Vol. 122, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, May 1975, p. 719-726 Copyright 0 1975 American Society for Microbiology
Endogenous Messenger Ribonucleic Acid-Directed Polypeptide Chain Elongation in a Cell-Free System from the Yeast Saccharomyces cerevisiae BYRON M. GALLIS1 AND ELTON T. YOUNG* Departments of Biochemistry* and Genetics, University of Washington, Seattle, Washington 98195 Received for publication 17 February 1975
An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respectively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 ,M pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes in vitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35SS]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.
eukaryotic cell-free systems, which have been shown to initiate and elongate polypeptides. Using knowledge gained from the characterization of other eukaryotic systems (1, 15, 23, 30), we attempted to develop an initiating, messenger-dependent protein-synthesizing system from yeast. It is shown that lysates of log-phase, vegetative yeast are neither messenger dependent nor capable of initiating the synthesis of polypeptides on added or endogenous mRNA.
The yeast Saccharomyces cerevisiae is a simple eukaryote which may provide a model system for study of control mechanisms in higher organisms. Knowledge of ribonucleic acid (RNA) metabolism in yeast (25, 38), as well as the extensive genetics of yeast (11, 27), allows the design of experiments that may be important in understanding the control of RNA synthesis in eukaryotes. Toward this end, we attempted to develop an in vitro protein synthesis system from yeast to use as an assay for the in vivo synthesis of yeast messenger RNA (mRNA). Several attempts (5, 33) have been made to develop in vitro protein-synthesizing systems from yeast, but it is not known whether these systems initiated polypeptide chains properly or could be made messenger dependent. The description of yeast extracts used for peptide synthesis preceded the characterization of other
MATERIALS AND METHODS Growth of yeast and preparation of yeast lysate. Strains of yeast (AP-1, 1/a; 131-209/9) were obtained from Anita K. Hopper. The doubling time was 75 min at 30 C in YEP medium (2 g of glucose, 20 g of peptone [Difco], and 10 g of yeast extract per liter plus 40 jig each of adenine and uracil per ml). Cells were initially grown to stationary phase, counted, and inoculated into four 1-liter cultures at 104 cells per ml. Fifteen hours later the cultures were harvested in
I Present address: Department of Virology, ISREC, 1011 Lausanne, Switzerland.
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log phase at a concentration of 2 x 107 to 4 x 107 cells per ml. Yeast cultures were rapidly poured into centrifuge buckets containing ice, and the cells were centrifuged in a Sorvall RC-3 centrifuge at 5,500 rpm for 5 min. The yield was about 8 to 10 g of cells. Cells were washed by suspension in 3 volumes of lysis buffer [10 mM KCl, 10 mM tris(hydroxymethyl)aminomethanehydrochloride (pH 7.5), 1.5 mM magnesium acetate] and centrifuged in a Sorvall RC2B ss-34 rotor at 10,000 rpm for 10 min. The cells were resuspended in 2 volumes of lysis buffer with twice their weight of acid-washed glass beads (0.45 to 0.55 mm diameter) and broken with three 15-s bursts in a Bronwill homogenizer. The lysate was centrifuged for 10 min at 30,000 x g. The supernatant fluid was removed and passed over a G-25 (medium) column (2.5 by 30 cm) equilibrated with 25 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5), 85 mM KCl, 3.3 mM magnesium acetate, and 1 mM dithiothreitol. Those fractions in the void volume containing more than 50 absorbancy (260 nm) units per ml were pooled, quick-frozen in 0.65-ml portions in a dry ice-acetone bath, and stored at -70 C. No loss of activity was observed over a period of several months. The S-30's contained 60 to 70 absorbancy (260 nm) units per ml. Preparation of the yeast S-30 was sometimes varied. In a preparation designed to inhibit possible proteolysis by trace metal-activated proteases, 1 mM ethylenediaminetetraacetate (tetrasodium salt) was substituted for 1.5 mM magnesium acetate in the lysis buffer. The ethylenediaminetetraacetate was subsequently removed by G-25 chromatography, which also equilibrated the lysate with 3.3 mM magnesium acetate. Alternatively, phenylmethane sulfonyl fluoride, an inhibitor of serine proteases, was added to a final concentration of 1 mM in combination with ethylenediaminetetraacetate to the lysis buffer, and the lysate was then processed as above. For preparations that were preincubated, the conditions were identical to those used for the Krebs ascites system (22, 23) subsequent to passage over the G-25 column. Preincubation was for 15 min at 30 C. Assay for amino acid incorporation. Each 100-Al reaction mixture contained 25 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5), 3.3 mM magnesium acetate, 85 mM KCl, 1 mM dithiothreitol, 1 mM adenosine 5'-triphosphate, 0.2 mM guanosine 5'-triphosphate, 5 mM creatine phosphate, 0.15 mg of creatine phosphokinase per ml, 40 ,AM concentrations of the 19 amino acids, one unlabeled amino acid as indicated, 60 IAI of S-30, and RNA as indicated. All incubations were at 30 C. Each reaction mixture contained 3.0 to 3.5 mg of ribosomes per ml. Reactions were terminated by adding 10 Al of reaction mixture to 1 ml of 5% trichloroacetic acid containing the nonradioactive isotope of the amino acid used for labeling. The precipitate was heated at 80 C for 10 min to hydrolyze amino acyl transfer RNA (tRNA) and collected on GF/C filters. Preparations of the Krebs ascites system and the rabbit reticulocyte polysomal RNA were as described (22, 23). Preparation of rib.oome high-salt factors. Ribosome high-salt wash factors were prepared by the
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methods of Schreier and Staehelin (30). Ribosome high-salt wash factors from yeast ribosomes were purified through the ammonium sulfate step; those from Krebs ascites ribosomes were purified through the diethylaminoethyl cellulose step. Preparation of tRNA's. tRNA's were prepared from mouse liver, yeast, and Escherichia coli by the method of von Ehrenstein (39). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a slight modification of that of Studier'(36). Chemicals. L-[35S]methionine (141 Ci/mmol) and L- [6-3H]serine (1.23 Ci/mmol) were purchased from New England Nuclear; adenosine 5'-triphosphate and guanosine 5'-triphosphate were from P. L. Biochemicals; phosphocreatine, phosphocreatine kinase, cycloheximide, puromycin, and polyuridylic acid [poly(U) I were from Sigma Chemical Co.; and amino acids were from Calbiochem.
RESULTS
Kinetics of amino acid incorporation. A time course of amino acid incorporation directed by endogenous mRNA shows that synthesis of polypeptides is linear for 4 min but reaches a maximum by 8 min (Fig. 1A). This is similar to the results of So and Davie (33) and Bretthauer et al. (5), who showed that amino acid incorporation in yeast lysates was linear for the first 5 to 10 min. The duration of time over which amino acid incorporation occurs in the yeast lysate is brief, however, relative to the duration of incorporation in other eukaryotic cell-free systems such as the Krebs II ascites (23), the wheat embryo (8), and the rabbit reticulocyte (17) cell-free systems. Effect of rabbit reticulocyte and yeast polysomal RNAs on amino acid incorporation. The brief duration of amino acid incor-
Minutes
FIG. 1. Kinetics of protein synthesis in the yeast cell-free system. Reactions (100 ul) contained 50 ,Ci of [3H]serine (1.23 Ci/mmol) per ml. Samples of the reaction mixture were counted at the times indicated; 1 pmol = 80 counts/min. (A) No preincubation; (B) preincubation for 15 min at 30 C. Symbols: 0, endogenous incorporation; A, 100 ug of yeast polyadenylic acid-containing RNA per ml; 0, 600 usg of rabbit reticulocyte polysomal RNA per ml.
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poration suggested that a labile component(s) became inactivated during the preparation of the yeast lysate or under conditions of protein synthesis. Therefore, various conponents required for protein synthesis were added to the yeast lysate to attempt to extend the duration of polypeptide synthesis. Addition of either rabbit reticulocyte or yeast polysomal RNAs to the yeast S-30 failed to stimulate or extend the duration of amino acid incorporation over and beyond the high, endogenous background activity (Fig. 1A). Some eukaryotic cell-free systems translate different mRNA's with different efficiencies (31). For example, rabbit reticulocyte lysates translate endogenous hemoglobin messenger more efficiently than heterologous mouse or duck hemoglobin messenger (17, 35). Thus, the possibility existed that the yeast ribosomes were translating endogenous mRNA more effeciently than added rabbit reticulocyte polysomal RNA containing hemoglobin messenger, but that the high, endogenous amino acid incorporation masked the translation of hemoglobin messenger. In the rabbit reticulocyte lysate, which contains high amounts of endogenous hemoblobin messenger, polypeptides are made from exogenous RNA's without stimulation of amino acid incorporation (16, 34). If the yeast S-30 is allowed to incorporate [35S jmethionine after addition of polysomal RNA from rabbit reticulocytes and the products are separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, no polypeptide is made that corresponds in molecular weight to marker hemoglobin (Fig. 2). The addition of this rabbit reticulocyte polysomal RNA to a preincubated Krebs II ascites system (23) causes the synthesis of polypeptides which co-migrate with the hemoglobin marker (Fig. 2), indicating that the mRNA is active. The yeast polysomal RNA is also highly active as we have shown by translating it in both the Krebs ascites cell-free system and in an extract from wheat embryos (8a). To attempt to make amino acid incorporation in the yeast cell-free system dependent upon added mRNA, the endogenous amino acid incorporation was lowered by preincubation under conditions of protein synthesis. This allows ribosomes to complete their cycle and degradation of endogenous mRNA to occur (23, 30). However, the protein synthetic activity directed by added mRNA, as well as the endogenous activity, decreases as the time of preincubation increases (22). Therefore, [3H]uridine mRNA from T7 bacteriophage-infected E. coli was added to the yeast system under conditions of protein synthesis to determine how brief an
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incubation period would be needed to degrade mRNA. In 15 min at 30 C, 50% of the [3H]uridine T7 mRNA became acid soluble (Fig. 3). This rate of solubilization is about the same as that observed in E. coli cell-free systems. Preincubation of the system under reaction conditions for 15 min at 30 C (see above) reduced amino acid incorporation by 80% (Fig. 1B, note scale change). Addition of rabbit reticulocyte polysomal RNA or yeast polyadenylic acidenriched mRNA (8a) still failed to stimnulate amino acid incorporation (Fig. 1B). Effect of tRNA and ribosome high-salt wash factors on amino acid incorporation. Several other components necessary for protein synthesis were added to the lysate to extend the duration of synthesis or to achieve a stimulation of amino acid incorporation. Since the system appeared to elongate polypeptide chains but not to initiate them (see below), several potentially labile components required for initiation, tRNA and initiation factors, were added to the system (Table 1). In some instances, tRNA (2, 3) and initiation factors (14, 24, 26, 28) have been shown to be labile components of the Krebs ascites system. However, neither tRNA purified from E. coli, S. cerevisiae, or mouse liver nor ribosome high-salt wash factors purified from yeast or Krebs ascites ribosomes stimulate or extend the duration of amino acid incorporation significantly in the yeast lysate (Table 1). Neither of these ribosome high-salt wash factor preparations, when tested in a Krebs ascites system containing Krebs salt-washed ribosomes, could be shown to be active. Since the data suggest that there is no initiation on exogenous mRNA, the system was studied further to determine whether or not initiation was occurring on endogenous mRNA. Dependence of amino acid incorporation upon cation concentration. There is little dependence of amino acid incorporation on potassium concentration over a range of 50 to 130 mM KCl. Maximum incorporation occurs over a range of 85 to 100 mM KCl. The optimal magnesium concentration is about 6 mM, but amino acid incorporation occurs over a range of 3 to 13 mM Mg2+. So and Davie (33) and Bretthauer et al. (5) found similar, broad magnesium optima for yeast in in vitro protein synthesis systems, but these data are in contrast to magnesium optima described for the Krebs ascites (22), the wheat embryo (29), and the rabbit reticulocyte (1) cell-free systems, in which amino acid incorporation occurs from about 2 to 4 mM Mg2+. Stimulation of polyphenylalanine synthesis by poly(U). The translation of natural mRNA's in eukaryotic cell-free systems is re-
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mRNA-DIRECTED POLYPEPITIDE CHAIN ELONGATION
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FIG. 3. Degradation of T7 mRNA with time under conditions of protein synthesis in the yeast cell-free system. A 100-,ul reaction mixture contained 13 gg (1,000 counts/min per Ag) of [3H]uridine T7 RNA. Aliquots of the reaction mixture were removed at the times indicated and precipitated with 5% trichloroacetic acid.
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thauer et al. (5) observed a similar stimulation by poly(U) in yeast extracts. Effects of cycloheximide and pactamycin on endogenous amino acid incorporation. To test whether or not the endogenous amino acid incorporation was due to polypeptide chain initiation and elongation or to elongation alone, antibiotics that inhibit either initiation or elongation were added to the system (Table 2). Pactamycin, which, at 1 AM, specifically blocks initiation in E. coli (7) and in mammalian extracts (18, 24), has no effect on amino acid incorporation in the yeast S-30 (Fig. 4A). However, 1 ,uM pactamycin inhibits rabbit reticulocyte polysomal RNA-directed amino acid incorporation in the Krebs ascites system (Fig. 4B). All translations were carried out at 3.3 mM Mg2+, since Kappen et al. (13) have shown that high magnesium overcomes pactamycin inhibition of initiation. At concentrations of pactamycin > 10 AM (20), chain elongation is also inhibited. Pactamycin at 50 ,uM (Fig. 4A) partially inhibits amino acid incorporation in the yeast S-30. On the other hand, cyclohexiTABLE 1. Effect of tRNA's or ribosome high-salt wash
stricted to a low, narrow range of magnesium factors on amino acid incorporationa concentrations, about 2 to 4 mM Mg2+ (22, 23, 29, 40). In contrast, the translation of the Serine ConcenincorAdditions tration synthetic messenger, poly(U), occurs over a (mg/ml) porated range of 5 to 20 mM Mg (23, 30) and its (pmol) translation is not dependent upon the presence None ..140 of initiation factors (32, 40). The addition of 136 poly(U) to the yeast cell-free system mimics the E. coli tRNA .0.4 Mg2+-dependent characteristics of the system Yeast ribosome high-salt wash factors ..................... 1 82 by using endogenous natural messenger as temribosome high-salt wash plate. The optimum magnesium concentration Yeast factors, E. coli tRNA ........ 1, 0.4 82 for phenylalanine incorporation is high, 6 to 8 Krebs ascites ribosome high-salt mM Mg2+, about the same as the optimum for wash factors ............ .... 1 138 phenylalanine incorporation directed by endog- Krebs ascites ribosome high-salt enous messenger. Both endogenous mRNA and wash factors, rabbit reticulopoly(U) are translated over the same broad cyte polysomal RNA ........ 1, 0.6 138 range of magnesium concentrations. These ob- Krebs ascites ribosome high-salt servations suggest that the endogenous stimulawash factors, yeast polyadenylic acid-containing RNA .. 1, 0.5 138 tion of amino acid incorporation is due to ..169 elongation of polypeptide chains, not initiation. None 0.3 168 The inability of the system to translate added Yeast tRNA .................. 0.3 187 messenger is not due to the lack of ribosomes or Mouse liver tRNA ............. a protein synthesis components necessary for Each 100-ul reaction mixture contained 50 of elongation, since addition of 400 ,g of poly(U) [3H]serine (1.23 Ci/mmol) per ml, 85 mM KCl,pCiand to the system stimulates phenylalanine incorpo- 3.3 mM magnesium acetate. 1 pmol = 80 counts/min. ration fivefold over endogenous synthesis. Bret- The S-30's were not preincubated. FIG. 2. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell-free products. (a,b) Krebs ascites system; (c,d) yeast lysate; (b,d) rabbit reticulocyte polysomal RNA added. Protein synthesis reaction contained 100 MCi of L-[35S]methionine (141 Ci/mmol) per ml. Reactions were terminated by addition of an equal volume of x2 Studier buffer [100 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 6.8; 2% sodium dodecyl sulfate; 20% glycerol; 0.28 3-mercaptoethanol, 0.1% bromophenol blue] and boiling for 2 min. A 10-Ml volum e of this mixture was placed in each slot of a 15% polyacrylamide slab gel and electrophoresis was performed. The gel was dried and autoradiographed.
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GALLIS AND YOUNG TABLE 2. Inhibition of protein synthesisa
.Concn Additios Additions (yMm)
Serine incorporated
%bition Inhi-
(pmol)
None Pactamycin ......... Pactamycin ......... None Cycloheximide ...... a
Each
1 50 10
90 90 90 105 13
0 0 34 0 87
100-Al reaction mixture contained 50 yCi of
[3H]serine (1.23 Ci/mmol) per ml, 85 mM KCl, and 3.3 mM magnesium acetate. 1 pmol The S-30's were not preincubated.
=
80 counts/min.
mide, which inhibits chain elongation in eukaryotic cell-free systems at 10 AM (9, 18), blocks incorporation in the yeast S-30 by 87% (Table 2). The sensitivity of the system to cycloheximide and its resistance to low levels of pactamycin suggest that the observed amino acid incorporation is due to chain elongation, not initiation on endogenous mRNA. Effects of inhibition of proteases on amino acid incorporation in the yeast lysate. Lysis of yeast cells for preparation of the S-30 could have resulted in protease degradation of ribosome subunits or factors necessary for translation. Lysis of log-phase growing yeast cells causes release of proteases (37). There are three general classes of proteases in yeast (25): acid proteases, which are completely inhibited at pH 7.5; active-serine alkaline proteases, which are inhibited by compounds such as phenylmethane sulfonyl fluoride; and trace metal-activated proteases, which are inhibited by 1 mM ethylenediaminetetraacetate. Therefore, yeast lysates, instead of being prepared in pH 7.5 buffer (see above) containing 1.5 mM magnesium acetate, were prepared by adding pH 7.5 buffer containing 1 mM ethylenediaminetetraacetate without a divalent cation. The lysate was passed over a G-25 column equilibrated with reaction buffer containing 3.0 mM Mg to remove the ethylenediaminetetraacetate and
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DISCUSSION The characteristics of protein synthesis in the yeast S-30 suggest that amino acid incorporation is due to the elongation of nascent polypeptide chains and not to de novo initiation of new polypeptides. There are three lines of evidence for this. Pactamycin, an inhibitor of polypeptide chain initiation, fails to block amino acid incorporation. Secondly, incorporation occurs maximally at magnesium concentrations that are inhibitory to translation of added natural mRNA's in other cell-free systems. These Mg concentrations are optimal for translation of poly(U), which may be translated without all of the components necessary for initiation (32, 40). Finally, the system fails to be stimulated by or to translate added natural mRNA's, which are translated upon addition to either a Krebs ascites system or wheat embryo cell-free system. The inability of the yeast S-30 to be stimulated by active homologous template RNAs indicates that the failure to initiate translation on exogenous RNA is not due solely to an incompatibility between mRNA and ribosomes or initiation factors. On the other hand, addition of poly(U), a synthetic polymer known not to require a complete initiation complex, stimulates the system, as was also shown by Bretthauer et al. (5) and by So and Davie (33). The inability of the yeast lysate to initiate could be due to the lability of ribosomes, initiation factors, tRNA, or mRNA. Yeast transfer and polyadenylic acid-enriched mRNA added to the yeast lysate appear to be funcA
B
6~ ~ ~ ~ ~1
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5
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restore the proper Mg concentration. No differFIG. 4. Effect of pactamycin on the kinetics of ence in amino acid incorporation or ability to amino acid incorporation in the yeast and Krebs initiate was found between this preparation and ascites cell-free systems. Reactions (100 jil) contained the preparation lysed in buffer containing 1.5 the same amount of [3H]serine as indicated in the mM magnesium acetate. Furthermore, when legend of Fig. 1 and the same magnesium and lysis was carried out in pH 7.5 buffer containing potassium concentrations, 3.3 mM Mg and 85 mM are optimal except that the both 1 mM ethylenediaminetetraacetate and 1 KCI; all ionic conditions was lowered to test the effect Mg concentrations mM phenylmethane sulfonyl fluoride, and these yeast of pactamycin. (A) Incorporation by yeast cell-free were subsequently removed by the G-25 chro- system directed by endogenous mRNA. (B) Incorpomatography, no change in the ability of the ration by Krebs ascites cell-free system directed by system to initiate was detected. However, this 600 ,g of rabbit reticulocyte polysomal RNA per ml. method also reduced endogenous incorporation Symbols: A, no pactamycin; 0, 1 uM pactamycin; 0, 50 ,M pactamycin. by 50%.
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tional components in other cell-free systems. Both the Krebs ascites and wheat embryo cell-free systems translate this yeast mRNA into polypeptides similar to those made in vivo (8a). When added to the wheat embryo cellfree system in the presence of yeast polyadenylic acid-enridhed RNA, yeast tRNA stimulates mRNA-directed incorporation twofold. The rate of RNA degradation in the yeast extract (Fig. 3) does not appear to be high enough to preclude stimulation by exogenous mRNA. Since yeast ribosomes are able to elongate polypeptides in vitro, the inability of the yeast S-30 to initiate may be due to inactive initiation factors, not ribosomes. We have not been able to prepare active initiation factors from yeast. One observation suggests that initiation factors that dissociate ribosomes are inactive in yeast lysates. When yeast polysomes are prepared in the absence of cycloheximide, most of the ribosomes are in the form of 80S monomers, not subunits (our unpublished data). Thus, initiation factors in yeast lysates are unable to effect a dissociation of monosomes into subunits (12, 19). Lack of free subunits would prevent initiation on either endogenous or added yeast polyadenylic acid-enriched mRNA (10). Yeast ribosomes were treated with a solution of 0.5 M KCl and 10 mM puromycin, which dissociates them into subunits (4, 6). However, a fractionated yeast in vitro system made from an S-100, with puromycin-treated rib.osomes and a ribosomal high-salt wash, does not initiate polypeptide synthesis with polyadenylic acid-enriched RNA from yeast. The lack of initiation may be due to other initiation factor functions that are inactive in the yeast extract. ACKNOWLEDGMENTS This work was supported by a Public Health Service Training Grant to the Department of Biochemistry, by American Cancer Society grant NP 124, and by Public Health Service grant AI 09456 from the National Institute of Allergy and Infectious Diseases. B.G. wishes to thank Jim and Anita Hopper for constant encouragement and continued interest throughout the course of this work and Jim Hopper for the gel analysis shown in Fig. 2. We thank G. B. Whitfield of the Upjohn Co. for his generous gift of pactamycin. LITERATURE CITED 1. Adamson, S. D., E. Hubert, and W. Godchaux. 1968. Factors affecting the rate of protein synthesis in lysate systems from reticulocytes. Arch. Biochem. Biophys.
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31.
32.
33. 34.
230:172-176. 35. Stewart, A. G., E. S. Gander, C. Moul, B. Luppis, and K. Scherrer. 1973. Differential translation of duck- and rabbit-globin messenger RNA's in reticulocyte-lysate systems. Eur. J. Biochem. 34:205-212. 36. Studier, F. W. 1973. Analysis of bacteriophage T7 early RNA's and proteins on slab gels. J. Mol. Biol. 79:237-248.
37. Sylv6n, B., C. A. Tobias, H. Malmgren, R. Ottoson, and B. Thorell. 1959. Cyclic variations in the peptidase and catheptic activities of yeast cultures synchronized with respect to cell multiplication. Exp. Cell Res. 16:75-87. 38. Udem, S. A., and J. R. Warner. 1972. Ribosomal RNA synthesis in Saccharomyces cerevisiae. J. Mol. Biol. 65:227-242. 39. von Ehrenstein, G. 1967. Isolation of sRNA from intact Escherichia coli cells, p. 588-596. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12A. Academic Press Inc., New York. 40. Wigle, D. T., and A. E. Smith. 1973. Specificity in initiation of protein synthesis in a fractionated mammalian cell-free system. Nature (London) New Biol. 242:136-140.
JOURNAL OF BACTERIOLOGY, May 1975, p. 719-726 Copyright 0 1975 American Society for Microbiology
Endogenous Messenger Ribonucleic Acid-Directed Polypeptide Chain Elongation in a Cell-Free System from the Yeast Saccharomyces cerevisiae BYRON M. GALLIS1 AND ELTON T. YOUNG* Departments of Biochemistry* and Genetics, University of Washington, Seattle, Washington 98195 Received for publication 17 February 1975
An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respectively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 ,M pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes in vitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35SS]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.
eukaryotic cell-free systems, which have been shown to initiate and elongate polypeptides. Using knowledge gained from the characterization of other eukaryotic systems (1, 15, 23, 30), we attempted to develop an initiating, messenger-dependent protein-synthesizing system from yeast. It is shown that lysates of log-phase, vegetative yeast are neither messenger dependent nor capable of initiating the synthesis of polypeptides on added or endogenous mRNA.
The yeast Saccharomyces cerevisiae is a simple eukaryote which may provide a model system for study of control mechanisms in higher organisms. Knowledge of ribonucleic acid (RNA) metabolism in yeast (25, 38), as well as the extensive genetics of yeast (11, 27), allows the design of experiments that may be important in understanding the control of RNA synthesis in eukaryotes. Toward this end, we attempted to develop an in vitro protein synthesis system from yeast to use as an assay for the in vivo synthesis of yeast messenger RNA (mRNA). Several attempts (5, 33) have been made to develop in vitro protein-synthesizing systems from yeast, but it is not known whether these systems initiated polypeptide chains properly or could be made messenger dependent. The description of yeast extracts used for peptide synthesis preceded the characterization of other
MATERIALS AND METHODS Growth of yeast and preparation of yeast lysate. Strains of yeast (AP-1, 1/a; 131-209/9) were obtained from Anita K. Hopper. The doubling time was 75 min at 30 C in YEP medium (2 g of glucose, 20 g of peptone [Difco], and 10 g of yeast extract per liter plus 40 jig each of adenine and uracil per ml). Cells were initially grown to stationary phase, counted, and inoculated into four 1-liter cultures at 104 cells per ml. Fifteen hours later the cultures were harvested in
I Present address: Department of Virology, ISREC, 1011 Lausanne, Switzerland.
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GALLIS AND YOUNG
log phase at a concentration of 2 x 107 to 4 x 107 cells per ml. Yeast cultures were rapidly poured into centrifuge buckets containing ice, and the cells were centrifuged in a Sorvall RC-3 centrifuge at 5,500 rpm for 5 min. The yield was about 8 to 10 g of cells. Cells were washed by suspension in 3 volumes of lysis buffer [10 mM KCl, 10 mM tris(hydroxymethyl)aminomethanehydrochloride (pH 7.5), 1.5 mM magnesium acetate] and centrifuged in a Sorvall RC2B ss-34 rotor at 10,000 rpm for 10 min. The cells were resuspended in 2 volumes of lysis buffer with twice their weight of acid-washed glass beads (0.45 to 0.55 mm diameter) and broken with three 15-s bursts in a Bronwill homogenizer. The lysate was centrifuged for 10 min at 30,000 x g. The supernatant fluid was removed and passed over a G-25 (medium) column (2.5 by 30 cm) equilibrated with 25 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5), 85 mM KCl, 3.3 mM magnesium acetate, and 1 mM dithiothreitol. Those fractions in the void volume containing more than 50 absorbancy (260 nm) units per ml were pooled, quick-frozen in 0.65-ml portions in a dry ice-acetone bath, and stored at -70 C. No loss of activity was observed over a period of several months. The S-30's contained 60 to 70 absorbancy (260 nm) units per ml. Preparation of the yeast S-30 was sometimes varied. In a preparation designed to inhibit possible proteolysis by trace metal-activated proteases, 1 mM ethylenediaminetetraacetate (tetrasodium salt) was substituted for 1.5 mM magnesium acetate in the lysis buffer. The ethylenediaminetetraacetate was subsequently removed by G-25 chromatography, which also equilibrated the lysate with 3.3 mM magnesium acetate. Alternatively, phenylmethane sulfonyl fluoride, an inhibitor of serine proteases, was added to a final concentration of 1 mM in combination with ethylenediaminetetraacetate to the lysis buffer, and the lysate was then processed as above. For preparations that were preincubated, the conditions were identical to those used for the Krebs ascites system (22, 23) subsequent to passage over the G-25 column. Preincubation was for 15 min at 30 C. Assay for amino acid incorporation. Each 100-Al reaction mixture contained 25 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5), 3.3 mM magnesium acetate, 85 mM KCl, 1 mM dithiothreitol, 1 mM adenosine 5'-triphosphate, 0.2 mM guanosine 5'-triphosphate, 5 mM creatine phosphate, 0.15 mg of creatine phosphokinase per ml, 40 ,AM concentrations of the 19 amino acids, one unlabeled amino acid as indicated, 60 IAI of S-30, and RNA as indicated. All incubations were at 30 C. Each reaction mixture contained 3.0 to 3.5 mg of ribosomes per ml. Reactions were terminated by adding 10 Al of reaction mixture to 1 ml of 5% trichloroacetic acid containing the nonradioactive isotope of the amino acid used for labeling. The precipitate was heated at 80 C for 10 min to hydrolyze amino acyl transfer RNA (tRNA) and collected on GF/C filters. Preparations of the Krebs ascites system and the rabbit reticulocyte polysomal RNA were as described (22, 23). Preparation of rib.oome high-salt factors. Ribosome high-salt wash factors were prepared by the
J. BACTERIOL.
methods of Schreier and Staehelin (30). Ribosome high-salt wash factors from yeast ribosomes were purified through the ammonium sulfate step; those from Krebs ascites ribosomes were purified through the diethylaminoethyl cellulose step. Preparation of tRNA's. tRNA's were prepared from mouse liver, yeast, and Escherichia coli by the method of von Ehrenstein (39). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a slight modification of that of Studier'(36). Chemicals. L-[35S]methionine (141 Ci/mmol) and L- [6-3H]serine (1.23 Ci/mmol) were purchased from New England Nuclear; adenosine 5'-triphosphate and guanosine 5'-triphosphate were from P. L. Biochemicals; phosphocreatine, phosphocreatine kinase, cycloheximide, puromycin, and polyuridylic acid [poly(U) I were from Sigma Chemical Co.; and amino acids were from Calbiochem.
RESULTS
Kinetics of amino acid incorporation. A time course of amino acid incorporation directed by endogenous mRNA shows that synthesis of polypeptides is linear for 4 min but reaches a maximum by 8 min (Fig. 1A). This is similar to the results of So and Davie (33) and Bretthauer et al. (5), who showed that amino acid incorporation in yeast lysates was linear for the first 5 to 10 min. The duration of time over which amino acid incorporation occurs in the yeast lysate is brief, however, relative to the duration of incorporation in other eukaryotic cell-free systems such as the Krebs II ascites (23), the wheat embryo (8), and the rabbit reticulocyte (17) cell-free systems. Effect of rabbit reticulocyte and yeast polysomal RNAs on amino acid incorporation. The brief duration of amino acid incor-
Minutes
FIG. 1. Kinetics of protein synthesis in the yeast cell-free system. Reactions (100 ul) contained 50 ,Ci of [3H]serine (1.23 Ci/mmol) per ml. Samples of the reaction mixture were counted at the times indicated; 1 pmol = 80 counts/min. (A) No preincubation; (B) preincubation for 15 min at 30 C. Symbols: 0, endogenous incorporation; A, 100 ug of yeast polyadenylic acid-containing RNA per ml; 0, 600 usg of rabbit reticulocyte polysomal RNA per ml.
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poration suggested that a labile component(s) became inactivated during the preparation of the yeast lysate or under conditions of protein synthesis. Therefore, various conponents required for protein synthesis were added to the yeast lysate to attempt to extend the duration of polypeptide synthesis. Addition of either rabbit reticulocyte or yeast polysomal RNAs to the yeast S-30 failed to stimulate or extend the duration of amino acid incorporation over and beyond the high, endogenous background activity (Fig. 1A). Some eukaryotic cell-free systems translate different mRNA's with different efficiencies (31). For example, rabbit reticulocyte lysates translate endogenous hemoglobin messenger more efficiently than heterologous mouse or duck hemoglobin messenger (17, 35). Thus, the possibility existed that the yeast ribosomes were translating endogenous mRNA more effeciently than added rabbit reticulocyte polysomal RNA containing hemoglobin messenger, but that the high, endogenous amino acid incorporation masked the translation of hemoglobin messenger. In the rabbit reticulocyte lysate, which contains high amounts of endogenous hemoblobin messenger, polypeptides are made from exogenous RNA's without stimulation of amino acid incorporation (16, 34). If the yeast S-30 is allowed to incorporate [35S jmethionine after addition of polysomal RNA from rabbit reticulocytes and the products are separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, no polypeptide is made that corresponds in molecular weight to marker hemoglobin (Fig. 2). The addition of this rabbit reticulocyte polysomal RNA to a preincubated Krebs II ascites system (23) causes the synthesis of polypeptides which co-migrate with the hemoglobin marker (Fig. 2), indicating that the mRNA is active. The yeast polysomal RNA is also highly active as we have shown by translating it in both the Krebs ascites cell-free system and in an extract from wheat embryos (8a). To attempt to make amino acid incorporation in the yeast cell-free system dependent upon added mRNA, the endogenous amino acid incorporation was lowered by preincubation under conditions of protein synthesis. This allows ribosomes to complete their cycle and degradation of endogenous mRNA to occur (23, 30). However, the protein synthetic activity directed by added mRNA, as well as the endogenous activity, decreases as the time of preincubation increases (22). Therefore, [3H]uridine mRNA from T7 bacteriophage-infected E. coli was added to the yeast system under conditions of protein synthesis to determine how brief an
721
incubation period would be needed to degrade mRNA. In 15 min at 30 C, 50% of the [3H]uridine T7 mRNA became acid soluble (Fig. 3). This rate of solubilization is about the same as that observed in E. coli cell-free systems. Preincubation of the system under reaction conditions for 15 min at 30 C (see above) reduced amino acid incorporation by 80% (Fig. 1B, note scale change). Addition of rabbit reticulocyte polysomal RNA or yeast polyadenylic acidenriched mRNA (8a) still failed to stimnulate amino acid incorporation (Fig. 1B). Effect of tRNA and ribosome high-salt wash factors on amino acid incorporation. Several other components necessary for protein synthesis were added to the lysate to extend the duration of synthesis or to achieve a stimulation of amino acid incorporation. Since the system appeared to elongate polypeptide chains but not to initiate them (see below), several potentially labile components required for initiation, tRNA and initiation factors, were added to the system (Table 1). In some instances, tRNA (2, 3) and initiation factors (14, 24, 26, 28) have been shown to be labile components of the Krebs ascites system. However, neither tRNA purified from E. coli, S. cerevisiae, or mouse liver nor ribosome high-salt wash factors purified from yeast or Krebs ascites ribosomes stimulate or extend the duration of amino acid incorporation significantly in the yeast lysate (Table 1). Neither of these ribosome high-salt wash factor preparations, when tested in a Krebs ascites system containing Krebs salt-washed ribosomes, could be shown to be active. Since the data suggest that there is no initiation on exogenous mRNA, the system was studied further to determine whether or not initiation was occurring on endogenous mRNA. Dependence of amino acid incorporation upon cation concentration. There is little dependence of amino acid incorporation on potassium concentration over a range of 50 to 130 mM KCl. Maximum incorporation occurs over a range of 85 to 100 mM KCl. The optimal magnesium concentration is about 6 mM, but amino acid incorporation occurs over a range of 3 to 13 mM Mg2+. So and Davie (33) and Bretthauer et al. (5) found similar, broad magnesium optima for yeast in in vitro protein synthesis systems, but these data are in contrast to magnesium optima described for the Krebs ascites (22), the wheat embryo (29), and the rabbit reticulocyte (1) cell-free systems, in which amino acid incorporation occurs from about 2 to 4 mM Mg2+. Stimulation of polyphenylalanine synthesis by poly(U). The translation of natural mRNA's in eukaryotic cell-free systems is re-
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mRNA-DIRECTED POLYPEPITIDE CHAIN ELONGATION
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(Q)
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16
FIG. 3. Degradation of T7 mRNA with time under conditions of protein synthesis in the yeast cell-free system. A 100-,ul reaction mixture contained 13 gg (1,000 counts/min per Ag) of [3H]uridine T7 RNA. Aliquots of the reaction mixture were removed at the times indicated and precipitated with 5% trichloroacetic acid.
723
thauer et al. (5) observed a similar stimulation by poly(U) in yeast extracts. Effects of cycloheximide and pactamycin on endogenous amino acid incorporation. To test whether or not the endogenous amino acid incorporation was due to polypeptide chain initiation and elongation or to elongation alone, antibiotics that inhibit either initiation or elongation were added to the system (Table 2). Pactamycin, which, at 1 AM, specifically blocks initiation in E. coli (7) and in mammalian extracts (18, 24), has no effect on amino acid incorporation in the yeast S-30 (Fig. 4A). However, 1 ,uM pactamycin inhibits rabbit reticulocyte polysomal RNA-directed amino acid incorporation in the Krebs ascites system (Fig. 4B). All translations were carried out at 3.3 mM Mg2+, since Kappen et al. (13) have shown that high magnesium overcomes pactamycin inhibition of initiation. At concentrations of pactamycin > 10 AM (20), chain elongation is also inhibited. Pactamycin at 50 ,uM (Fig. 4A) partially inhibits amino acid incorporation in the yeast S-30. On the other hand, cyclohexiTABLE 1. Effect of tRNA's or ribosome high-salt wash
stricted to a low, narrow range of magnesium factors on amino acid incorporationa concentrations, about 2 to 4 mM Mg2+ (22, 23, 29, 40). In contrast, the translation of the Serine ConcenincorAdditions tration synthetic messenger, poly(U), occurs over a (mg/ml) porated range of 5 to 20 mM Mg (23, 30) and its (pmol) translation is not dependent upon the presence None ..140 of initiation factors (32, 40). The addition of 136 poly(U) to the yeast cell-free system mimics the E. coli tRNA .0.4 Mg2+-dependent characteristics of the system Yeast ribosome high-salt wash factors ..................... 1 82 by using endogenous natural messenger as temribosome high-salt wash plate. The optimum magnesium concentration Yeast factors, E. coli tRNA ........ 1, 0.4 82 for phenylalanine incorporation is high, 6 to 8 Krebs ascites ribosome high-salt mM Mg2+, about the same as the optimum for wash factors ............ .... 1 138 phenylalanine incorporation directed by endog- Krebs ascites ribosome high-salt enous messenger. Both endogenous mRNA and wash factors, rabbit reticulopoly(U) are translated over the same broad cyte polysomal RNA ........ 1, 0.6 138 range of magnesium concentrations. These ob- Krebs ascites ribosome high-salt servations suggest that the endogenous stimulawash factors, yeast polyadenylic acid-containing RNA .. 1, 0.5 138 tion of amino acid incorporation is due to ..169 elongation of polypeptide chains, not initiation. None 0.3 168 The inability of the system to translate added Yeast tRNA .................. 0.3 187 messenger is not due to the lack of ribosomes or Mouse liver tRNA ............. a protein synthesis components necessary for Each 100-ul reaction mixture contained 50 of elongation, since addition of 400 ,g of poly(U) [3H]serine (1.23 Ci/mmol) per ml, 85 mM KCl,pCiand to the system stimulates phenylalanine incorpo- 3.3 mM magnesium acetate. 1 pmol = 80 counts/min. ration fivefold over endogenous synthesis. Bret- The S-30's were not preincubated. FIG. 2. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell-free products. (a,b) Krebs ascites system; (c,d) yeast lysate; (b,d) rabbit reticulocyte polysomal RNA added. Protein synthesis reaction contained 100 MCi of L-[35S]methionine (141 Ci/mmol) per ml. Reactions were terminated by addition of an equal volume of x2 Studier buffer [100 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 6.8; 2% sodium dodecyl sulfate; 20% glycerol; 0.28 3-mercaptoethanol, 0.1% bromophenol blue] and boiling for 2 min. A 10-Ml volum e of this mixture was placed in each slot of a 15% polyacrylamide slab gel and electrophoresis was performed. The gel was dried and autoradiographed.
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GALLIS AND YOUNG TABLE 2. Inhibition of protein synthesisa
.Concn Additios Additions (yMm)
Serine incorporated
%bition Inhi-
(pmol)
None Pactamycin ......... Pactamycin ......... None Cycloheximide ...... a
Each
1 50 10
90 90 90 105 13
0 0 34 0 87
100-Al reaction mixture contained 50 yCi of
[3H]serine (1.23 Ci/mmol) per ml, 85 mM KCl, and 3.3 mM magnesium acetate. 1 pmol The S-30's were not preincubated.
=
80 counts/min.
mide, which inhibits chain elongation in eukaryotic cell-free systems at 10 AM (9, 18), blocks incorporation in the yeast S-30 by 87% (Table 2). The sensitivity of the system to cycloheximide and its resistance to low levels of pactamycin suggest that the observed amino acid incorporation is due to chain elongation, not initiation on endogenous mRNA. Effects of inhibition of proteases on amino acid incorporation in the yeast lysate. Lysis of yeast cells for preparation of the S-30 could have resulted in protease degradation of ribosome subunits or factors necessary for translation. Lysis of log-phase growing yeast cells causes release of proteases (37). There are three general classes of proteases in yeast (25): acid proteases, which are completely inhibited at pH 7.5; active-serine alkaline proteases, which are inhibited by compounds such as phenylmethane sulfonyl fluoride; and trace metal-activated proteases, which are inhibited by 1 mM ethylenediaminetetraacetate. Therefore, yeast lysates, instead of being prepared in pH 7.5 buffer (see above) containing 1.5 mM magnesium acetate, were prepared by adding pH 7.5 buffer containing 1 mM ethylenediaminetetraacetate without a divalent cation. The lysate was passed over a G-25 column equilibrated with reaction buffer containing 3.0 mM Mg to remove the ethylenediaminetetraacetate and
J. BACTERIOL.
DISCUSSION The characteristics of protein synthesis in the yeast S-30 suggest that amino acid incorporation is due to the elongation of nascent polypeptide chains and not to de novo initiation of new polypeptides. There are three lines of evidence for this. Pactamycin, an inhibitor of polypeptide chain initiation, fails to block amino acid incorporation. Secondly, incorporation occurs maximally at magnesium concentrations that are inhibitory to translation of added natural mRNA's in other cell-free systems. These Mg concentrations are optimal for translation of poly(U), which may be translated without all of the components necessary for initiation (32, 40). Finally, the system fails to be stimulated by or to translate added natural mRNA's, which are translated upon addition to either a Krebs ascites system or wheat embryo cell-free system. The inability of the yeast S-30 to be stimulated by active homologous template RNAs indicates that the failure to initiate translation on exogenous RNA is not due solely to an incompatibility between mRNA and ribosomes or initiation factors. On the other hand, addition of poly(U), a synthetic polymer known not to require a complete initiation complex, stimulates the system, as was also shown by Bretthauer et al. (5) and by So and Davie (33). The inability of the yeast lysate to initiate could be due to the lability of ribosomes, initiation factors, tRNA, or mRNA. Yeast transfer and polyadenylic acid-enriched mRNA added to the yeast lysate appear to be funcA
B
6~ ~ ~ ~ ~1
60
0
4
8
12
16
0 Minutes
5
10
15
restore the proper Mg concentration. No differFIG. 4. Effect of pactamycin on the kinetics of ence in amino acid incorporation or ability to amino acid incorporation in the yeast and Krebs initiate was found between this preparation and ascites cell-free systems. Reactions (100 jil) contained the preparation lysed in buffer containing 1.5 the same amount of [3H]serine as indicated in the mM magnesium acetate. Furthermore, when legend of Fig. 1 and the same magnesium and lysis was carried out in pH 7.5 buffer containing potassium concentrations, 3.3 mM Mg and 85 mM are optimal except that the both 1 mM ethylenediaminetetraacetate and 1 KCI; all ionic conditions was lowered to test the effect Mg concentrations mM phenylmethane sulfonyl fluoride, and these yeast of pactamycin. (A) Incorporation by yeast cell-free were subsequently removed by the G-25 chro- system directed by endogenous mRNA. (B) Incorpomatography, no change in the ability of the ration by Krebs ascites cell-free system directed by system to initiate was detected. However, this 600 ,g of rabbit reticulocyte polysomal RNA per ml. method also reduced endogenous incorporation Symbols: A, no pactamycin; 0, 1 uM pactamycin; 0, 50 ,M pactamycin. by 50%.
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mRNA-DIRECTED POLYPEPTIDE CHAIN ELONGATION
tional components in other cell-free systems. Both the Krebs ascites and wheat embryo cell-free systems translate this yeast mRNA into polypeptides similar to those made in vivo (8a). When added to the wheat embryo cellfree system in the presence of yeast polyadenylic acid-enridhed RNA, yeast tRNA stimulates mRNA-directed incorporation twofold. The rate of RNA degradation in the yeast extract (Fig. 3) does not appear to be high enough to preclude stimulation by exogenous mRNA. Since yeast ribosomes are able to elongate polypeptides in vitro, the inability of the yeast S-30 to initiate may be due to inactive initiation factors, not ribosomes. We have not been able to prepare active initiation factors from yeast. One observation suggests that initiation factors that dissociate ribosomes are inactive in yeast lysates. When yeast polysomes are prepared in the absence of cycloheximide, most of the ribosomes are in the form of 80S monomers, not subunits (our unpublished data). Thus, initiation factors in yeast lysates are unable to effect a dissociation of monosomes into subunits (12, 19). Lack of free subunits would prevent initiation on either endogenous or added yeast polyadenylic acid-enriched mRNA (10). Yeast ribosomes were treated with a solution of 0.5 M KCl and 10 mM puromycin, which dissociates them into subunits (4, 6). However, a fractionated yeast in vitro system made from an S-100, with puromycin-treated rib.osomes and a ribosomal high-salt wash, does not initiate polypeptide synthesis with polyadenylic acid-enriched RNA from yeast. The lack of initiation may be due to other initiation factor functions that are inactive in the yeast extract. ACKNOWLEDGMENTS This work was supported by a Public Health Service Training Grant to the Department of Biochemistry, by American Cancer Society grant NP 124, and by Public Health Service grant AI 09456 from the National Institute of Allergy and Infectious Diseases. B.G. wishes to thank Jim and Anita Hopper for constant encouragement and continued interest throughout the course of this work and Jim Hopper for the gel analysis shown in Fig. 2. We thank G. B. Whitfield of the Upjohn Co. for his generous gift of pactamycin. LITERATURE CITED 1. Adamson, S. D., E. Hubert, and W. Godchaux. 1968. Factors affecting the rate of protein synthesis in lysate systems from reticulocytes. Arch. Biochem. Biophys.
125:671-683. 2. Aviv, H., I. Boime, and P. Leder. 1971. Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system. Proc. Natl. Acad. Sci. U.S.A. 68:2303-2307. 3. Benveniste, K., J. Wilczek, and R. Stern. 1973. Translation of collagen mRNA from chick embryo calvaria in a cell-free system derived from Krebs II ascites cells.
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Nature (London) New Biol. 246:303-305. 4. Blobel, G. 1971. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc. Natl. Acad. Sci. U.S.A. 68:390-394. 5. Bretthauer, R. K., L. Marcus, J. Chaloupka, H. 0. Halvorson, and R. M. Bock. 1963. Amino acid incorporation into protein by cell-free extracts of yeast. Biochemistry 2:1079-1084. 6. Brown, G. E., A. J. Kolb, and W. M. Stanley. 1974. A general procedure for the preparation of highly active eukaryotic ribosomes and ribosomal subunits, p. 368-387. In K. Moldave and L. Grossman (ed.), Methods in enzymology, vol. 30, part F. Academic Press Inc., New York. 7. Cohen, L. B., A. E. Herner, and I. H. Goldberg. 1969. Inhibition by pactamycin of the initiation of protein synthesis. Binding of N-acetylphenylalanyl transfer ribonucleic acid and polyuridylic acid to ribosomes. Biochemistry 8:1312-1326. 8. Davies, J. W., and P. Kaesberg. 1973. Translation of virus messenger RNA: synthesis of bacteriophage QB proteins in a cell-free extract from wheat embryo. J. Virol. 12:1334-1341. 8a. Gallis, B. M., J. P. McDonnell, J. E. Hopper, and E. T. Young. 1975. Translation of poly(riboadenylic acid) [poly(A)] enriched messenger RNA's from the yeast, Saccharomyces cerevisiae, in heterologous cell-free systems. Biochemistry 14:1038-1046. 9. Godchaux, W., S. D. Adamson, and E. Hubert. 1967. Effect of cycloheximide on polyribosome function in reticulocytes. J. Mol. Biol. 27:57-72. 10. Gottlieb, M., N. H. Lubsen, and B. D. Davis. 1974. Preparation and assay of ribosome dissociation factors from Escherichia coli and rabbit reticulocytes and the assay for free ribosomes, p. 87-94. In K. Moldave and L. Grossman (ed.), Methods in emzymology, vol. 30, part F. Academic Press Inc., New York. 11. Hartwell, L. H. 1970. Biochemical genetics of yeast. Annu. Rev. Gen. 4:373-396. 12. Kaempfer, R., and J. Kaufman. 1972. Translational control of hemoglobin synthesis by an initiation factor required for recycling of ribosomes and for their binding to messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 69:3317-3321. 13. Kappen, L. S., H. Suzuki, and I. H. Goldberg. 1973. Inhibition of reticulocyte peptide-chain initiation by pactamycin: accumulation of inactive ribosomal initiation complexes. Proc. Natl. Acad. Sci. U.S.A. 70:22-26. 14. Lebleu, B., U. Nudel, E. Falcott, C. Prives, and M. Revel. 1972. A comparison of the translation of mengo virus RNA and globin mRNA in Krebs ascites cell-free extracts. FEBS Lett. 25:97-103. 15. Lockard, R. E., and J. B. Lingrel. 1969. The synthesis of mouse hemoglobin ,B-chains in a rabbit reticulocyte cell-free system programmed with mouse reticulocyte 9S RNA. Biochem. Biophys. Res. Commun. 37:204-212. 16. Lockard, R. E., and J. B. Lingrel. 1972. Mouse hemoglobin messenger ribonucleic acid. Translational capacities of rabbit and duck reticulocyte cell-free systems programmed with mouse 9S ribonucleic acid. J. Biol. Chem. 247:4174-4179. 17. Lockard, R. E., and J. B. Lingrel. 1973. Translation of mammalian globin messenger RNA's in an avian reticulocyte cell-free system. Biochim. Biophys. Acta 229:148-152. 18. Lodish, H. R., D. Houseman, and M. Jacobson. 1971. Initiation of hemoglobin synthesis. Specific inhibition by antibiotics and bacteriophage ribonucleic acid. Biochemistry 10:2348-2356. 19. Lubsen, N. H., and B. D. Davis. 1972. A ribosome dissociation factor from rabbit reticulocytes. Proc. Natl. Acad. Sci. U.S.A. 69:353-357. 20. Macdonald, J. S., and I. H. Goldberg. 1970. An effect of
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