Molec. Gem Genetics 112, 14-27 (1971) © by Springer-Verlag 1971

DNA-Dependent in vitro Synthesis of Enzymes of the Galaetose Operon of Escherichia coli

WALDEMAI~ WETEKAM, KARIN STAACK, and I~UTtI E ~ I ~ G

Institut fiir Genetik der Universitgt zu Koln, 5000 K61n 41. Weyertal 121

Received April 19, 1971

Summary. Two active enzymes of the galactose operon of Escherichia coli, uridyl trans- ferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact gMactose operon of Escheri- chia coli.

The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3': 5'-monophosphate.

D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of gMactokinase, indicating that the repressor of the galactose operon is active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.

Introduction

Numerous phage specific enzymes have been synthes ized in cell-free sys tems for p ro te in synthes is d i rec ted b y the respect ive phage nucleic acids (e.g. SMser, Gestc land, and Bolle, 1967; Schweiger and Gold, 1969; Young, 1970; Her r l ich

and Schweiger, 1971). Over the pa s t years , Z u b a y and his coworkers have deve loped a sys tem for

the efficient synthes is of an ac t ive bac te r ia l enzyme, f l -galactosidase of Escherichia coli (reviewed in : Z u b a y and Chambers , 1969; Zubay , Chambers , and Cheong, 1970). I t is of pa r t i cu l a r in te res t t h a t this synthes is is r egu la ted b y the repressor of the lactose operon. Fu r the rmore , the inves t iga t ion of the in v i t ro sys tem has led to the e luc ida t ion of a novel mechan i sm of pos i t ive cont ro l t h a t is p r e s u m a b l y effective in the regu la t ion of the synthes is of all those inducible enzymes t h a t are sub jec t to ca tabo l i t e repress ion: a p ro te in factor , referred to as CAP or CRP factor , has been i so la ted t h a t specif ical ly binds cyclic adenosine 3 ' : 5 ' -mono- phospha te and media tes the s t i m u l a t o r y effect of this me tabo l i t e on the t rans- c r ip t ion of the lactose operon (Zubay, Schwartz , and Beckwi th , 1970 ; E ron et al., 1971 ; de Crombrugghe et al., 1971).

The regu la t ion of the galae tose operon of Escherichia coli has been s tud ied in de ta i l and different t ypes of mu ta t i ons have been charac te r ized t h a t affect the express ion of th is operon (But t in , 1963a, b; J o r d a n and Saedler , 1967; Jo rdan , Saedler , and Star l inger , 1968 ; Saedler et al., 1968 ; Shapiro, 1969 : A d h y a and Shapiro, 1969; Shapi ro and Adhya , 1969; F i e the n and Star l inger , 1970).

DNA-Dependent in vitro Synthesis of Enzymes of the Galactose Operon 15

We were therefore interested to s tudy the regulat ion of the galactose operon in the in vitro system. I n this communica t ion we report the DNA dependent in vitro synthesis of two active enzymes of the galactose operon, of galactokinase and of ur idyl transferase 1.

We have found tha t this synthesis, at least to a large extent , is subject to regulatory mechanisms: it is s trongly dependent on the presence of cyclic AMP and i t is s t imula ted by D-fucose, known as an inducer of the galactose operon from in vivo studies (Butt in, 1963a). This la t ter f inding shows tha t under these condit ions the repressor of the galactose operon is active. This conclusion is verified by the effect of regulatory muta t ions in this system.

Materials and Methods Bacterial Strains Used/or Preparation o/S-30 Extracts. For most experiments the S-30

extract was prepared from a T1 resistant derivative of strain Fi 165 (k) (Fiethen and Star- linger, 1970). It is a tryptophan requiring derivative of Escherichia eoli K12, H/rH. It con- tains a deletion that covers the entire galactose operon with the exception of a promoter-distal segment of the kinase gene. This deletion had been isolated by Shapiro, 1967, as $165 and had been transferred by t)1 transduction into this strain Fi 165 (k). All strains carrying this deletion of the galactose region are devoid of the activity of any one of the enzymes of the galactose operon. This strain does contain, however, the wildtype allele of the structural gene for the galactose repressor, galR +.

An S-30 extract containing a defective galactose repressor was prepared from S 165 gaIR2. It is Shapiro's original strain mentioned above, into which the regulator mutation galR2 (Saedler et al., 1968) was transferred by PI transduction (unpublished experiments by J. Besemer).

Strains o/ Galaetose Transducing Phage Lambda and Lysogenic Bacteria Used /or DNA Preparation. All phage strains used for the preparation of DNA containing the galactose operon are derivatives of the heat-inducible, lysis-inhibited strain of lambda dg No. 70/4 that carries the wildtype alleles of this operon (Michaelis et al., 1969). A derivative of this phage carrying an operator constitutive mutation was isolated by J. Besemer (unpublished experiments) as a homogenote of the operator constitutive mutation galO/o isolated by Fiethen (1969), by the method described by Fiethen and Starlinger (1970). DNA carrying a deletion of the entire kinase gene and of a portion of the transferase gene was isolated from lambda dg~61 isolated and characterized to end in deletion group VI of the transferase gene by Pfeifer and Oellermann (1967).

Mass-lysates containing transducing particles in addition to non-defective phage lambda were prepared by heat induction of the following strains of doubly lysogenic bacteria:

N IO/T1 (~cls57t6S" "~dgclS5716s), N IO/T1 (2cls57t6S , ~dg161clS57teS) , galJ~ ~ A gal (2cls57tes, 2dgOcls57t6s). N 10 is an epimeraseless derivative of E. coli K12 W8 (Pfeifer and Oellermann, 1967).

The bacterial host galR ~ 2gal (a derivative of E. coli H]r H) facilitates testing the galO~o marker carried on its prophage (J. Besemer, unpublished experiments).

Growth and Lysis o/ Cells/or Preparation o/S-30 Extracts. Conditions were as described by Zubay, Chambers, and Cheong (1970), with the following modifications: Cultures were not grown in a fermenter, but aerated by compressed air at 30 °C in 10 litre batches or by vigorous shaking of 500 ml portions on a New Brunswick reciprocal shaker. Cultures were harvested before reaching stationary phase with a yield of ca. 5 g of packed cells/litre of medium. After

1 Abbreviations are as defined in the Journal of Biological Chemistry. In addition, the following abbreviations have been used: 2dg: galaetose transducing defective phage lambda; cyclic AMP: cyclic adenosine-3': 5'-monophosphate; TCA: trichloroacetic acid; UDPG: uridine diphosphoglucose; enzymes: transferase (uridyl transferase) UDPglucose: c¢-D-galac- rose-l-phosphate uridylyltransferase (EC 2.7.7.12); kinase (galactokinase) ATP: D-galactose 1-phosphotransferase (EC 2.7.1.6).

16 W. Wetekam, K. Staack, and R. Ehring:

lysis of cells by one passage through a French pressure cell (Aminco Corp.) at a pressure of 600 kg/cm 2 the lysate was centrifuged twice at 30000 x g and preincubated as described by Nirenberg (1963). Dialysis was as indicated by Zubay, Chambers, and Cheong (1970), except for a shorter dialysis period (for a total of 3 to 4 hours against three changes of I litre each of buffer III). The extract was stored at -- 55 °C. For some preparations dithiothreitol was replaced by dithioerythritol at the same final concentration without significant effect.

Protein concentration was determined by the method of Lowry et al. (1951), with bovine serum albumin as standard.

Preparation o / D N A . All phage strains used carry the lambda mutations cI 857 and t68 which permit induction of phage production by heat treatment of lysogenic bacteria and concentration of induced cells before lysis. Preparation of mass-lysates was as described by Michaelis et al. (1969). Doubly lysogenic ceils were grown at 30 °C in a medium containing per litre of deionized water 16 g Bacto-trypton, 10 g yeast-extract, 5 g NaC1, 4 g glucose (sterilized separately) M g S Q (1 mM final concentration), tris-chloride, pH 7.4 (0.10 M final concentration). When the cultures reached an absorbance of 2 at 546 nm (I cm light-path), they were quickly warmed to 44 °C, held at that temperature for 2-3 minutes, aerated for 15 minutes at 37 °C and for 4 to 6 hours at 30 °C. Cells were collected by centrifugation and resuspended to yield 1/10 of the original volume in phage suspension medium containing l g NaC1 and 0.10 g gelatine per litre of 0.01 M tris-chloride, pH 7.4. After lysis of the cells by addition of chloroform and DNase-treatmcnt (2 tzg/ml final concentration) for 15 minutes at 37 °C the lysate was freed of debris by centrifugation at 27000 × g for 20 minutes. Phage particles were sedimented by ccntrifugation at 78000 × g for two hours (Spinco Rotor 30) and resuspended in phage suspension medium in ca. 1/20 of the volume of the lysate. 0.75 g of solid CsC1 (Merck p.a.) was added for each ml of phage suspension and the solution was adjusted to give a refractive index value of 1.381. After centrifugation at 23000 r.p.m, either in a Spinco Rotor SW 50 for 24 hours or in a Spinco Rotor 30 for 40 hours the transducing particles were separated from the non-defective particles as a band of lower buoyant density which was collected and dialyzed against several changes of phage suspension medium.

The phage suspension was diluted into 0.10 M sodium phosphate buffer pH 7.2, con- taining NaC1 at 0.10 M final concentration to give an absorbance of 13 at 260 nm (1 cm light path) and sodium dodecylsulfate was added to give a final concentration of 0.33%. DNA was released by three successive extractions at 4 °C of the phage suspension with freshly distilled phenol as described by Zubay, Chambers, and Cheong (1970). After dialysis against three changes of 0.01 M sodium ethylene diamine-tetra-acetate, pH 8.0, for a total of 16 hours and against two changes of 0.01 M tris-acetate, p i t 8.0, the DNA concentration was estimated from the extinction at 260 nm using the nominal extinction coefficient of 0.02 cm-0/~zg (Skalka, Burgi, and Hershey, 1968).

Protein Synthesis Incubation. The conditions were as given by Zubay, Chambers, and Cheong (1970) -°, with the exception that S-30 and DNA were prepared from strains appro- priate for the synthesis of galactose enzymes. The standard cell-free system for protein synthesis contained per milliliter:

Tris-acetate, pH 8.2 Dithiothreitol Potassium acetate Each of twenty amino acids Cytidine 5'-triphosphate Guanosine 5'-triphosphate Uridine 5'-triphosphate Adenosine 5'-triphosphate Trisodium phosphoenol pyruvatc Ammonium acetate Cyclic adenosine-3/: 51-mono -

phosphate D-fucose Transfer RNA Pyridoxine HC1

44 ~zmoles 1.4 fzmoles

55 fzmoles 0.22 [zmoles 0.55 ~moles 0.55 ~moles 0.55 ~moles 2.2 ~zmoles

21 ~zmoles 27 ~moles

0.5 tzmoles 5 y.moles

100 ~*g 27 ~zg

DNA-Dcpendent in vitro Synthesis of Enzymes of the Galactose Operon 17

Triphosphopyridine nucleotide 27 tzg Flavine adenine dinucleotide 27 ~g Folinie acid 27 ~g p-aminobenzoie acid 11 ~g Magnesium acetate 14.7 txmoles ~ Calcium chloride 7.4 ~moles ~dg DNA 50 ~g S-30 6500 ~g

All deviations from this standard system are indicated in the legends of figures and tables. Cyclic adenosine 3':5'-monophosphate and D-fucose at the concentrations indicated were present in all experiments unless otherwise stated.

l~outinely, incubation in a total volume of 0.20 or 0.40 ml was for 30 minutes at 37 °(3 with shaking and was terminated by dilution into ice-cold buffer, 1 mM triethanol-amine- tIC1, adjusted with NaOH to pH 8.0, to which dithiothreitol was added prior to use to a final concentration of 1.3 m ~ (Gulbinsky and Cleland, 1968). Details for the specific experi- mental conditions are given in the legends of figures and tables.

Enzyme Assays. a) Uridyl transferase was assayed by the method of Buttin (1963a), in which the product of the reaction, uridine-5'-diphospho-i4C-galaetose, is measured as charcoal- adsorbable radioactivity. For the test incubation 0.05 ml of a substrate mixture was mixed with an equal volume of suitably diluted protein synthesis mixture (as indicated for each experiment in the legend) to give the final concentrations of 1 mM i4C galactose-l-phosphate (0.5 ~Ci/~mole) and 4.3 mM uridine-5'-diphosphoglucose in 0.125 M glyeylglycine buffer, pH 7.5. Mter incubation at 37 °C for 30 minutes 0.10 ml of ice-cold triehloroacetie acid was added to the incubation mixture, followed by 2 ml of a suspension of 3 g charcoal per litre of a solution of 7 g Na 2 HPO4.2 HsO, 3 g KH2PO~, 1 g NHaC1 in distilled water, adjusted to pH 6.0. After standing for at least one hour in the cold, the suspension was filtered through glass-fibre filters (Whatman, GF/B) and the charcoal was washed on the filter with four portions 5 ml each of deionized water. The filters were dried and radioactivity retained on charcoal was measured in a gas-flow counter (Nuclear Chigaco). The counting efficiency for these conditions was determined with a commercial preparation of uridine-5'-diphospho- l*C-galatose. Zero-time blank values were obtained for each experiment in which triehloro- acetic acid was added to the assay system prior to the addition of the substrate mixture.

One unit of transferase activity is defined as that amount of enzyme that converts one ~mole of galaetose-l-phosphate per hour to uridine diphosphogalactose (i.e. to a charcoal adsorbable form) under these conditions.

b) Galaetokinase was tested in a system similar to that described by Wilson and Itogness (1966). 0.05 ml of a substrate mixture was mixed with 0.05 ml of the protein synthesis mixture (diluted as indicated in the legends) to give the final concentrations of 1.5 mM ~4C-galaetose (0.5 ~Ci/~mole), 1.5 mM adenosine 5'-triphosphate, 4 mM MgC12, 3.2 mM NaF and 1 m_~ dithiothreitol in 0.10 mM tris-chloride, pH 8.0. After incubation at 37 °C, generally for 30 minutes, aliquots (20 ~1) of the assay mixture were spotted onto discs (2.5 cm diameter) of anion exchange paper (Whatman DE 81). After 30 seconds each disc was immersed in 80% ethanol. At the end of the experiment the discs were washed with deionized water and dried for scintillation counting with "Omnifluor" (NEN, 4 g per litre of toluene) scintillation fluid. For each experiment, zero-time blank values were obtained by applying 20 ~1 aliquots of a complete assay mixture to filter discs without any incubation.

One unit of galactokinase activity is defined here as that amount of enzyme that phos- phorylates 1 ~mole of galactose per hour under these conditions.

For conversion of enzyme activity into the corresponding amounts of galaetokinase poly- peptide, it has been assumed that one unit of kinase activity measured under our conditions corresponds to 0.5 units as defined by Wilson and Itogness (1966), because these authors

2 For reasons not yet understood, some preparations of S-3O show a different requirement for magnesium ion than that indicated by Zubay and Chambers (1969). We have determined the optimum magnesium ion concentration for each preparation and used this in the experi- ments. This optimal concentration appears to permit specific synthesis as seen from the effect of D-fucose in Fig. 4A.

2 lVfolec. Gem Genetics 112

18 W. Wetekam, K. Staack, and R. Ehring:

have used a galaetokinase test system containing a lower concentration of gnlaetose (1.0 mM) 2-mereaptoethanol in place of dithiothreitol and an incubation at p t I 7.8 at a lower tempera- ture (30 °C).

Only the values given in Fig. 1 of this paper have not been corrected in any way in order to demonstrate the properties of the DNA-dependent protein synthesis system. All other data presented in figures and tables have been corrected as follows: since the blank values obtained for both enzymes for zero minutes of test incubation are independent of the con- centration of the protein synthesis mixture in the assay system over the concentration range used here, they have been subtracted from the directly measured values of the experimental samples before these were corrected for the dilution of the protein synthesis mixture. The amount of radioactive product formed was then used to calculate units of enzyme activity (~moles of product formed per hour of enzyme test incubation). The protein synthesis mixture had been diluted by a factor of five or ten before mixing with an equal volume of substrate solution. Therefore, each transferase test (0.10 ml final volume) represented 10 txl or 5 tzl of the protein synthesis mixture, whereas a 20 ~l aliquot evaluated for each kinase assay con- tains 2 ~l or 1 tzl of the protein synthesis mixture. For all figures and tables with the excep- tion of Fig. 1 the enzyme activity synthesized during the incubation period indicated is expressed for 1 ml undiluted protein synthesis mixture.

As shown in Fig. 1, the galactokinase activity does not rise significantly above the zero- time blank value in a complete protein synthesis mixture incubated either in the absence of DNA or in the presence of DNA that is defective for the galactose genes. This is true also for the transferase activity (cf. legend Fig. 2). Therefore, no blank values other than those obtained for zero minutes of enzyme test incubation have been taken into account. The only exception is Fig. 2, in which blank values were subtracted that had been obtained from a complete synthesis mixture that was incubated with DNA that did not contain the galactese genes (see legend of Fig. 2).

The conditions used for protein synthesis and enzyme assays as well as the blank values that have been subtracted are specified in the legends of figures and tables. Most blank values for the galaetekinase were lower than that shown in Fig. 1. This is attributed to the use of a new batch of laC-galactese.

Incorporation o/ laC-leucine into polypeptide material was measured as described by Chambers and Zubay (1969), by the addition of l~C-leucine to a protein synthesis mixture of standard composition (0.36 txCi/t~mole of leucine). After 70 minutes of incubation at 37 °C the incorporation was terminated by the addition of 3 ml of ice-cold 10% trichloroacetic acid to 0.2 ml or 0.4 ml of the synthesis mixture. After standing in ice for 15 minutes the mixtures were heated to 95 °C for 20 minutes and after cooling the precipitates were collected on membrane filters (Sartorins Membranfilter No. 11306) and were washed with 5 portions of 5 ml each of 5% trichloroacetic acid (57irenberg, 1963). The TCA-insoluble radioactivity was measured by scintillation counting of the dried filters with Omnifluor scintillation fluid.

All pH-adjustments of buffers refer to measurements at room temperature regardless of the temperature used in the experiment.

Substrates and other reagents were obtained from the companies indicated in parenthesis: Bacteriological growth media (Difco Laboratories); L-amino acids (Mann Research Labora- tories); substrates for protein synthesis system (Boehringer, Mannheim); dithiothreitol, dithioerythritol, Trizma Base, substrates for enzyme assays, pyridoxine-ttC1, p-aminobenzoie acid, bovine serum albumin (Sigma Chemical Company); u-D-fueose (Roth, Karlsruhe); deoxyribonuelease, ribonuelease, (Worthington Biochemical Corporation); folinic acid ("Leucovorin", Lederle); radioactive subtrates for enzyme assays and leucine incorporation (Radiochemical Centre, Amersham). Actinomycin (Merck, Sharp, and Dohme) was a gift of Dr. P. Overath.

Resu l t s and Discuss ion

T h e cel l - f ree e x t r a c t t h a t c o n t a i n s r i b o s o m e s a n d al l t h e p r o t e i n f ac to r s

r e q u i r e d fo r p r o t e i n b iosyn thes i s , c o m m o n l y r e fe r r ed to as "S -30" , was p r e p a r e d f r o m a s t r a in of Escherichia coli t h a t c o n t a i n s a d e l e t i o n of t h e ga l ac to se r e g i o n a n d is t h e r e f o r e d e v o i d of t h e a c t i v i t y of a n y one of t h e e n z y m e s of t h e g a l a e t o s e

ope ron .

DNA-Dependent in vitro Synthesis of Enzymes of the Galactose Operon 19

7,0"

3 6.0-

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3.0- i::::

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o I~ 2~ 3'o Lo ~o ~o~i. Fig. 1. Kinetics of in vitro synthesis of galactokinase. Protein synthesis was at 37°C under standard conditions (magnesium acetate 14.7 aM) with 6.9 mg/ml of S-30 extract and with 49 [zg/ml of 2dg DNA (e -e) or without the addition of DNA (©--o) . At the time intervals indicated, 0.05 ml aliquots were removed from the 1 ml synthesis mixtures and added to 0.45 ml of ice-cold buffer. Kinase assays were immediately started by mixing 0.05 ml samples of these dilutions with 0.05 ml of substrate mixture. After 60 minutes of enzyme test incuba- tion at 37°C 20 ~zl samples were removed from the enzyme assay mixtures and applied to filters for measurement of radioactive galactose-l-phosphate as described in "Material and Methods". In a separate experiment, a protein synthesis mixture of identical composition (final volume 0.30 ml) was incubated for 60 minutes at 37 °C with 47 tLg/ml of DNA from the kinase deletion strain ~dg161. The enzyme assays were performed as described above, with the exception that 0.05 ml of the undiluted protein synthesis mixture was added to 0.05 ml of the substrate mixture (zx--z~). In this figure the directly measured results have been plotted, whereas all data presented in subsequent figures and tables have been corrected as described in "Material and Methods". To facilitate comparison, it may be added that after subtraction of the blank indicated for zero minutes of incubation in this figure and after correction for the dihition of the protein synthesis mixture the maximum value measured in this experiment for the complete system (e e) corresponds to 6.04 nnits of galacto- kinase synthesized in 50 minutes per milliliter of undiluted protein synthesis mixture. The increase over the zero-time blank observed for the control with DNA from the kinase deletion (A--A) amounts to 0.03 units of apparent enzyme activity obtained after 60 minutes

incubation of one milliliter of undiluted protein synthesis mixture

DNA was extracted from purified t ransducing phage l ambda tha t carries the wildtype alleles of the galactose genes. If this DNA is added to the bacterial ext ract in the presence of all substrates and co-factors required for DNA-depen- den t prote in synthesis, t hen the appearance of act ive galactokinase can be followed by a s t andard enzyme assay procedure (Fig. 1). At various t imes after the s tar t of the react ion di lute al iquots of the prote in synthesis system were tes ted for galactokinase act ivi ty. Fig. 1 is the only figure in which the direct ly measured values have been plot ted in order to demons t ra te the characteristics of this

2*

20 W. Wetekam, K. 8taaek, and R. Ehring:

system : on the ordinate are given milli-units of galactokinase activity (i.e. nmoles of product formed per 60 minutes of enzyme test incubation) obtained per test. In addition, on the right hand scale the actually measured radioactivity of the product retained per filter has been indicated. Except for the control experiment plotted as triangles these activities represent 1 ~1 of the original protein synthesis mixture.

We have not yet investigated in detail the early stages of the reaction and therefore we cannot yet estimate the time required for the first active enzyme to appear. I t may be noted that the kinase gene is the third gene of the galaetose operon. If the synthesis is specific with respect to the start of transcription, then the synthesis of a polycistronie messenger would have to be complete before the appearance of the enzyme.

In this figure, it is also shown that the reaction is entirely dependent on DNA that contains an intact galactose operon: the control, incubated without the addition of DNA and tested at the same dilution as the complete system (open circles) does not show galactokinase activity significantly different from the low zero-time blank value. The more crucial control is taken from a separate experi- ment (triangles), in which DNA was added to the complete system at the same concentration as in the experimental sample, but this DNA carries a deletion of the kinase gene and therefore cannot direct the synthesis of that enzyme. A very low unspecific blank reaction can be observed in this case. I t is independent of DNA and is observed only, because the aliquots taken for the enzyme assay were tested at a tenfold higher concentration than used for the experimental samples. I t may be noted that such DNA's tha t do not contain an intact kinase gene and therefore fail to promote kinase synthesis do stimulate the overall protein synthesis, measured as 14C-leucine incorporation into TCA-insoluble material, to a comparable degree as does the DNA from ~ l d t y p e 2dg.

I t should be noted that only Fig. 1 contains directly measured values to which no corrections have been applied. All other data presented in the following figures and tables have been corrected for a zero-time value and for the dilution of the protein synthesis mixture, as is described in the "Material and Methods" section.

Concomitantly with the synthesis of galaetokinase the synthesis of uridyl transferase (referred to as " t ransferase" in this paper), the product of the second gene of the galactose operon, can be observed in this system (Fig. 2). Both enzymes have been tested from the same incubation mixtures in which they have been synthesized in response to increasing amounts of added 2dg-DNA. They show the same dependence on DNA concentration with 50 ~g/ml, the standard con- centration used by Zubay, Chambers, and Cheong (1970), being in the saturating concentration range.

All values given in this figure have been corrected for an appropriate blank value. I t may be noted that a control primed with DNA carrying a deletion of the transferase gene yields less than 2 % of the transferase rate obtained under optimal conditions. Even the values observed with the lowest concentrations of ~dg DNA used here are definitely above the unspecific blank reaction. Therefore this system provides a sensitive assay for the biological activity of DNA con- raining the galactose operon.

DNA-Dependent in vitro Synthesis of Enzymes of the Galaetose Operon 21

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6 21~ 3'0 io ~ ~o 7'0 concentration DNA (~.g/m[)

-51)

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Fig. 2. Synthesis of transferase (o o) and galac%okinase (e e) in response to varying DNA concentrations. Synthesis was under standard conditions (magnesium acetate 14.7 raM) with 6.9 mg/ml of S-30 extract and DNA from ;trig at varying concentrations as indicated. After incubation at 37 ° C for 30 minutes in a total volume of 0.20 ml synthesis was terminated by the addition of 0.80 ml of buffer to each synthesis mixture. Those samples incubated with 50 and 69 ~g DNA/ml were further diluted by a fatter of 2. Enzyme assays were started by the addition of 0.05 ml of each dilution to 0.05 ml of substrate mixture and terminated after 30 minutes of incubation at 37°C by the addition of 0.10 ml of TCA to each transferase test or by applying 20 ~1 of the kinase assay mixture to filters as described. A blank value obtained for zero minutes of incubation was substracted from each value (95 c.p.m./trans- ferase test and 100 c.p.m./kinase test, corresponding to 7% and 6%, respectively, of the directly measured maximum values in the experiment. The S-30 extract used in this experi- ment had lost activity upon storage and the values obtained in a control incubation with DNA that does not contain intact galactose genes, was significant as compared to the activities obtained with low concentrations of Xdg DNA. Therefore, in contrast to all other data pre- sented in this paper, this additional blank value has been subtracted from all values shown in this figure, after they had been corrected for the dilution of the protein synthesis mixture. These blank values represent ca. 50% of the corrected values given in this figure for the

incubation with 5 Fg of DNA

The active transferase molecule is presumed to conta in two identical subuni ts , whereas the galaetokinase of Escherichia coli consists of a single polypept ide chain (Saito, Ozutsumi, and Kurahashi , 1967 ; Wilson and Hogness, 1966). Association of snbuni t s does no t appear to be a l imit ing step in the synthesis of active t rans- ferase in the in vitro system, because the relat ive activit ies of transferase and galactokinase are similar to those observed in extracts of wildtype cells.

The appearance of act ive enzyme is t rue de-novo prote in synthesis t ha t requires the synthesis of messenger-RNA. I t is completely prevented by inh ib i to r s of DNA-dependen t R N A synthesis and of protein biosynthesis (Table 1).

22

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W. Wetekam, K. Staack, and R. Ehring:

0.2 ' 0'.4 ' 0'5 ' 0'8 ' 1.0

m M cyc[. A M P

Fig. 3. Stimulation of galactokinase synthesis by cyclic AMP. Synthesis was under standard conditions with the exception that the concentration of cyclic AMP was varied as indicated. After incubation for 60 minutes at 37 °C of the synthesis mixture that contained 14.7 mM magnesium acetate, 6.5 mg/ml of S-30 extract, 50 ~g/ml of )ldg DNA, the kinase assays were started immediately by mixing 0.05 ml aliquots of the undiluted protein synthesis mixtures with 0.05 ml of the substrate mixture. The enzyme assays were incubated for 45 minutes at 37 °C and the reaction was terminated by applying 20 ~1 of each test incuba- tion to filters as described before. A blank value obtained for zero minutes of incubation was subtracted from all values which were then converted to units of galactokinase activity synthesized per milliliter of undiluted protein synthesis mixture in a 60 minute incubation. The blank value of 160 c.p.m, subtracted amounted to 3 % of the directly measured maximum

value obtained in the experiment a

Table 1. E//ect o] inhibitors o/Dl~A-dependent RNA synthesis and o/protein synthesis The inhibitors were added in the final concentration indicated before the start of the

synthesis reaction. Protein synthesis was under standard conditions with 50 ~g/ml 2dg DNA, 7.5 mg/ml of S-30 extract at a concentration of 14.7 m ~ magnesium acetate. The protein synthesis (total volume 0.20 ml) was terminated by the addition of 0.80 ml of buffer to each synthesis mixture. Enzyme assays were performed with 0.05 ml aliquots of these 1 : 5 dilutions, exactly as described for Fig. 2. Before the values were corrected for the dilution of the protein synthesis mixture, a blank value obtained for zero minutes of incubation was sub- tracted, 83 c.p.m, for the transferase tests and 61 c.p.m, for the kinase tests, corresponding to 2% and 1.5%, respectively, of the directly measured values for the synthesis in the absence of inhibitor.

Enzyme activity (units/ml of

Concentration synthesis mixture) of inhibitor

galacto- trans- kinase ferase

No addition 4.9 9.3 DNase (1 izg/ml) 0.1 0.02 Actinomycin (2 Ezg/ml) 0.1 0.04 Rifampicin (1 tzg/ml) 0.1 0.05 RNase (1 ~g/ml) 0.1 0.03 Chloramphenicol (100 9g/ml) 0.1 0.07

3 The 8-30 extract used in this experiment was of an unusually low specific activity.

DNA-Dependent in vitro Synthesis of Enzymes of the Galactose Operon 23

u1

..c:

t o

"E

' , .9 a

3-

2-

1-

t t t J , i

0 Z, 8 12 16

mM Mg '+

J J i K i i i i L 1

4 8 12 16

mM Mg ++

E

2=

o

o 0 t

20 20 b

Fig. 4. Effect of D-fucose on the synthesis of galactokinase by either an extract containing active galaetose repressor or by a repressor deficient extract. The S-30 extract was either prepared from a galR+ strain (a) or from a galR- strain (b) and tested in the presence (. . ) or absence ( o - - © ) of D-fueose. Protein synthesis was under standard conditions with 7.35 mM magnesium acetate 2, 25 ~g/ml (a) or 20 t~g/ml (b) of DNA from 2dg and 6.5 mg/ml of S-30 extract. Cyclic AMP (5 X 10 -~ M) was present in all incubations, D-fucose, when present, was added to a final concentration of 5 x 10 -a M. After incubation for 30 minutes at 37°C 1.6 ml of ice-cold buffer was added to each protein synthesis mixture (total volume 0.4 ml). Kinase assays were started by mixing 0.05 ml aliquots of these dilutions with 0.05 ml of the substrate mixture. After 30 minutes of enzyme test incubation 20 t~l samples were applied to filters as described. Before correcting for the dilution, a blank value of 100 e.p.m. (a) or 80 c.p.m. (b) was subtracted from all values. I t amounted to 2.5% (a) or 2% (b) of

the directly measured maximum value in each experiment

A cont ro l expe r imen t was pe r fo rmed to examine the re l i ab i l i ty of a c t i v i t y tes t s as a measure of enzyme synthes ized in v i t ro :

A crude ex t r ac t was p repa red f rom induced wi ld type cells of Escherichia coli a n d a d i lu te a l iquot was a d d e d to a p ro te in synthes is i ncuba t ion mix tu re a t the end of the synthes is react ion. I t was found t h a t under the condi t ions used in our exper imen t s t h a t pe rmi t the a s say of d i lu te a l iquots of the synthes is mix ture , the recovery of a d d e d enzyme a c t i v i t y amoun t s to ca. 70% of the control t h a t has been d i lu t ed in buffer in the absence of p ro te in synthes is mix tu re .

The nex t exper imen t s concern the regu la t ion of th is in v i t ro synthesis . Most of th is synthes is is specific wi th respect to cont ro l of t r ansc r ip t ion . W e conclude this f rom the s t rong dependence of the synthes is on the presence of cyclic A M P (Fig. 3). The sa tu ra t ing concen t ra t ion (0.5 to 1 mM) is t he same as t h a t found b y Chambers and Z u b a y (1969) for the synthes is of f l -galactosidase, ind ica t ing t h a t the same cont ro l mechan i sm is opera t ive . The ex t en t of s t imula t ion b y eylie A M P varies in di f ferent exper iments (cf. Table 2). 10-20% of the m a x i m a l synthes is is observed in the absence of cyclic AMP.

The synthes is of ga lac tose enzymes in th is in v i t ro sys tem is cont ro l led b y the ga lac tose repressor : D-fucose, known as an inducer of the galac tose operon f rom in v ivo s tudies (But t in , 1963a, b), s t imula tes the synthes is of ga lae tokinase , if i t

24 W. Wetekam, K. Staack, and R. Ehring:

Table 2. E]/ect o/ cyclic AMP and D-/ucose on synthesis o/galactokinase directed by wildtype and galO c DNA

DNA was either from ~dg that contains the wildtype form of the galactose operon or from a phage carrying an operator-constitutive mutation galO c. The final DNA con- centrations used are indicated. D-fucose (final concentration 5 × 10aM) and cyclic AMP (5 × 10-4M) were present only, when indicated. The protein synthesis incubation was with 7.35 mM magnesium acetate and 6.5 mg/ml of S-30 extract prepared from the strain R+gal that contains active galactose repressor. After incubation at 37 ° C for 30 minutes the synthesis mixtures (0.4 ml total volume) were diluted by the addition of 1.6 ml of buffer and galacto- kinase assays of these dilutions were performed exactly as described for Fig. 2. A blank value obtained for zero minutes of incubation was subtracted from all values before correcting for the dilution. The blank value was 90 c.p.m, corresponding to 3.5% of the maximum value directly measured for the experiment with ~dg DNA, and 130 c.p.m. corresponding to 2 % of the maximum value directly measured in the experiment with galO c DNA. The difference in absolute activities between these experiments is attributed to the use of different preparations of S-30 extract.

Galactokinase synthesized Ratio

DNA Cyclic (units/ml synthesis of activ- AMP mixture) ities

-~ Fueose -- Fucose

Wildtype ~- 4.5 2.4 1.9 (20 izg/ml) - - 0.8 0.6 1.3

O c ~ 9.1 9.9 0.9 Mutant -- 1.1 1.2 0.9 (25 ~g/m])

is t e s t ed wi th low concen t ra t ions of DNA. This s t i m u l a t o r y effect is t rue derepres- sion. I t is no t observed, if e i ther a s t ra in t h a t carr ies a m u t a t i o n galR 2 in the s t ruc tu ra l gene for t he ga lae tose repressor is used as a source for the bac te r i a l ex t r ac t (Fig. 4) ~ or if t he D N A is i so la ted f rom a t r ansduc ing phage t h a t carr ies an ope ra to r cons t i tu t ive m u t a t i o n in the ga lae tose operon (Table 2).

Zubay , Chambers , a n d Cheong (1970) have eva lua t ed the effect of inducer a t d i f ferent D N A concen t ra t ions to e s t ima te the concen t ra t ion of func t iona l lactose repressor in t he in v i t ro sys t em p r e p a r e d f rom a w i ld type s t ra in .

Fol lowing th is approach , we f ind under our condi t ions 50% repress ion of ga lac tok inase synthes is w i th a D N A concen t ra t ion of ca. 20 txg/ml (Table 3). Assuming t h a t repressor b inds so effect ively to D N A t h a t unde r these condi t ions t he concen t ra t ion of free repressor is negligible, th is is i n t e rp re t ed to mean t h a t unde r these condi t ions 50% of al l DIqA molecules are b o u n d to repressor and 50 % of the D N A is free, wi th the concen t ra t ion of repressor being equal to half the concen t ra t ion of D N A .

Using a va lue of 28 × l0 s d a l t o n as the molecu la r weight for D N A of t rans- ducing phage l a m b d a ( tha t is 90 % of the molecu la r weight g iven b y Burg i a n d H e r s h e y (1963), for t he D N A of infect ious phage l ambda) , we ob t a in a concent ra- t ion of ca. 4 × 10 -1° M for t he func t iona l ga lac tose repressor in the in v i t ro sys tem.

D~qA-Dependent in vitro Synthesis of Enzymes of the Galactose Operon 25

Table 3. E]/eet o/D-/ucose on galactokinase synthesis directed by di]/erent amounts o] wildtype DNA

The conditions for the protein synthesis were as described for Table 2 with the exception that cyclic AM~ (5 × 10-aM) was present in all synthesis mixtures. D-fucose (5 × 10-aM) was added as indicated. DNA from 2dg carrying the wildtype galactose operon was added at varying concentrations as indicated. Synthesis for 30 minutes in a total volume of 0.4 ml and kinase assays after 1:5 dilution of the synthesis mixtures were exactly as described for Table 2. The blank value obtained for zero minutes of incubation represented 2% of the directly measured maximum value in the experiment.

DNA Galactokinase synthe- Ratio txg/ml sized (units/ml of activ-

synthesis mixt.) ities

-~- :Fucose -- :Fucose

10 3.7 1.1 3.3 25 4.9 2.5 1.9 40 5.9 3.6 1.6 50 4.5 4.3 1.1

This value is similar to the concentration of 6.5 × 10 -1° M observed by Zubay, Chambers, and Cheong (1970) for the functional lactose repressor in the in vitro system.

I t is approximately half the concentration predicted from in vivo studies by Germaine and Rogers (1970). From the study of the escape synthesis of galactose enzymes, these authors estimated 17 molecules of galactose repressor present in each cell. Combining this figure with the number of cells extracted in the prepara- tion of the S-30 extract, the final concentration of galactose repressor in the protein synthesis system would be expected to approximate 10 -9 M.

In summary, we have described the in vitro synthesis of two gene products of the galactose operon. The DNA dependence of the synthesis of both enzymes can be measured with equal accuracy, so that this system appears suited for the in vitro study of the effect of polar mutations. The synthesis of the operator proximal enzyme of this operon, UDPglucose epimerase, has also been observed in this system (unpublished experiments). Work is in progress to adapt test methods suited for routine measurements of this enzyme in the in vitro system.

The yield of active uridyl transferase and of galactokinase in the in vitro system may be compared to the activities measured by the same test methods in crude extracts from growing cultures of fully induced wildtype Escherichia coll. After 60 rain incubation the activities obtained in the standard synthesis system expressed in units per mg of S-30 protein amount to 5-10 % of the specific activities of each of the two enzymes observed in extracts from growing cells.

The number of copies of DNA containing the galactose operon is higher by a factor of approximately 100-200 in the in vitro system than in a culture of cells from which an extract containing an equivalent enzyme activity can be prepared.

Using the data obtained by Wilson and Hotness (1969), for purified galacto- kinase, the activity measured after one hour of synthesis under conditions des- cribed in Fig. 1 amounts to ca. 1.1 × 1013 galactokinase polypeptide chains. For

26 W. Wetekam, K. Staack, and R. Ehring:

each D N A molecule added , a p p r o x i m a t e l y 10 ac t ive ga lac tok inase molecules have been ob ta ined .

This ac t ive ga lac tok inase syn thes ized corresponds to 7.6 × 10 -7 g of p ro te in syn thes ized in one mil l i l i ter of t he in v i t ro sys tem. This is a ca. t enfo ld h igher y ie ld t h a n t h a t ca lcu la ted for the in v i t ro synthes is of f l -galactosidase (Chambers and Zubay , 1969; Zubay , Chambers , a n d Cheong, 1970). This difference m a y be a t t r i b u t e d to the h igher degree of comp lex i t y of the s t ruc ture of /~-galactosidase.

Acknowledgement. We wish to thank Professor P. Starlinger for stimulating discussions, helpful advice and critical reading of the manuscript. Most of the bacterial and phage strains used were kindly supplied by Professor Starlinger. We are very much indepted to Dr. J. Besemer for contributing the recently constructed strains carrying mutations in the regulator gene or in the operator region of the galactose operon.

This work was supported by the Deutsche Forschungsgemeinschaft through SFB 74.

Note Added in Proo]. After submission of our manuscript independent evidence concerning the regulation of galaetokinase synthesis in a cell-free system by cyclic adenosine-3'-5' mono- phosphate has been published by J. S. Parks, M. Gottesman, R. Perlman and L Pastan: Regulation of galactokinase synthesis by cyclic adenosine 3',5'-monophosphate in cell-free extracts of Escherichia coll. J. biol. Chem. 246, 2419-2424 (1971).

R e f e r e n c e s

Adhya, S.L., Shapiro, d .A. : The galactose operon of E. coli K12. I. Structural and pleio- tropic mutations of the operon. Genetics 62, 231-247 (1969).

Burgi, E., ~ershey, A. D. : Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309 (1963).

Buttin, G.: Mechanismes r6gulateurs dans la biosynth~se de.s enzymes du m6tabolisme du galactose chez Escherichia coli K12. I. La biosynth~se induite de la galactokinase et l'in- duction simultan6e de la s6quence enzymatique. J. molec. Biol. 7, 164-182 (1963a). II . Le d6terminisme g~n6tique de la r6gulation. J. molec. Biol. 7, 183-205 (1963b).

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Crombrugghe, B. de, Chen, B., Gottesman, IV[., Pastan, 1., Varmus, H.E. , Emmet, M., Perlman, R. L. : Regulation of hw tuRNS synthesis in a soluble cell-free system. Nature New Biology (Loud.) 230, 37-40 (1971).

Eron, L., Arditti, R., Zubay, G., Connaway, S., Beckwith, J. R. : An adenosine 3':5'-cyclic monophosphate-binding protein that acts on the transcription process. Proc. nat. Acad. Sci. (Wash.) 68, 215-218 (1971).

Fiethen, L. : Untersuchungen fiber 0e-Mutanten im Galactose Operon yon Escherichia coll. Diss. Universit~t zu KSln (1969).

- - Starlinger, P.: Mutations in the galaetose-operator. Molec. Gen. Genetics 108, 322-330 (1970).

G~rmaine, G.R., Rogers, P.: Role of gal repressor depletion in 2dg transduction escape synthesis. J. molec. Biol. 47, 121-135 (1970).

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DNA-Dependent in vitro Synthesis of Enzymes of the Galaetose Operon 27

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Communica ted by H. G. W i t t m a n n

Waldemar Wetekam Karin Staaek Ruth Ehring Institut ffir Genetik der Universitat BRD-5000 KSln 41, Weyertal 121 Deutschland