[18] Purification of TF1—A template-specific DNA-binding protein and transcription inhibitor from bacteriophage SPO1-Infected Bacillus subtilis

204

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

[ 1 8 ] P u r i f i c a t i o n of T F 1 - - A T e m p l a t e - S p e c i f i c DNA-Binding Protein and Transcription Inhibitor from B a c t e r i o p h a g e S P O l - I n f e c t e d Bacillus subtilis 1 By G. G. JOHNSON and E. P. GEIDUSCH~.K

rATP ] [ rAp ] rCTP | DNA polymer~e | rCp | rGTP[ d- SPO1 TF1 [rGp|, -I- [PPd~ rUTPJ~ LrUpl Materials Buffers and Other Solutions

A: 10 mM Tris.HCl, pH 7.5, 50 mM KC1, 0.1 mM EDTA, 300 ~g/ml lysozyme B: 10 mM Tris.HC1, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol C: 10 mM Tris hydroxide, 10 mM cacodylic acid, .adjusted to pH 6.0 with HCI, 0.1 mM EDTA CD: Buffer C with 0.1 mM dithiothreitol E: 10 mM Tris.HC1, pH 7.5, 5 mM MgCl2 G: 10 mM glycine adjusted to pH 10 with NaOH, 0.1 mM dithiothreitol Ammonium sulfate: Enzyme grade (NH4)2SO4 solution, saturated at room temperature and adjusted to pH 7 with NH4OH. Media. CHT50 contains, per liter: glucose, 5 g; casein hydrolyzate, 1.25 g; L-tryptophan, 50 mg; CaC12, 5 mmoles; (NH4)2S04, 2 g; K~HP04, 14 g; KH2PO4, 6 g; sodium citrate, 1 g; MgS04, 0.8 mmoles. The low sulfate synthetic medium s contains, per liter: L-valine, 20 mmoles; L-arginine, 2.4 mmoles; L-leucine, 7.0 mmoles; L-threonine, 4.2 mmoles; L-serine, 6.0 mmoles; L-glutamic acid, 25 mmoles; L-alanine, 14 mmoles; I~asparagine, 10 mmoles; D-glucose, 25 mmoles; MgCl~, 0.4 mmole; KH2PO4, 10 mmoles; Na2S04, 40 umoles; CaC12, 0.1 mmole; FeCI~, 3.6 umoles; NH4CI, 10 mmoles; MnCl2, 0.1 mmole; and NH4N03, 1.2 mmoles. Bacteria. Bacillus subtilis 168M (trp-) are the host for wild-type phage SP01; they are nonpermissive for sus mutants. They are used for growing

1Our research on this subject was supported by grants of the National Institutes of Health, the National Science Foundation, and the former Life Insurance Medical Research Foundation. 2 j. E. Donnellan, E. H. Nags, and H. S. Levinson, J. Bacteriol. 87, 332 (1964).

[18]

TF1

PREPARATION

205

wild-type phage SPO1 and as a source of TF1 after infection with phage. B. subtilis HA 101B (leu- his:us met[u, su+) are the permissive host for sus mutants of phage SPO1; they are phenotypically leu- and at a selective disadvantage to su- revertants in complete media. Accordingly, they are best maintained on minimal medium supplemented with leucine only. Phage. These are grown as described elsewhere. 3,4 When phage SPO1 are to be used for the preparation of DNA, they are purified from the original lysate either by precipitation with polyethylene glycol (Note 1, under Special Considerations), or by differential centrifugation (Note 2). Phage are then further purified by isopycnic centrifugation in CsC1, density 1.50, in buffer E or by velocity centrifugation in a step gradient composed of layers of various concentrations of CsC1 in buffer E (Note 3). The CsC1 purified phage are dialyzed into buffer E. Phage S P 0 1 D N A . This is prepared by phenol extraction 5 Chromatographic Materials. DEAE-ceilulose (Whatman DE52) and SE Sephadex G-50 are pretreated by two cycles of washing with 0.5 M HC1 and 0.5 M NaOH. Phosphocellulose is washed in the same way but with 0.1 M HCI. These materials are then equilibrated with their respective column buffers. When appropriate (as to size), columns are prepared in disposable plastic syringes on glass fiber filter supports (Whatman GF/C). These filters are boiled in water for 10 minutes prior to use in order to remove impurities.

Assay of TF1 Activity The purification of TF1 can be followed conveniently by measuring its effect on SP01 DNA-dependent RNA synthesis catalyzed by bacterial RNA polymerase. The characteristic property of TF1 which distinguishes it from other inhibitors of transcription in vitro is its specific action on hmU-containing SP01 DNA and on the DNA of closely related h m U containing phages. 6 Accordingly, controls showing noninhibition of RNA synthesis on other, non-hm U-containing templates should be included. The standard assay contains, per milliliter, 100 ~moles of Tris.HCt, pH 7.5, 10 ~moles of MgC12, 0.8 ~mole of spermidine, 1 t~mole each of three unlabeled ribonucleoside 5'-triphosphates (usually ATP, GTP, and CTP), 0.12 ~mole of labeled 3H- or 14C-labeled ribonucleoside 5'-triphosphate, usually UTP, 2.5 ~g DNA, E. coli or B. subtilis RNA polymerase and varying quantities of TF1 (standard assay A). The assay medium may addi3S. Okubo, B. S. Strauss, and M. Stodolsky, Virology 24, 552 (1964). 4L. P. Gage and E. P. Geiduschek, J. Mol. Biol. 57, 279 (1971). 5j. D. Mandell and A. D. Hershey,Anal. Biochem. 1, 66 (1960). s D. Wilson and E. P. Geiduschek,Proc. Nat. Acad. Sci. U.S. 62, 514 (1969).

206

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

tionally contain 0.10 M KCI and 1 mg/ml Triton X-100 (standard assay B). The use of Triton is mainly of importance with highly purified TF1 because of the latter's pronounced surface adhesiveness (see below). In either standard assay, reactants are added as follows: DNA is added to the reaction medium at 0 °. TF1 is added next at 0 °, and the mixture is equilibrated at 30°. RNA synthesis is started by the addition of RNA polymerase. It has been verified that the interaction of purified TF1 with SP01 DNA in assay medium B is sufficiently rapid that further preincubation of TF1 with DNA is not necessary. The total volume of the standard assay usually is 250 ~1. Reaction is terminated after 10 minutes by the addition of one volume

100

,

80

60 Z

40

•

2 2O

I

5

I

iO TFI (pg/ml)

I

i

15

20

FTG. 1. SPO1 transcription-inhibition assay of TF1 in standard incubation solvents A ( O ) and B (O). Assays were performed in acid-washed, 7 X 50 m m glass tubes. In order to minimize losses of TF1 due to surface adsorption before addition to the incubation tube, serial dilutions of TF1 were made on a Teflon surface; a single glass micropipette was used for the addition of increasing concentrations of TF1 to the different assay tubes of assay A; a separate pipette was used for assay B. The solvent and diluent for TF1 is buffer G with 0 % or 1% Triton X-100. (Each assay ultimately contains 0.1 volume of buffer G, which only barely changes the composition from t h a t listed under Materials.) [14C]ATP was the labeled ribonucleoside 5'-triphosphate in this experiment.

[18]

TF1 PREPARATION

207

0.2% SDS, 20 mM EDTA. The mixture is immersed in a boiling water bath for 3 minutes, cooled, and 0.8 ml 10% TCA and 50 ~g denatured carrier DNA are added. After 15 minutes on ice, the precipitated nucleic acids and proteins are filtered on glass fiber (Whatman GF/C) filters and counted in toluene-based scintillation fluid. TF1 fractions containing large quantities of C13CCOOH precipitable material quench the radioactivity of 3H. Such samples are best analyzed with 32p-labeled ribonucleoside 5'-triphosphate. Alternatively, the precipitated nucleic acids may be treated with one of the proprietary solubilizing agents before counting in toluene-based scintillation fluid (Note 4). One unit of TF1 activity is now defined as the quantity of protein inhibiting SPO1 DNA-directed RNA synthesis by 50% in standard assay B. Activity was originally defined with respect to assay A6; the units are not substantially different. Further discussion relevant to this question follows on page 214. Fig. 1 shows a titration curve of TF1. Growth of Phage SPOl-Infected B. subtilis 168M (see Note 5) Good yields of TF1, which is synthesized after phage SPO1 infection of B. subtilis, can be obtained from cultures grown in a variety of media. The procedure described here yields relatively large quantities of cells. Bacteria are grown at 37 ° in CHT50 supplemented with 4 g of yeast extract and 2.5 g of Difco nutrient broth per liter (the yeast extract and nutrient broth are separately autoclaved twice before use). They are infected with 1012 phage/liter when the cell density reaches 200 Klett units (measured with a number 66 filter). This cell density corresponds to As00nm = 2. When wild-type phage SPO1 are used, the culture is collected 20 minutes after infection and poured onto ice supplemented with salts of the med i u m - t h a t is, with a concentrated solution to give approximately, per liter, 2 g of (NH4)~SO4, 14 g of K2HPO4, 6 g of KH2PO4, 1 g of Na citrate, 0.8 mmoles of MgSO4 and 5 mmoles CaCI~ upon total melting of theiee. The cells are collected by eentrifugation, suspended in buffer A, frozen, and stored without washing at - 7 0 ° until further use. Preparation of TF1

Step 1. Breakage of Cells. A quantity of bacteria containing 5 g of protein, suspended in 250 ml of buffer A, is quickly frozen by swirling in a 2-liter flask, and set in a Dry Ice-ethanol bath. After at least 30 minutes, the frozen bacteria are warmed at 37 ° until visibly lysed and are cooled to 4 °. They are then disrupted further by vigorous sonieation. For example, when using a Branson Model L "Sonifier" converter equipped with a mierotip at maximum power output, it has been convenient to process 100-ml aliquots in a 250-ml beaker immersed in ice, by sonieating for five

208

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

2-minute intervals. In this and all subsequent stages of the purification, the temperature is maintained at 0-4 ° . The remainder of the procedure is described for a sonicated cell suspension containing 5 g of protein.

Step 2. Streptomycin Precipitation of Nucleic Acids and of Some Protein. Streptomycin sulfate solution, 75 ml, is added, dropwise, with stirring (final concentration 2.3% w/v). After it has settled for 15 minutes, the precipitate is centrifuged and discarded. The precipitate contains DNA, RNA, ribosomes, and some proteins, including RNA polymerase. Lower concentrations of streptomycin sulfate may allow TF1 to coprecipitate with nucleic acids. Step 3. Ammonium Sulfate Precipitation. To the supernatant from step 2 (Note 6), 1.22 volumes of room temperature-saturated, neutralized (NH4)2SO4 solution are slowly added with stirring (final concentration: 55% saturation). The resulting precipitate is removed by centrifugation and discarded. An additional 1.1 volumes of saturated (NH4)~SO4 solution (Note 7) are slowly added to the supernatant (final concentration: 70% saturation). After an additional 30 minutes, the precipitate is collected by centrifugation and the supernatant is discarded. The pellet is freed, as thoroughly as practicable, of adhering supernatant liquid and is dissolved in 20 ml of buffer B with 0.1 M KC1. The solution is equilibrated with the same solvent by Sephadex G-25 gel chromatography or by dialysis (a precipitate can form during dialysis and is removed by centrifugation). Step ~. DEAE-Cellulose Chromatography. The TFl-containing solution from step 3 (approximately 24 ml) is applied to a 2.5 cm diameter, 8 cm high (45 ml) DEAE-cellulose column previously equilibrated with buffer B containing 0.10 M KC1. The flow rate is 50 ml/hour, and the column is rinsed with 30 ml of the same buffer. Other proteins can be eluted by applying a salt gradient to the column, but TF1 is in the voided material together with approximately 25% of the applied protein, as determined by the Lowry phenol method ~ and is approximately 25% pure when prepared from wild-type phage SPOl-infected cells 20 minutes after infection at 37 ° . Step 5. SE-Sephadex Chromatography. The pooled TFl-containing fractions from step 4 are applied to a 1.75 cm in diameter, 7 cm high column (17 ml) of SE-Sephadex G-50 previously equilibrated with buffer CD containing 0.10 M KC1 at a flow rate of 50 ml/hour. The column is rinsed with 20 ml more of this buffer and 50% of the protein fails to bind. The adsorbed proteins are then eluted with a linear 0.1-0.5 M KC1 gradient in 160 ml buffer CD. TF1 elutes in a symmetric peak centered at 0.32 M O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[18]

TF1 PREPARATION

209

-200 -iO0 -0

200--20

!

j x.-

,oo- / ,,Ic

0

4

~

,:.

0.2-

~. ~,

g

o

,7

.:"

0.4

0 . . . . . . . .I0. . . .

20-

.O

-

•

30

x

40

FRACTION NUMBER

FIG. 2. Chromatography on SE-Sephadex. Protein from step 4, 145 mg, was applied in buffer CD with 0.10 M KC1 and eluted with a 0.1 to 0.5 M KCI gradient in the same buffer. Protein, 130 mg, was recovered in the combined fractions. The volume per fraction was 5 ml. TF1 assay A was used. K C I (Fig. 2). T h e peak fractions off the S E - S e p h a d e x c o l u m n are a p p r o x i m a t e l y 9 0 % or more pure w h e n p r e p a r e d from w i l d - t y p e phage SPO1 20 m i n u t e s after infection.

Step 6. DEAE-Cellulose and Phosphocellulose Chromatography (Notes 8, 9, and 10). Pooled fractions of T F 1 from t h e S E - S e p h a d e x c o l u m n are dialyzed i n t o buffer C D i n r e g e n e r a t e d cellulose casings, a p p l i e d to a s m a l l c o l u m n (2 ml) of D E A E - c e l l u l o s e a n d washed with buffer C D . T h e v o i d e d fractions which c o n t a i n T F 1 are pooled a n d applied to a 1.35 c m i n - d i a m TABLE I SUMMARY OF PURIFICATIONOF TF1

Fraction I. Sonicate II. Streptomycin sulfate supernatant III. 55-70% saturated (NHD~SO~ IV. 0.10 M KC1, DEAE void V. SE-Sephadex VI. DEAE void VII. Phosphocellulose

Protein recovery

Activity recovery" (%)

mg/ml

---

20 5.5

23.4

100

23

28.8 14.7 16 41

68 61 39 34

Volume (ml) 250 415

a Recovery of TF1 is calculated relative to fraction III.

% 100 46 11

5 1.67

2.9 0.5

0.45

0.37

210

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

eter, 6 cm high (10 ml) column of phosphocellulose previously equilibrated with the same buffer, at a flow rate of 15 ml/hour. The column is rinsed with buffer CD and eluted with a 0 to 0.5 M KC1 gradient (160 ml) at a flow rate of 15 ml/hour. TF1 elutes in an asymmetric peak centered at 0.27-0.33 M KC1. The course of the purification of TF1 is summarized in Table I.

Special Considerations NOTE 1: To 10 liters of lysate, add 50 mg crude pancreatic DNase and suspend 20 g Celite. After 15 minutes the mixture is filtered through filter paper under suction; 292 g of NaC1 and 1000 g of polyethylene glycol 6000 are dissolved in the filtrate, which is then stored at 0-4 ° for 16 hours. The finely suspended but precipitated phage are sedimented in the cold (e.g., 10 minutes at 1.3 X 104 gma.). The sedimented phage are overlayered with buffer E for 16 hours and then resuspended. NOTE 2: The filtered lysate is centrifuged for 10 minutes at 5000 gmax. The decanted supernatant is centrifuged for 180 minutes at 27,500 gmaxand the supernatant is discarded. The pelleted phage are resuspended as in procedure A and cleared of debris by centrifugation at 5000 g~ax for 10 minutes. NOTE 3: The step gradient is made up by superimposing 6 CsC1 solutions of decreasing density and using these as a substrate for a relatively wide zone of resuspended phage. For example, 2 ml of resuspended phage may be layered on 0.5-ml zones of (in order of increasing density) 20, 30, 40, 50, 60, 70% saturated CsCl in buffer E and centrifuged in a bucket rotor for 30 minutes at 9 X 104 g. . . . The zone of phage is collected and dialyzed against buffer E. NOTE 4: The procedure used by us is the following: 0.10 ml of water is added to the dry filter in a scintillation vial followed by 0.9 ml of NCS solubilizer (Amersham-Searle proprietary quaternary ammonium base reagent). After digestion for 1 hour at 37 °, 14 ml of toluene based scintillation fluid are added. NOTE 5: The production of TF1 can be enhanced by the use of phage SPO1 s u s mutants which do not lyse and which overproduce certain of the viral proteins. F4 s is one such mutant, and TF1 has been prepared from cells collected 2 hours after infection with mutant F4. NOTE 6: Alternatively, 35.1 g of solid (NH4)2SO4 can be added per 100 ml of solution from step 2, to give 55% saturation, and the pH can be adjusted to 7 with 1 M NH~OH. NOTE 7: Alternatively, 12.1 g of solid (NH4)2SO4 can be added per 8D. Fujita, B. Ohlsson-Wilhelm,and E. P. Geiduschek,J .

Mol. Biol.

57, 301 (1971).

[18]

TF1 PREPARATION

211

100 ml of solution from step 2, to give 70% saturation, and the p H can be adjusted to 7 with 1 M NH4OH. NOTE 8: At this point, an alternative purification scheme utilizes the retention of TF1 on DEAE-celhlose at high pH. The TFl-containing fractions from the SE-Sephadex column are dialyzed into buffer G and applied to a column of DEAE-cellulose equilibrated with buffer G. The column is washed with buffer G and TF1 elutes after the void volume but without application of a salt gradient. In one instance, approximately 6 mg of TF1 in 20 ml of buffer G was applied to a 1.5 cm in diameter, 5 cm high (10 ml) column of DEAE-celhlose, and more than 70% of the activity of TF1 was e h t e d as described. NOTE 9: The yield at this step can be low, probably as a consequence of the surface adhesiveness of TF1. This is particularly a problem when small preparations of TF1 (for example, radioactively labeled TF1) are to be made. The inclusion of 1% Triton X-100 in buffer CD can improve the yield of TF1 in phosphocellulose chromatography. In one such instance, 500 tLg of purified 35S-labeled and unlabeled TF1 were applied to a 18-ml column (1.5 cm in diameter, 10 cm high) and eluted at 0.33 M KC1 in a single, slightly asymmetric peak. NOTE 10: aSS-Labeled T F 1 . Bacteria are grown in the low-sulfate medium to a density of As0o nn, = 1. T h e y are then infected with 2 X 1012 S P 0 1 sus F4 phage per liter and labeled with 35 mCi of Na235SO4. Ten micromoles per liter of Na~S04 are added 5 minutes later. When cells are collected 60 minutes after infection, approximately 30% of the radioactivity has been incorporated into protein. According to the course of the subsequent purification of TF1 we judge that more than 1% of the incorporated ass has entered TF1. In one instance, TF1 was purified from cells that had been labeled as above and mixed with a 20-fold excess of unlabeled cells prepared in the same way. The purification procedure described in this article was followed for steps 1-3 (Notes 6 and 7), 4, and 5. The final purification was b y chromatography on DEAE-celhlose at p H 10 (Note 8). Purified TF1 had a specific activity of 5 X 104 dpm/t~g. Tests for the Purity of TF1 TF1 is a small basic protein, whose amino acid composition is shown in Table II. The purity of TF1 preparations can be gauged b y several criteria~; the experimental details of these determinations are not given here: (a) Gel electrophoresis in SDS at pH 8.8 ~° and in 6.25 M urea at g G. G. Johnson and E. P. Geiduschek, J. Biol. Chem. 247, 3571 (1972). 10U. K. Laemmli, Nature (London) 227, 680 (1970).

212

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

TABLE II A~INO ACID Co~,PosrrION OF TFI Amino

Mole

Molecules

acid

percentage

per unit~

Gly Ala

5.8 10.6

6 11

Ser ~

6.4

7

Thr° Met Pro Valb Ileb Leu

8.4 1.9 3.6 7.6 4.8 8.2 4.0 1.0 6.3 16.3 2.0 13.5 100.4

9 2 4 8 5 9 4 1 7 17 2 14 106

Phe

Tyr Asx Glx Arg Lys

Extrapolated to 0 hour hydrolysis. hydrolysis. c Trp, Cys, and His are absent from TF1. b 48-Hour

p H 3.211 yield single bands of protein. (b) Isoelectric focusing in p H 8-10 ampholine yields a single narrow band of uniform specific activity at p H 9.8. (c) TF1 is retarded on Sephadex G-200 chromatography at low ionic strength and elutes in a single broad zone of uniform specific activity, close to the included volume. At higher ionic strength, for example in buffer C with 0.25 M KC1, TF1 elutes just ahead of chymotrypsinogen marker (molecular weight 25,700) as a single zone of uniform specific activity. Surface Adhesiveness

of TF1

Purified TF1 adheres strongly to a variety of surfaces. This creates difficulties for work with small quantities or dilute solutions of TF1, for example, in measurements of TF1 binding to DNA, or in the preparation of small quantities of radioactively labeled TF1. Two kinds of solutions to this problem have been sought: Selection of a least adhesive surface and addition of agents that modify surface adhesiveness of the protein. T h e current status of this problem is described below. Teflon Binds Less TF1 Than Other Surfaces. Table I I I shows that Teflon II S. Panyim and R. Chalkley, Arch. Biochem. Biophys. 130, 337 (1969).

[18]

TF1 PREPARATION

213

TABLE III SURFXCE ADSORPTXONOF ['~S]TF1~ Surface

TF1 recovery (%)

Glass Glass (silanized) Polycarbonate Polypropylene Polyethylene Cellulose nitrate Polyallomer (Beckman) Stainless steel Teflon

11 10 11 16 27 7 17 16 79

A droplet of TF1, 0.14/~g in 0.035 ml (4 ~g/ml, 6300 cpm/gg), is exposed to the surface for 60 minutes at room temperature (22-24°). Recovery of nonadsorbed TF1 is determined from an 0.025-ml aliquot of the droplet. The solvent is 10 mM phosphate (Na), pH 7.5, with 0.15 M KC1. binds a small quantity of TF1 less avidly than a variety of other surfaces. T h e transfer of TF1 for this experiment was done with a glass micropipette which had been rinsed with 3H-TF1 until it delivered a constant amount of radioactivity in a calibrated volume. Various cleaning regimes of the glass surface with aqua regia, dichromate-sulfuric acid, or detergent, did not substantially improve the outcome of experiments like t h a t shown in Table III. Experiments with centrifuge tube materials showed that TF1 also binds strongly to "polyallomer" (Beckman) and to nitrocellulose. Pt, Ta, and gold-plated steel were also found to bind TF1. The surface adhesiveness of TF1 m a y be partly temperature dependent (more severe at higher than at lower temperatures) but this question has not, so far, been extensively investigated. SPO1 D N A changes the binding of TF1 to glass. For quantitative determinations, for example of TF1 binding to DNA, this poses further difficulties. Triton X-IO0 Reduces the Surface Adhesiveness of TF1. TF1 is stable in Triton solution (indeed, it is not irreversibly denatured in sodium dodecyl sulfate or concentrated sodium perchlorate). One percent Triton strongly reduces the binding of TF1 to glass; an example of this effect is s h o ~ in Table IV. R N A polymcrase inhibition and D N A binding can be assayed in the presence of 0.1% Triton. 1~ Accordingly, purification and assay of TF1 in Triton X-100-containing buffers can be helpful (see Note 9).

1~G. G. Johnson and E. P. Geiduschek, manuscript in preparation (1973).

214

CONFORMATION OF NUCLEIC ACID STRUCTURE

[18]

T A B L E IV GLASS SURFACE ADSORPTION OF [~sS]TFla % Triton X-100

TF1 recovery (%)

0.10 0.01 0. 001

78 64 21

" Experimental details as described in the legend to Table III.

Some Quantitative Aspects of the Transcription-Inhibition Assay for TF1 Effect of Triton X-IO0. A comparison of the inhibition of E. coli RNA polymerase by TF1 in the absence and in the presence of Triton X-100 (standard assays A and B) is shown in Fig. 1. The (fortuitously) coincident titration curves result from two opposing effects. The higher ionic strength of assay B decreases sensitivity of SPO1 transcription to inhibition by TF1. Triton X-100 increases sensitivity to inhibition by TF1. The latter effect is plausibly associated with the decreased binding of TF1 to glass. While D N A competes such binding to some extent, the competition is not complete under the conditions of assay A. For the purified preparation of TF1 shown in Fig. 1, one unit of TF1 activity equals 5.4 ~g of protein. Effect of Concentration of Reactants. The binding of TF1 to SPO1 DNA at the temperature and ionic strength of the transcription-inhibition assay is incomplete. The available, but not yet extensive, data on T F 1 - D N A binding lead to the following estimate: when a limiting quantity of TF1 is added to 2.5 ug of SPO1 DNA in 1 ml of assay medium B, less than 40% of the TF1 is bound to DNA. 12 Accordingly, at high concentrations of ligands, transcription inhibition occurs at lower proportions of added TF1 to DNA. Specific Activity of TF1. Preparations of TF1 that are homogeneous by the criteria described in the section on tests for purity of TF1 have specific activities of 150 units per milligram of Folin protein equivalent (bovine serum albumin, Armour, fraction V) or greater. However, variations of specific activity can be achieved by manipulations whose significance has not yet been analyzed. For example, a homogeneous phosphocellulose fraction of TF1 (fraction 7, Table I) in buffer CD plus 0.3 M KC1, specific activity 150 units/rag, was dialyzed by Sephadex G-200 chromatography into low ionic strength phosphate buffer and yielded TF1 with a specific activity of 200 units/rag, with no apparent loss of protein. Other Methods of Detecting TF1. An assay for specific inhibition of transcription of SPO1 DNA by bacterial RNA polymerase, requires relatively

[18]

TF1 PREPARATION

215

large quantities of TF1 and is quantitative only in the absence of competing molecular species. For these reasons TF1 activity is not assayed in crude extracts until nucleic acids and RNA polymerase have been removed (Table I, step 2). In addition, small quantities of low concentrations of TF1 may be difficult to purify because of nonspecific surface adhesion losses. Qualitative and quantitative immunological methods are useful for determining the presence of TF1 in preparations where the quantity or purity of TF1 is limiting. Two of these methods, the ring or interracial test and interference with the quantitative precipitation of [35S]TF1 by rabbit antibody to purified TF1, have been used. 13

13D. H. Campbell, J. S. Garvey, N. E. Cremer, and D. H. Sussdorf, "Methods in Immunology," 2nd ed., p. 236 and p. 246. Benjamin, New York, 1970.