THE JOUBNAL OF BIOLOGICAL CHEMISTRY Vol. 252, No. 19, Issue of October 10, pp. 6889-6894, 1977

Prrnled rn U.S.A.

DNA-directed in Vitro Synthesis of P-Galactosidase STUDIES WITH PURIFIED FACTORS*

(Received for publication, April 14, 1977)

HSIANG-FU KUNG, BETTY REDFIELD, BENJAMIN V. TREADWELL, BARNET ESKIN, CARLOS SPEARS, AND

HERBERT WEISSBACH

From the Roche Institute of Molecular Biology, Nutley, New Jersey 07110

The phage DNA-directed synthesis of P-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP re- ceptor protein; N’“-formyltetrahydrofolate Met-tRNA’ transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl- tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and L, (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. Proc. 35,15X’). Under these conditions, P-galactosidase syn- thesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other re- quired components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl- tRSA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for P-galactosidase syn- thesis. The present report describes the purification from E. coli extracts of three fractions, called L,, 4, and L,, that are needed to restore enzyme synthesis.

In order to better understand the regulation of gene expres- sion in prokaryotes, we have initiated a program whose goal is to identify the components required for the DNA-directed in

vitro synthesis of P-galactosidase. Our previous studies, which relied on fractionation of the Escherichia coli extracts, showed that enzyme synthesis was dependent on ribosomal protein L12, IF-l,’ IF-3, RNA polymerase, CAMP receptor protein, EF-

Tu, and tRNA (l-5). In addition, two other factors (L and LJ which are necessary for /3-galactosidase synthesis have been purified from E. coli extracts (1, 2).

Although fractionation of the E. coli extract has yielded

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘hduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: IF-l, initiation factor 1; IPTG, iso- propylthiogalactoside; ONPG, orthonitrophenylgalactoside; ppGpp, guanosine 5’-diphosphate-3’-diphosphate; CRP, cyclic AMP receptor protein; EF, elongation factor; PEG 6000, polyethylene glycol 6000; RF, release factor; AA-tRNA, aminoacyl-tRNA.

considerable information on the requirements for enzyme syn- thesis in this system, there are limitations to this approach. The search for other required factors would be greatly facili- tated, however, if most of the known factors required for transcription and translation were available in a highly puri- fied form. It has now been possible to obtain many of the 30 known factors’ plus L and L, in a high state of purity. Al- though the purified factors do not support the DNA-directed synthesis of /?-galactosidase, this system could be used to look for additional required components. However, the short supply of some of the aminoacyl-tRNA synthetases was a limiting factor in these studies. It was noted that many of the AA- tRNA synthetases could be replaced by an Ehrlich ascites supernatant, making it possible to conserve the limited quan- tities of purified synthetases. The present study shows that in the presence of an ascites extract and other purified factors, at least three additional fractions are needed to restore P-galac- tosidase synthesis.

MATERIALS AND METHODS

Bacteria

Escherichia coli Z19i” (contains mutant gene is and the M-15 modified P-galactosidase gene on both chromosome and episome) was kindly provided by Dr. G. Zubay, Columbia University. The organism was grown at 28” to a density of approximately 1 x lo” cells/ml in a medium described by Zubay et al. (6). The harvested cells were stored at -20” until they were used for the preparation of the S-30 extract (1).

Bacteriophage and DNA Preparations

Bacteriophage hh80dlacp’ ~1857 t68 was prepared by heat induc- tion of E. coli RV (hh80dlacpcI857 t68 and hh80cI857t68) kindly provided by Dr. I. Pastan, National Institutes of Health. The phages were purified by banding in a cesium chloride density gradient, and phage DNA was extracted by the method of Thomas and Abelson (7).

Enzymes

RNA polymerase, L, and L, were prepared from the S-30 extract as described by Kung et al. (1, 2). CRP and initiation factors were prepared from a 1 M NH&l wash of ribosomes according to proce- dures described previously (8-11). EF-Tu, EF-Ts, EF-G, N1”-formyl- tetrahydrofolate Met-tRNA’ transformylase (12-161, and most of the AA-tRNA synthetases were prepared by previously described meth- ods (for a review of AA-tRNA synthetase purifications, see Refs. 17 and 18). However, several of the factors were prepared elsewhere and

’ The known factors are RNA polymerase, CRP, 20 AA-tRNA synthetases, N”‘-formyl-tetrahydrofolate Met-tRNA transformylase, IF-l, IF-2, IF-3, EF-Tu, EF-Ts, EF-G, RF-l, and/or RF-2.

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6890 In Vitro Synthesis of @Galactosidase with Purified Factors

the authors are deeply indebted to a large number of scientists who have aided this study.3

Assays

Assays for the activities of RNA polymerase, CRP, initiation factors, elongation factors, N’O-formyltetrahydrofolate Met-tRNA’ transformylase, and AA-tRNA synthetases have been described pre- viously (1, 8-18). The /3-galactosidase synthesizing system and assay were the same as described previously (l-5) and details are pre- sented in the legend to Table I.

Preparation and Fractionation of E. coli S-30 Extract

An S-30 extract from E. coli Z19i’l was prepared by the method of Zubay et al. (6), except that the preincubation step was omitted. The ribosomal wash, washed ribosomes, and a supernatant fraction were prepared from the S-30 extract as described previously (1, 31. The 0.25 M salt eluate from DEAE (0.25 M DEAE-fraction) containing 20 AA-tRNA synthetases, elongation factors, and other factors required for protein synthesis as well as the 1 M salt eluate from DEAE (1 M DEAE-fraction) containing RNA polymerase and L factor were pre- pared by DEAE-cellulose chromatography of the supernatant frac- tion according to the procedure described by Kung et al. (1).

Preparation of Ascites Extract

An ascites S-30 extract was prepared from Ehrlich ascites cells according to the method described by Housman et al. (19). The S-30 extract was centrifuged for 1 h at 100,000 x g and the supernatant was removed and stored in liquid nitrogen (ascites S-1001.

Purification of Additional Factors Required for p-Galactosidase Synthesis

The purification of three additional fractions required for the in vitro synthesis of P-galactosidase are described below. The assay for these factors was based on their ability to stimulate P-galactosidase synthesis under conditions which will be described under “Results”.

Purification of L,, -L, was partially purified from the salt wash of ribosomes (1). The ribosomal wash (300 ml, 3050 mg of protein) was fractionated by (NH&SO, and the protein precipitating between 50 and 80% saturation (950 mg of protein) was dialyzed and dissolved in 56 .nl of Buffer I (0.02 M Tris/Cl (pH 7.5), 1 mM dithiothreitol, 1 mM sodium EDTA, and 10% glycerol) and applied to a DE52 column (2.5 x 30 cm) equilibrated with Buffer I. The active fraction was in the column pass-through (100 mg of total protein). This solution was then applied to a CM-cellulose column (CM 23, 1.5 x 20 cm) and equilibrated with 0.01 M Tris/Cl (pH 7.41, and the column was washed with this buffer until the absorbance of the effluent at 280 nm was below 0.02. Protein was eluted with a 300-ml linear gradient of NH&l (0 to 0.35 M) in 0.01 M TrislCl, pH 7.4. The active fractions which appeared between 0.15 and 0.3 M NH&l (total protein, 30 mg) were pooled, diluted 3-fold, and placed on a phosphocellulose column (1.5 x 20 cm) equilibrated with 0.01 M Tris/Cl (pH 7.41 containing 0.1 M NH&l. The protein was eluted with 300 ml of a linear gradient of

3 Most of the factors described in Footnote 2 have been purified in our Insitute with the invaluable help of the Biopolymer Laboratory of the Roche Research Center. One exception is the release factors which were kindly supplied by Drs. Jeanne Ratliff and Thomas Caskey of Baylor College of Medicine. In addition, we are deeply indebted to a large number of scientists in this country and abroad who have at times supplied us with individual factors. These include: arginyl-, isoleucyl-, leucyl-, phenylalanyl- and seryl-tRNA synthe- tases (Dr. Robert Waterson, Emory University), lysyl-tRNA synthe- tase (Dr. A. Mehler, University of Wisconsin), tryptophanyl-tRNA synthetase (Dr. K. Muench and M. Lee, University of Miami), tyrosyl-tRNA synthetase (Dr. C. J. Bruton, Imperial College of Science and Technology, London, and Dr. P. Schimmel, Massachu- setts Institute of Technology), phenylalanyl-tRNA synthetase (Dr. M. Stulberg, Oak Ridge National Laboratory), valyl- and glutamyl- tRNA synthetase (Dr. D. Siill, Yale University), histidinyl- and cysteinyl-tRNA synthetases (Dr. W. Konigsberg, Yale University), and RNA polymerase (Dr. R. Burgess, University of Wisconsin). EF- Tu, EF-Ts, and EF-G were kindly supplied by Drs. D. Miller and N. Brot of the Roche Institute of Molecular Biology. The Ehrlich ascites cells were kindly supplied by Ms. M. Buck of the Chemotherapy Department of Hoffmann-La Roche, Inc.

TABLE I

In vitro synthesis of Pgalactosidase with different fractions

The basic system (70 $1 contained: 3.5 pmol of Trislacetate, pH 8.2; 0.85 pmol of magnesium acetate; 2.9 pmol of potassium acetate; 1.9 pmol of NH, acetate; 0.1 wmol of dithiothreitol; 0.016 pmol of each of 20 amino acids; 0.039 pmol each of CTP, GTP, and UTP; 0.16 pmol of ATP; 1.4 pmol of phosphoenolpyruvate; 0.5 pg of pyruvate kinase; 0.035 pmol of CAMP; 60 pg of Escherichia coli B tRNA; 0.4 pmol of isopropylthiogalactoside; 3 nmol of ppGpp; 60 pmol of W’O- methenyltetrahydrofolate; 3 mg of polyethylene glycol 6000, 8 pg of hh80dlacp’DNA and protein components. The protein fractions used are as follows: Experiment 1, S-30 extract, 450 pg of protein; Experi- ment 2, 1.2A,,, of NH&l-washed ribosomes, ribosomal wash (100 kg of protein), a 0.25 M DEAE-salt eluate (220 pg of protein) (11, and a 1 M DEAE-salt eluate (15 yg of protein) (11; Experiment 3, 1.2 A,,,, of NH,Cl-washed ribosomes, RNA polymerase (2 pgl, L (1.5 pg), 1 hg each of IF-l, IF-2, IF-3, CRP, 4, EF-Ts, RF-l, RF-Z, and 20 AA- tRNA synthetases, 10 pg of EF-Tu, 5 pg of EF-G, and 2 pg of transformylase. The reaction was carried out at 37” for 90 min. At the end of the incubation, 1 ml of orthonitrophenylgalactoside (ONPG) reagent (106 mg of ONPG, 297 ml of 0.1 M sodium phosphate buffer, pH 7.3, and 3 ml of 14 M mercaptoethanol) was added. The assay tubes were incubated at room temperature for the length of time sufficient to develop significant yellow color and the rate of optical density change was determined at 420 nm.

Experiment system

1 s-30 2 DEAE-fractions 3 Purified factors

Aah

2.0-3.0 - 1.7-2.5

co.1

NH&l (0.1 to 0.5 M) in 0.01 M TrislCl, pH 7.4, and the active fractions (total protein, 15 mgl appeared between 0.25 and 0.4 M NH&l. The pooled fractions were concentrated to a small volume by Amicon membrane ultrafiltration (UM-2) and dialyzed against Buffer II (0.01 M Tris acetate (pH 8.21, 0.06 M potassium acetate, 0.014 M magnesium acetate, and 1 mM dithiothreitol).

Purification of L, Fraction-The 0.25 M DEAE-fraction (1) was used as the source of this factor. About 1.35 g of protein were fractionated by the addition of (NH&SO,, and the material precipi- tating between 40 and 75% (NH&SO, saturation (1 g of protein) was dialyzed and dissolved in 0.025 M KPO, buffer, pH 6.9, containing 20 mM P-mercaptoethanol (Buffer III). This solution was applied to a DE52 column (2.8 x 40 cm1 equilibrated with Buffer III. The column was washed with 1 liter of Buffer III and then eluted with 3 liters of a linear gradient of 0.025 to 0.25 M KPO, buffer (pH 6.9) containing 20 mM P-mercaptoethanol. L, eluted between 0.012 and 0.14 M KPO, and the proteins in the pooled fraction (350 mg of protein) were precipitated by the addition of (NH&SO, to 85% saturation and the precipitate was dissolved in Buffer II. A portion of the precipitate (43 mg in 2 ml1 was placed on a Sephadex G-75 column (2.8 x 90 cm1 equilibrated with Buffer II. The active material (35 mg of protein) was present in the excluded peak and these fractions were pooled and concentrated by Amicon ultrafiltration (UM-10). The solution was applied to a hydroxylapatite column (Hypatite C, 0.9 x 10 cm) and the protein was eluted with 30-ml portions of 0.01,0.05,0.1, and 0.2 M KPO, buffer (pH 6.5). L, activity was present in the 0.2 M KPO, buffer (pH 7.5) eluate and this fraction was concentrated by Amicon membrane ultrafiltration (PM-lo). The concentrated sample (3 mg of protein) was dialyzed against Buffer II and then applied to a DEAE- Sephadex column (A-50.0.9 x 8 cm1 equilibrated with Buffer II. The column was washed with 20 ml of Buffer II and the protein was eluted with 200 ml of a linear gradient of 0 to 0.35 M KC1 in Buffer II. The active fractions were eluted between 0.05 and 0.1 M KC1 (total protein, 1.6 mg). The pooled fractions were concentrated by Amicon membrane ultrafiltration (UM-10) and the concentrate was dialyzed against Buffer II.

Purification of L, - Fraction L, was purified from a 100,000 x g supernatant of an E. coli S-30 extract. To 100 ml of supernatant containing 3 g of protein were added 1 volume of cold acetone and 9 volumes of cold methanol. The precipitated material was lyophilized for about 30 min and the residue was suspended in 100 ml of Buffer II. Any denatured material was removed by centrifugation and the

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In Vitro Synthesis of @Galactosidase with Purified Factors 6891

supernatant was fractionated by (NH&SO,. The protein precipitat- ing between 50 and 85% saturation C-300 mg) was dialyzed and dissolved in 10 ml of Buffer II. Cold methanol was added and the material precipitating between 12.5% and 42% methanol (-200 mg protein) was dissolved in 5 ml of Buffer II and a portion (2 ml) was applied to a DE52 column (0.1 x 10 cm) equilibrated with Buffer II and washed with this buffer. The active fraction was eluted from the column with Buffer II containing 0.2 M KCl.

Polyacrylamide Gel Electrophoresis

The procedure of Weber and Osborn (20) using 10% gels containing 0.1% sodium dodecyl sulfate was employed. Gels were stained with Coomassie blue as described by Shapiro et al. (21).

Protein Determination

Protein was determined by the method of Warburg and Christian (22) or the procedure of Lowry et al. (231. Crystalline bovine serum albumin was used as standard.

RESULTS

Supply of Purified Factors -In order to define the require- ments for P-galactosidase synthesis in vitro, two approaches have been used. Initially, the cell extracts, such as the ribo- somal salt wash and high speed supernatant (S-200), were fractionated in an attempt to obtain evidence for new factors. These studies led to the isolation of two factors, L and I+, that were required for P-galactosidase synthesis (1, 2). A second approach was to obtain the factors that are known to be required for transcription and translation in a high state of purity and then determine what additional components are needed.

The list of required factors that we consider essential for the synthesis of any protein in a DNA-directed system are RNA polymerase, N’“-formyltetrahydrofolate Met-tRNA’ transfor- mylase (transformylase), 20 AA-tRNA synthetases, IF-l, IF-2,

IF-3, EF-Tu, EF-Ts, EF-G, RF-l, and/or RF-2. In addition, the synthesis of P-galactosidase requires CRP, L, and I.+. This total of 33 factors does not include the enzymes that remove the formyl moiety and NH,-terminal methionine from the nascent polypeptide chain since it is not certain that these reactions are required to obtain enzymatically active /3-galac- tosidase. Also not included in this list are a large number of factors (24-34) that have been reported to affect transcription or translation in a variety of other systems (e.g. p factor (241, H factor (25, 26), M factor (27), X factor (28), Y factor (291, rescue factor (30), EF-P (311, peptidyl-tRNA hydrolase (32,33), and ribosome release factor (34)). Recent studies (35) indicate that L, may be ribosome release factor.

A summary of the present state of our supply of purified factors is shown in Figs. 1 and 2, where the purified proteins have been examined on polyacrylamide disc gels in the pres- ence of sodium dodecyl sulfate. Fig. 1 shows the gel patterns of 16 AAtRNA synthetases that have been obtained in a rela- tively high state of purity. Asparaginyl-, cysteinyl-, glutamyl- and prolyl-tRNA synthetases are not included in Fig. 1, but our estimates of the purity of these proteins range between 30 and 60%. It should be noted that minor contaminating bands are seen in several of the synthetase preparations (e.g. methi- onyl-, glycyl-, and aspartyl-tRNA synthetasesl. However, the two major bands in the glycyl-tRNA synthetase represent subunits since this enzyme is of the (Y& type. Some of the AA- tRNA synthetases, such as alanyl, asparaginyl, aspartyl, and cysteinyl synthetases, have not been purified to homogeneity before from Escherichia coli extracts and, therefore, more detailed studies will be required to elucidate the subunit com- position of these factors. In Fig. 2 the gel patterns of the other known factors required for transcription and translation are shown. Release factors 1 and 2 have been estimated to be

RNAP 1 L 1 TF 1 IF-2 1 Tu 1 G CRP La IF-I IF-3 Ts

I Asp I Gly I Ile I Lys I Ser I Thr I Tyr I Arg Gln His Leu Met Phe Trp Val

FIG. 1 (left). Sodium dodecyl sulfate gel electrophoresis of 16 AA- tRNA synthetases. The procedure was that of Weber and Osborn (20). The gels (0.5 x 12 cm) contained 10% acrylamide, 0.1% sodium dodecyl sulfate, and 0.1 M sodium phosphate, pH 7.0. Samples (10 Fgl were prepared by heating for 2 min at 90” in the presence of 1% sodium dodecyl sulfate, 1% P-mercaptoethanol, and 50% glycerol. Electrophoresis was run at room temperature at a current of 8 mA/ gel for 5 h. The gels were removed from the tubes and stained for 2 h

in 0.25% Coomassie brilliant blue. Excess stain was removed by exposing the gel to a solution of 7% acetic acid and 5% methanol at 37” for 18 h.

FIG. 2 (right). Sodium dodecyl sulfate gel electrophoresis of other protein factors required for gene expression. For details of the gel procedure see the legend to Fig. 1. RNAP, RNA polymerase; TF, transformylase.

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greater than 60% pure” but are not presented in Fig. 2 because of the small quantities available. In Fig. 2, the factors with the most noticeable contaminants are L factor, IF-2, EF-Ts, and EF-G.

tRNA Acylation and P-Galactosidase Synthesis with an Ascites Extract-As seen in Table I, no significant P-galacto- sidase synthesis was obtained when the purified proteins (a total of 33, including L and L,J were used, in place of an unfractionated S-30 extract or a partially fractionated system (ribosomes, ribosomal wash, and 0.25 and 1.0 M salt eluates from DE52 chromatography). This result was not unexpected since it was assumed that, in addition to the available purified factors, other components would be required for the expression of the lactose operon. This was confirmed by the finding that the addition of a small quantity of E. coli supernatant or ribosomal wash to the purified factors restored activity (data not shown).

The obvious approach was to fractionate the cell extract and purify those components required to restore enzyme synthesis. However, the isolation from the cell extract of other compo- nents necessary to restore activity presented a serious techni- cal problem since large amounts of purified factors would be required if one used the components described in Experiment 3 of Table I. Although many of the purified factors were present in sufficient quantities to carry out such studies, only small quantities of some of the AA-tRNA synthetases were availa- ble. This limited the number of experiments that could be done without rapidly exhausting the supply of these proteins. One solution to this problem would be to obtain an extract that contained a large number of the AA-tRNA synthetases but was not contaminated with most other factors that were re- quired for P-galactosidase synthesis. Animal cell extracts were considered since it is known that some E. coli tRNAs are acylated by eukaryotic synthetases but that many of the other eukaryotic factors required for transcription and translation do not function in a heterologous prokaryotic system. There are, however, reasons that prevent the use of most animal extracts in the bacterial system. These include the presence of inhibitors and high endogenous p-galactosidase-like activity in the extracts. We, therefore, looked for a suitable eukaryotic cell extract that could acylate E. coli tRNA, but had low /?- galactosidase-like activity. An Ehrlich ascites extract proved to be satisfactory. As shown in Table II, the addition ofE. coli tRNA to a supernatant from Ehrlich ascites cells stimulates the incorporation of 13 amino acids into a cold trichloroacetic acid-insoluble material. Those amino acids in which the en- dogenous acylation was not stimulated by E. coli tRNA were aspartate, glutamate, glutamine, histidine, phenylalanine, proline, and tyrosine. There seemed to be no inhibitors present in the ascites extract and 45 pg of an ascites S-100 extract contained no detectable /3-galactosidase activity after a 24-h incubation with the substrate o-nitrophenylgalactoside.

Since most of the AA-tRNA synthetases in the partially fractionated E. coli extract were present in the 0.25 M DEAE- salt eluate, it was hoped that the ascites S-100 extract, supple- mented with a few limiting factors, could substitute for the 0.25 M DEAE-eluate. Table III shows the results of such an experiment. In a system containing ribosomes, ribosomal wash, a 0.25 M DEAE-salt eluate, and a 1 M DEAE-salt eluate, only the 0.25 M DEAE-fraction could be replaced, in part, with the ascites S-100. EF-Tu had to be added, but the seven AA- tRNA synthetases lacking in the ascites S-100, as well as EF-

4 Results of Drs. J. Ratliff and T. Caskey, Baylor University.

6892 In Vitro Synthesis of pGalactosidase with Purified Factors

Ts, EF-G, release factors, etc., which are also present in the 0.25 M DEAE-fraction were not needed. Preliminary experi- ments showed that the unfractionated ribosomal wash had sufficient quantities of most of the factors missing in the ascites extract. This explained why P-galactosidase synthesis could proceed at a very significant rate when the ascites extract was used in place of the 0.25 M DEAE-extract.

P-Galactosidase Synthesis Using Ascites Extract and Puri- fied Factors -With the ascites extract supplying many of the AA-tRNA synthetases, it was now possible to test whether the ribosomal wash and 1 M DEAF-salt eluate could be replaced by purified factors. The list of purified components and the dependencies on the individual factors for P-galactosidase syn- thesis are shown in Table IV. A low but significant amount of P-galactosidase synthesis was obtained (A,,,,lh = 0.07) in such a system. Although this value represented only 3 to 5% of that seen with the unfractionated extracts, it was possible to obtain many factor dependencies. It should be noted that the seven AA-tRNA synthetases which were lacking in the ascites ex- tract were added as a group, but three of these (phenylalanyl, prolyl, and tyrosyl synthetases) used in the experiments in Table IV were only about 40% pure. Good dependencies (>3- fold stimulation) are seen with the ascites extract, the group of seven AA-tRNA synthetases, RNA polymerase, L, Let, IF fac- tors, EF-Tu, EF-G, and CRP. Release factors and EF-Ts gave only a slight stimulation (I-fold or less) and no dependency was obtained for transformylase. Despite the inability to dem-

TABLE II

Aminoacyl-tRNA synthetase activities in Ehrlich ascites supernatant

The reaction mixture (100 ~1) contained 50 nmol of TrislHCl buffer (pH 7.5), 2.0 pmol of ATP, 5.0 pmol of MgCl,, 50 pmol of p- mercaptoethanol, 2 to 5 nmol (52 to 700 cpm/pmol) of labeled amino acid, 38 ng of ascites supernatant, and, where indicated, 2 A,,,, units of Escherichia coli tRNA. The incubations were done at 37” for 10 min. The reaction was stopped by addition of 10% cold trichloroacetic acid and placement in ice for 10 min, and the solutions were filtered through nitrocellulose filters. The filters were washed with 10 ml of cold 10% Cl,CCOOH, dissolved in a naphthaleneldioxane fluor and radioactivity was determined in a scintillation spectrometer. The asterisks denote those amino acids in which there was no significant acylation dependent on E. coli tRNA.

Acylation Amino acid

No tRNA added +E. coli tRNA

Alanine Arginine Asparagine Aspartate* Cysteine Glutamate* Glutamine* Glycine Histidine* Isoleucine Leucine Lysine Methionine Phenylalanine* Proline* Serine Threonine Tryptophan Tyrosine* Valine

0

0 0 0

0 2.8 0 2.8 0 0 2.3 0.4 1.1 5.4 2.1 2.4

15.8 0 3.2 4.0

14.1 66.2 15.5

0 17.9

4.2 0

16.7 0

83 103

15.2 50

3 2.7

16.4 52 34.8

4.2 49

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TABLE III TABLE V

Replacement of Escherichia coli extracts by ascites extract

The complete system and experimental details are described in Table I, Experiment 2, except that 5 ~8 of EF-Tu were added to the incubations. An ascites S-100 extract (45 pg of protein) was added where indicated.

Exper- iment system Addition &,dh

1 Complete 1.7 2 -Ribosomal wash co.01 3 -Ribosomal wash Ascites S-100 co.01 4 -0.25 M DEAE-fraction co.01 5 -0.25 M DEAE-fraction Ascites S-100 0.40 6 - 1.0 M DEAE-fraction co.01 7 - 1 .O M DEAE-fraction Ascites S-100 co.01

Effect of L,, L,, and L, on in vitro synthesis of @galactosidase

The basic system contained the protein components described in Table IV (complete system). Other components of the incubation are described in the legend of Table I. Lu (3.5 pg), L, (3.8 pg), and L, (5 pg) were purified as described under “Materials and Methods” and added where indicated.

Additions to basic system &dh

Lo + L, + L 0.58 - + L, + L, 0.23 L,j + - + L, 0.27

L + L, + - 0.11 - - - co.1

TABLE IV

Factor dependencies for Pgalactosidase synthesis using ascites

extract

The complete list of components added is described in Table I (Experiment 3) except that an ascites S-100 (45 pg) was used in place of 13 AA&RNA synthetases (see Table II). Protein components were omitted as indicated.

Protein components % Activity”

Complete 100 Ascites S-100 <lo Other synthetases’ 110 -RNA polymerase <lo

1; 22

-I;-1 30

15 -IF-2 <lo -IF-3 <lo -EF-Tu <lo - EF-Ts 70 -EF-G 15 -RF-l, RF-2 >50 -CRP <lo -Transformylase 83

o The activity of the complete system was 0.07 A,,,Jh. b The other synthetases were added as a group and included

aspartyl, glutamyl, glutaminyl-, histidinyl-, phenylalanyl-, prolyl-, and tyrosyl-tRNA synthetases. Between 1 to 3.5 pg of the individual AA-tRNA synthetases were added.

w b pn LY pg LA

FIG. 3. The effect of the concentration of L,, L,, and L, on the in uitro synthesis of p-galactosidase. The experimental conditions are similar to those described in Table V. A, effect of L,< concentration in the presence of 3.8 Kg of L, and 5 pg of L,. B, effect of L, concentra- tion in the presence of 3.5 pg of L,j and 5 pg of L,. C, effect of L, concentration in the presence of 3.5 pg of L,j and 3.8 /*g of L,.

cult to know at this time whether one or more required pro- teins are present in these extracts.

DISCUSSION

onstrate some dependencies, the results in Table IV indicate that the ascites extract supplemented with synthetases and

other purified factors could be used as a basic system to look for other required factors.

Other Factors Required for PGalactosidase Synthesis -

Three other fractions (referred to as L,, L,, and L,, see “Mate- rials and Methods”) were found to stimulate /3-galactosidase synthesis using the basic system described in Table IV. A typical experiment showing the effect of the factors is seen in Table V. P-Galactosidase synthesis yielding values of 0.5 to 1.0 A&h could be routinely achieved with these fractions. The stimulation of enzyme synthesis by each fraction also provided for an assay for the individual factor. Fig. 3 shows the effect of L,) , L, , and L, concentrations on the synthesis of p-galactosid- ase. L,$ is found in the ribosomal salt wash and co-purifies with IF-l, during the early steps of the procedure. L, and L, are present in the supernatant (S-200) and have been partially purified as described under “Materials and Methods.” L, is stable to alcohol precipitation which provides a useful step for its purification. Since these fractions are not pure, it is difi-

The present experiments describe the first reported attempt to obtain the DNA-directed in vitro synthesis of an enzymati-

tally active protein in a system composed of purified factors. Altogether, 33 factors, in addition to ribosomes, were added and, although the purified system was not very active for p- galactosidase synthesis, it provided a starting point to search for other factors that are required. As shown in this study, at least three other fractions (4, L,, and L,) are needed and experiments are in progress to isolate the active components in these fractions.

The difficulties of assembling a system that requires no less than 35 proteins, as well as ribosomes, cannot be overempha- sized. We have managed to accumulate 33 of the factors (RNA polymerase, CRP, transformylase, 20 AA-tRNA synthetases, IF-l, IF-2, IF-3, EF-Tu, EF-Ts, EF-G, RF-l, RF-2, L, and L,,) in a reasonable state of purity which has made the present study possible. At least 26 of the proteins have now been purified in our own laboratory and very likely, as these experiments progress, it will be necessary for us to be in a position to purify all of the required components. A constant problem has been to maintain a supply of the factors in an active form, and this has been especially difficult with the 20 AA-tRNA synthe- tases. At any given time it is usually necessary to resort to

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6894 In Vitro Synthesis of PGalactosidase with Purified Factors

using partially purified preparations of some of the synthe- tases because the purified proteins have lost activity or the supply has been low. This finally necessitated the search for extracts that could replace the E. coli synthetases. The finding that an Ehrlich ascites extract could substitute for many of the E. coli synthetases has made it possible to continue this study. We know that the ascites extract does not supply most of the other known prokaryotic transcription and translation factors needed for p-galactosidase synthesis, but it is possible that there are other required components that the ascites extract is supplying. For the present time the system using the ascites extract has provided a useful tool to search for other factors in the E. coli extracts that are needed for fi-galactosidase synthe- sis.

(1973) J. Biol. Chem. 248, 50125015

Most of the factors used in the present study show one major band on disc gel electrophoresis in the presence of sodium dodecyl sulfate. However, it should be stressed that many of the factors show the clear presence of other contaminants when examined closely (see Figs. 1 and 2). Without removal of these trace materials by further purification, we have no way at this time to determine whether the contaminating materi- als may also be required for enzyme synthesis. A good example of this was seen with L,j, which was first detected as an impurity in the IF-l preparation. In addition, L, was also first observed as a contaminant in the tyrosyl-tRNA synthetase preparation. Our previous results indicate that L (1) and L, are very likely single entities. However, L,$, L,, and LA may each contain more than one active component. It is also impor- tant to be aware of the large number of factors, in addition to the 30 we consider basic to any coupled transcription-transla- tion system, that have been reported to affect gene expression (24-34). It seems reasonable that some of our “new” factors will be identical to them. Recent studies have shown that L,, has ribosome release factor activity (35) and it appears that the major component in the L, preparation is identical to a previ- ously described ribosome release factor (34).

The immediate goal of this project is to reconstitute the factors required for P-galactosidase synthesis, i.e. to obtain enzyme synthesis in a defined system. This should enable us to elucidate the role of the various factors that are required for this process and also to ultimately determine whether there are specific factors required for the expression of other genes on the bacterial chromosome. Finally, such a defined system should provide more insight into the mechanism that the cell uses to couple the processes of transcription and translation.

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H F Kung, B Redfield, B V Treadwell, B Eskin, C Spears and H WeissbachDNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors.

1977, 252:6889-6894.J. Biol. Chem. 

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