHz-terminal Peptides al and fil as in the internal peptides, indicating new chain synthesis. This result was the same whether or not the ribosomes were removed from the reaction mixture after the incubation. (c) NHz-Terminal valine analysis showed that the NHz-terminal assembly of fi chains in either normal or thalassemic valine was labeled to the extent expected for uniformly labeled reticulocytes. hemoglobin (Table III).
In previous studies with cell-free E. coli systems, we have E$ect of Limiting the tRNA Concentration In order to deter-examined the effect of tRNA species on the rate of translation mine the effect on the rates of synthesis of the a and /3 hemo-of artificial mRNA templates (11). It was suggested that AGA globin chains that is produced by an alteration in the concentra-and AGG might be "regulatory" codons in E. coli, i.e. codewords tion of unfractionated tRNA in the reaction mixture, the following recognized by species of tRNA present in rate-limiting amounts. experiment was performed. Two reaction mixtures were in-In order to determine whether or not individual species of tRNA cubated simultaneously: one (0.5 ml) contained 3H-r-leucine might be capable of influencing the rate of natural mRNA (1000 mCi per mmole) and 0.05 Z&60 unit of rabbit reticulocyte translation in mammalian cells, the synthesis of hemoglobin was tRNA per 50 ~1 ("excess" tRNA) ; the other (1.0 ml) was identical examined in the tRNA-dependent rabbit reticulocyte cell-free except for 14C-L-leucine (311 mCi per mmole) and only 0.002 system. When only a limiting amount of tRNA is available for Azoo unit tRNA per 50 ~1 ("limiting" tRNA).
The two mix-hemoglobin synthesis, the data of Fig. 9 indicate that a: chain tures were pooled after a 45.min incubation at 37", the heme production is reduced to 50% of the production of /3 chains. was removed by acid-acetone precipitation, and the globin chains When a saturating amount of unfractionated tRNrZ is used to were separated by carboxymethyl cellulose column chroma-supplement the system, approximately equal QI and fi chain tography.
As shown in Fig. 9, although the synthesis of both synthesis was produced (8). It therefore appears that one (or chains was depressed under conditions of "limiting" tRNA, (Y more) species of tRNA, which is used to a greater extent for a! chain synthesis was reduced to 50% of the production of fl than for fi chain synthesis, has become rate-limiting.
In previous chains.
studies a similar result was obtained when cell-free hemoglobin I3ffect of Adding tRNA from Heterologous Sources-Rabbit synthesis was carried out in the presence of saturating levels of liver tRNA appears to stimulate the rate of hemoglobin synthesis tRNA, but where several tRNA species were deleted (8). The to an equal extent as rabbit reticulocyte tRNA at limiting tRNA fractionated tRNA was produced by combining portions from a concentrations, as shown in Fig. 10. However, a higher rate of Freon reversed phase chromatography of unfractionated tRNA. synthesis is achieved in the presence of saturating reticulocyte Unequal chain synthesis with fractionated tRl;A or limiting tRNA than liver tRNA. E. coli tRNA is unable to stimulate concentrations of unfractionated tRNA lends support to the the synthesis of hemoglobin, as shown in Fig. 10. .4 partial ex-hypothesis that a different combination of tRNA species is planation for this result is seen in Table IV. Rabbit reticulocyte utilized in translating o(-and P-hemoglobin mRNA molecules in aminoacyl-tRNA synthetases are unable to acylate some species vivo. Even though the 01 and /3 chains of rabbit hemoglobin of E. coli tRNA.
If unfractionated E. coli tRNA is first acylated both contain all 20 amino acids, the (Y chain must contain at with fl. coli aminoacyl-tRNA synthetases, purified, and then least one codon which is used either less often or not at all in p added to the rabbit reticulocyte reaction mixture, stimulation of chain synthesis. This possibility exists because most amino hemoglobin synthesis does 0ccur.r acids are recognized by more than one codeword (26). In fact, evidence exists which suggests that the leucine codon UUG might DISCUSSION be utilized only once in the rabbit o( chain and not at all in the The cell-free protein-synthesizing system described in this fi chain (20,27). Preliminary evidence suggests that the tRNA paper is unique in that it is the first nonbacterial assay system responsible for the inhibition observed in our studies, however, reported that is dependent on added tRNA to translat,e natural might be a species of tRNA specific for glycine. This conclusion messenger RNA.
The products of the reaction are primarily is based on the observation that stimulation of r4C-valine transfer complete a-and fl-globin chains; i.e. initiation of new chains is from aminoacyl-tRN,4 into hemoglobin, produced by suppleactive. This assay system is being utilized to study the role of menting a tRNA-limiting system with additional tRNA plus tRNA in hemoglobin biosynthesis, the biological activity of aminoacyl-tRNA synthetases, is lost if the one amino acid, individual tRNA species, and the mechanism of initiation in the glycine, is omitted from the reaction mixture.l cell-free synthesis of hemoglobin.
The present experiments do not examine control in vivo of A role of tRNA in regulating the rate of translation of hemo-normal rabbit reticulocyte hemoglobin synthesis. However, since globin mRNA was first postulated by Itano (19). A theoretical the rates of translation of the hemoglobin mRNAs can be diffoundation for such a role was shown when Weisblum et al. (20) ferentially slowed in vitro either by reducing the total amount demonstrated that leucine tRT;IA can be separated into two frac-of tRNA present or by deleting a tRNA fraction, it is not untions which appear to insert leucine into different positions in reasonable to postulate that similar effects might take place the hemoglobin chains. This work has been extended to show within the intact cell either as normal control mechanisms or as that a similar phenomenon appears to exist for tRNAs carrying a result of mutations. serine (21), arginine (22), or glutamic acid (23). Since different The experiments examining hemoglobin synthesis in the presspecies of tRNA might be responsible for inserting an amino ence of unfractionated tRNA prepared from heterologous sources acid into different positions in the hemoglobin chains, the presence are not easy to interpret. It is possible that liver and reticuloof a limiting amount of one species might produce a reduced cyte tRNA contain the identical types and amounts of tRNA rate of mRNA translation at a given point. Winslow and species in vivo, but not after purification. Certainly, the purifica-Ingram (24) have shown, in fact, that the rate of assembly of tion of rabbit liver tRNA requires several different steps from hemoglobin chains in human bone marrow cells may contain a those described under "Experimental Procedure" for the purifica-"control point" in the synthesis of each chain beyond which tion of reticulocyte tRNA since the former must be separated the growth of the polypeptide chain is reduced. Clegg et al. from active nucleases, membranes, nuclear contents, etc. It is (25), however, were unable to detect a control point during the also possible, however, that liver cells and reticulocytes each contain their own spectrum of tRNA species and that these species reflect the synthetic requirements of that cell type. The data of Fig. 10 and Table 4 indicate that at least some E. cali tRNA species are distinctly different from rabbit species. Previous work from this laboratory examining heterologous acylation reactions between E. co& rabbit liver, and human spleen sources is consistent with the present data (15). Under investigation is the possibility that at least some E. coZi tRNA species cannot be directly utilized in cell-free mammalian protein synthesis, but might participate indirectly by transferring their amino acids to reticulocyte tRNA species.*