J. Mol. Biol. (1976) 107, 271-291

Internal Proteins of Bacteriophage T7 Pin.re

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Division of Biology California Institute of Technology Pasadena, Calif. 91125, U.S.A. (Received 12 April 1976, and in revised form 12 July 1976) Using electron microscopy it has been shown t h a t there is a protein-containing structure (core) inside bacteriophage T7 which is surrounded b y DNA..4. similar core is fotmd inside two different DNA-free T7 capsids + isolated from lysates of T7-infected Escherichia coli. One of these eapsids (capsid I) appears to p l a y an i m p o r t a n t role in D N A packaging. Stereo electron microscopy has been used to show t h a t the cores inside capsid I particles closely a p p r o x i m a t e cylinders with an axial hole. A t higher resolution some cores appear to be m a d e of discs of subtmits arranged perpendicular to the core axis. Evidence is presented indicating t h a t the core is a t t a c h e d to the outer envelope of the T7 capsids and T7 phage. The core in T7 phage is joined to the outer envelope at the same place as the tail. W h e n D N A is released from T7 phage using h e a t shock, two types of DNAfree capsid are formed; one has no tail (capsid IVA) a n d the second has a tail (capsid IVB). Capsids I V A and I V B do not have cores. B y comparing the protein compositions of T7 phage, the various T7 eapsids and T7 tails, it has been shown t h a t proteins P13, P14, P15 and P16w are possible components of the core. The protein co~mnon to the T7 head, core a n d tail appears to be P8. Core proteins are also found in some of the a a p s i d - D N A complexes described b y Serwer (1974a,b). Evidence is presented indicating t h a t the major protein of the envelope of T7, P10, is involved in binding D N A to capsids. 1. I n t r o d u c t i o n L y s a t e s o f b a c t e r i o p h a g e T 7 - i n f e c t e d Escherichia coli c o n t a i n t w o t y p e s o f p h a g e capsid:~. One o f t h e s e (capsid I) is a s s e m b l e d in t h e a b s e n c e o f T7 D N A s y n t h e s i s ; t h e second c a p s i d (capsid I I ) is n o t p r o d u c e d in t h e absence o f D N A s y n t h e s i s (Studier, 1972). This has p r o m p t e d t h e h y p o t h e s i s t h a t c a p s i d I is c a p a b l e o f p a c k a g i n g D N A a n d serves as a p r e c u r s o r to t h e h e a d of T7 phage. B y t h i s h y p o t h e s i s c a p s i d I is c o n v e r t e d to c a p s i d I I d u r i n g a n a b o r t i v e a t t e m p t to p a c k a g e D N A . T h e r e is evidence t h a t o t h e r b a c t e r i o p h a g e s p a c k a g e D N A via a p r e c u r s o r p h a g e e a p s i d similar to c a p s i d I (reviewed b y Casjens & K i n g , 1975). B e c a u s e of t h e a p p a r e n t i m p o r t a n c e o f e a p s i d I t Present address: Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, U.S.A. :~Any structure that contains a protein envelope related to the envelope which surrounds DNA in T7 phage will be referred to as a capsid. A capsid may or may not have proteins in addition to envelope proteins. wProteins coded for by an identified gene will be designated by P followed by the gene number as determined by comparison with the gel patterns presented by Studier (1972). Proteins which have not yet been assigned genes are designated by capital letters. 271

272

P. S E R W E R

d u r i n g D N A p a c k a g i n g a s t r u c t u r a l analysis o f c a p s i d I was u n d e r t a k e n using e l e c t r o n m i c r o s c o p y . D u r i n g t h e course o f this w o r k it was f o u n d t h a t b o t h c a p s i d I a n d c a p s i d I I h a v e a n i n t e r n a l core. F u r t h e r e x p e r i m e n t s r e v e a l e d t h a t a core is also p r e s e n t inside T7 phage. T h e s t r u c t u r e a n d c o m p o s i t i o n o f t h e cores a r e i n v e s t i g a t e d a n d possible roles t h e cores m a y p l a y d u r i n g D N A p a c k a g i n g are discussed.

2. Materials and Methods (a) Phage and bacterial strains T7 phage were obtained from Dr F. W. Studier. The host for T7 was E. coli BB/T1. (b) Media and buffers Stocks of T7 phage were grown in M9 medium (Kellenberger & Sechaud, 1957). Phage assays were done with top and b o t t o m agar contmning T broth : 10 g Bacto Tryptone, 5 g NaC1, 1 1 water. Buffers used were: (1) TTG- bmCfer : T7.buffer (Serwer, 1974a) with gelatin omitted (0"5 ~-NaC1, 0.01 M-Tris (pH 7.4), 0"001 M-MgC12); (2) Tris/Mg buffer: 0.2 ~-NaC1, 0.01 ~i-Tris (pH 7.4), 0.001 ~I-MgC12; (3) T r i s / E D T A buffer; 0.1 ~-NaC1, 0.01 M-Tris (pH 7.4), 0.001 M-EDTA. (c) Preparation of T7 phage, capsid I and capsid I I A n overnight culture of E.e oli BB was diluted 1 : 200 into 30 1 of M9 meditun and was grown at 30~ to 3 • 108 bacteria/ml. The bacteria were infected at a multiplicity of 0.05 to 0-1 with T7 phage and were incubated until lysis. To precipitate T7 phage and capsids Carbowax 6000 (Union Carbide) and NaCI were added to the lysate to final concentrations of 9% and 0.5 M, respectively (Yamamoto et al., 1970). Precipitation was continued a t 4~ for 2 to 3 days axLd then 900/0 of the supernatant was siphoned away. The Carbowax precipitate was pelleted (GSA rotor, 5500 revs/min for 20 min), a n d was resnspended in T 7 G - buffer. Cell debris was pelleted (GSA rotor, 5500 revs/min for 20 min). The pelleted debris was twice extracted with T 7 G - buffer and the supcrnatants were pooled giving a final volume of about 150 ml. This was then precipitated overnight a t 4~ with 9~/o Carbowax and pelleted as described above. The pellet was suspended in about 15 ml of T 7 G - buffer. This concentrated mat~erial is very viscous because of the presence of DNA. Six ml per gradient were centrifuged for 3 h (SW41 rotor, 33,000 revs/min at 18~ through a cesium chloride step gradient containing 1"0 ml of 1.74 g/ml; 1.0 ml of 1-51 g/ml; 1"5 ml of 1"45 g/ml; 1-0 ml of 1.27 g/ml; 1"5 ml of 1.22 g/ml. The phage b a n d (1.51 g/ml) and capsid b a n d (1.27 g/ml) were collected separately b y di'ipping through the tube bottom. Both phage and intracellular capsids were further purified by b u o y a n t density banding in a CsC1 density gradient as described in the legend to Fig. 4. Capsid I (density = 1.286 g/cm 3) was partially separated from capsid I I (density = 1.268 g/cm 3) at this stage. F u r t h e r purification of capsids I and I I was done b y sedimenting through a 5% to 250/0 sucrose gradient in T 7 G - buffer (SW41 rotor, 25,000 revs/min for 120 min at 18~ The capsid bands, which were visible, were collected from the top of the centrifuge tube using a hypodermic syringe and a blunt 18-gauge needle. Capsid I I was found in 2 bands differing b y 7 % in sedimentation rate. Capsids in the slower b a n d will be referred to as capsid I I h ; eapsids in the faster b a n d will be referred to as capsid I I B . Capsid I sediments about 1.48 times as fast as capsid I I A . Both phage and capsids were dialyzed into Tris/Mg buffer. Recovery of phage infectivity was 22 to 27%. (d) Polyacrylamide/sodium dodecyl sulfate gel eleetrophoresis The proteins comprising the various structures studied were determined using polyacrylamide/sodium dodeeyl sulfate gel electrophoresis basically according to the procedures described b y Studier (1973). A 2-layer separating gel was used: 7 ml of a 16 or 17% (w/v) solution of acrylamide was poured in the b o t t o m of a slab gel-forming device. Above this was poured a b o u t 17 ml of 10% (w/v) aerylamide and the 2 layers were polymerized together under a layer of water. The stacking gel (5~ was poured and proteins were

P H A G E T7 I N T E R N A L

PROTEINS

273

submitted to electrophoresis at 100 to 150 V. Gels were stained for 1.5 h at room temperature in 0.2% (w/v) Coomassie brilliant blue, 10% (v/v) methanol, 10~o (v/v) acetic acid and were destained in 10~/o (v/v) acetic acid at 4~ Photographs were made of gels in 10% (v/v) acetic acid with TriX Pan film, using an orange filter.

(e) Electron microscopy (i) Preparation of DNA-capsid complexes for electron microscopy using a protein monolayer technique was done according to Serwer (1974a), except t h a t 20 ~1 of 5 ~ammonium acetate were used, instead of 10 ~1 of 6 M-ammonium acetate. (ii) Grids for negative staining of specimens were prepared using essentially the technique of Gordon (1972). Parlodion films with holes were prepared using the teclmique of Huxley & Zubay (1960). The films were picked up on 400-mesh grids and were heavily covered with carbon to provide stability in the electron beam. A microscope slide was dipped in 0"7% Parlodion in amyl acetate, dried and then covered with a light coat of carbon. The light carbon film was barely visible, but its exact thickness is not known. This latter film was floated on water, carbon side up, and was picked up on the grids covered with fenestrated films. From 10 min to 1 h before use the grids were washed for 80 s in amyl acetate (to remove the Parlodion) and then for 10 s in acetone. This leaves a thin carbon film over holes in a h e a v y carbon film. Micrographs were taken of samples located over holes in the heavy carbon film where contrast was optimal. Such carbon films have a higher adsorption efficiency for T7 phage than films prepared b y simply evaporating carbon onto a Parlodion film. Negative staining was performed with either 1% (w/v) uranyl acetate or 1% (w/v) sodium phosphotungstate (pH 7.6). Details of the staining protocol used are given in the Figure legends. (iii) Electron microscopy was performed on a Philips 301 electron microscope equipped with a goniometer. The goniometer had a range of --60 ~ to ~-60 ~ (f) Centrifugation Several types of velocity, buoyant density and combination velocity-buoyant density sedimentations were used, as described in the Figure legends. Gradients which had sufficient material in them to scatter a visible amount of light were photographed using Kodak Commercial Film (4127). Buoyant densities of cesium chloride solutions were determined from their refractive indices using the formula of Ifft et al. (1961 ).

3. Results (a) Observation of a core in capsids I, I I and T7 phage Capsids I a n d I I were isolated f r o m lysates o f wild-type T7-infected E. coli as described in Materials a n d Methods and were observed in the electron microscope after negative staining. Inside 90% of the capsid I particles and 78% of the eapsid I I particles (300 counted) is a core, whose dimensions v a r y from 100 to 250 A (Figs 1 (a) a n d 1 (b), respectively). The envelope of capsid I has a smaller outside diameter, is thicker and less angular in profile t h a n the envelope of capsid I I . A n internal core has also been observed inside T7 phage using two different techniques for specimen preparation. W h e n T7 phage are t r e a t e d with glutaraldehyde at p H 7.2 and are t h e n observed in the electron microscope, 93% (204 counted) of the phage envelopes a p p e a r to have lost their DI~A (Fig. 2(a)). Less t h a n 0.2% of t h e phage in this preparation a p p e a r e m p t y when observed w i t h o u t glutaraldehyde treatm e n t (see Fig. 7(a)). The D N A ejected from the phage envelope is seen as negatively stained fibrils near the emptied envelopes. M a n y of the emptied envelopes h a v e tails visible and some appear to have an internal core (black arrows in Fig. 2(a)). W h e n e v e r a core and a tail are present on the same particle t h e y appear to be connected a n d to

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F i e . 1. Electron microscopy of capsid I and capsid II. Capsids I and I I were purified as described in Materials a n d Methods. For electron microscopy a 3 to 5 ~1 droplet of sample was placed on a carbon support film and was incubated at room temperature for 1 min. Excess liquid was withdrawn with a Pasteur pipet and a droplet of 1% sodium phosphotungstate (pH 7.6) was placed on the film before the sample dried. The grid was then dried b y withdrawing the excess stain with filter paper. (a) Capsid I ; (b) capsid I I A ; (c) selected eapsid II. Particles 1 and 2 are eapsid I I A ; the remaining particles are capsid IIB. Thus far no systematic difference in appearance or protein composition between capsid I I A and I I B has been detected. Magnification 224,000 • The arrowhead points to a vesicle which is less penetrated by negative stain than is capsid I. Such vesicles are also more heterogeneous in size t h a n is eapsid I. To determine whether these vesicles were made of T7 protein, a capsid I preparation was reacted with anti-T7 phage serum and was then negatively stained. Vesicles larger and smaller t h a n capsid I do not bind detectable quantities of antibody at serum concentrations suificient to completely cover capsid I with antibodies. Thus, /~hese vesicles are probably components of E. celi. The n u m b e r of vesicles is usually from 1 to 5% of the number of capsid I particles.

FIG. 2. Electron microscopy of the core in T7 phage. (a) Negatively stained T7 phage after t r e a t m e n t with gintaraldehydc at p H 7.2. To 3 ~1 of T7 phage ( ~ 4 0 0 ~g]ml) was added 1 ~1 of 12-5~/o glutaraldehyde in 0.5 l~-sodium phosphate, p H 7.2. The phage was fixed for 5 m i n a t room temperature in a 20 ~1 micropipet (to prevent evaporation). A droplet of fixed phage was placed on a carbon support film and was allowed to stand for 1 rain. The film was washed with 4 drops of distilled water and then with 2 drops of 1 ~o uranyl acetate. The excess stain was removed with filter paper and the grid was allowed to dry. (b) Positively stained T7 phage from a sample prepared as in Fig. 7(a). (c) Negatively s~ained core protein from heat-shocked T7 phage. Material from the top of the sucrose gradient in Fig. 4(b) ( ~ 80 ~g/ml) was dialyzed into Tris]Mg buffer. This material was t h e n fixed and negatively stained as in (a). (d) Negatively stained capsid VA. I)NA in the capsid-DNA complex containing capsid VA was digested with DNAase I (1 ~g/ml, 30 min, 25~ in 0.05 •-NaC1, 0-01 ~-Tris (pH 7.4), 0.01 M-MgC12). For electron microscopy 3 to 5~1 of the DNAase-digested material was placed on a carbon support film and was incubated for 1 rain. The film was then washed with 4 drops of distilled water and t h e n 2 drops of 1 ~o sodium phosphotungstate, p H 7-6. The excess stain was removed with filter paper a n d the sample was allowed to dry. Magnification 224,000 •

276

P. SERWER

be coaxial. Sometimes core-tail complexes are apparently dislodged from their normal location and are observed outside the phage envelope (white arrow in Fig. 2(a))~. The core of phage T7 can be observed with DNA,still packaged inside the phage envelope. When T7 phage are negatively stained with uranyl acetate there are always some phage with positively stained D N A surrounded b y little or no stain (Fig. 2(b)). An electron transparent region is often seen inside the positively stained phage (black arrows in Fig. 2(b)). This electron transparent region is presumably the core of T7. T h a t the electron transparent region does not result from negative staining of tails folded under the phage is suggested b y the following observations: (I) the electron transparent region is sometimes longer t h a n a T7 tail; (2) one can sometimes see a tail extending from the electron transparent region (arrowheads in Fig. 2(b)). The fact t h a t the core can be observed in this way suggests t h a t D N A is at least partially excluded from the core. The cores of capsid I and of T7 phage m a y play an i m p o r t a n t role in packaging D N A in bacteriophage T7. Therefore, the structure and composition of these cores has been further investigated. (b) Stereo electron microscopy of the capsid I core The cores of different capsid I particles do not have the same appearance. At least some of the variability in appearance arises from observation of the core in different orientations. This is sho~m by observing a single capsid I core at several angles using a goniometer. I n Figure 3 are capsid I particles which have been observed at angles of --60, --45, --30, 0, + 3 0 , + 4 5 , + 6 0 degrees. I n one orientation, cores in particles A to D (Fig. 3) are roughly circular with a central hole (A, --60~ B, --60~ C, + 3 0 ~ D, +45~ When cores in particles A to D are tilted they elongate in a direction perpendicular to the axis of tilt. Ignoring the finer details apparent in the cores of particles A to D, these cores therefore appear to be roughly cylindrical with a hole along the axis of the cyhnder. The circular appearance of some cores thus arises from viewing the core parallel to the core axis (to be referred to as the end view). When the electron beam is perpendicular to the core axis (to be referred to as t h e side view) one end of the core appears to be attached to the outer envelope of capsid I with the core axis often perpendicular to the envelope (A, 0~ B, + 3 0 ~ C, --30~ D, --45~ E; F). The core measures 170 to 200/~ in length from the inside edge of the outer envelope; the width of unflattened cores (see below) is 125 to 150A; the central hole is 30 to 50 J~ in diameter. When cores which are seen in a side view at 0 ~ are rotated about an axis parallel to the core axis (particles E and F, Fig. 3) the length of the core remains constant, as expected if the cores are basically cylindrical. However, the diameter of the core in particle F decreases with increasing tilt angle, indicating t h a t the core is flattened in a direction perpendicular to the grid surface. The diameter of the core in particle E varies to a much smaller extent and is therefore less flattened. I t is kno~a~ t h a t the outer envelope of the capsid I particles is also quite flattened because ~fDNA can also be ejected from phage T4 using glutaraldehyde. 17o internal core was observed in T4. Ejection of DNA from both T7 and T4 is pH-dependent. T7 ejects at pH 7.2 but after glutaraldehyde fixation at pH 5.5, using the same buffers as in Fig. 2(a), only 11~o (31 of 274) capsids appear empty. Phage T4 ejects DNA at pH 9-0, but not at pH 7.2. Both T4 and T7 are stable without glutaraldehyde treatment at these pH values and salt conditions. DNA is also ejected from T7 when T7 is fixed with 1~/o osmium tetroxide or 1~o potassium permanganate at pH 7.2 and room temperature.

P H A G E T7 I N T E R N A L

PROTEINS

277

FIG. 3. Stereo electron microscopy of the capsid I core. Capsid I was prepared for electron microscopy as in Fig. 1. Electron micrographs were made at different angles of tilt, using a goniometer with a range of -}-60 ~ The tilt axis is vertical for all particles. Magnification 197,000 • c a p s i d I envelopes which a r e circular in outline a t 0 ~ b e c o m e " e l l i p t i c a l " w h e n t i l t e d a n d t h e m i n o r axis o f t h e " e l l i p s e " decreases ~dth i n c r e a s i n g angle o f t i l t (all particles, F i g . 3). I f one assumes t h a t t h e capsid I p a r t i c l e s e m b e d d e d in n e g a t i v e s t a i n a r e solids o f r e v o l u t i o n , t h e d a t a fl'om stereo electron m i c r o s c o p y i n d i c a t e t h a t t h e d i a m e t e r of m o s t o f t h e s e p a r t i c l e s in t h e p l a n e o f t h e s u p p o r t film is 1-4 t o 2.0 t i m e s t h e h e i g h t o f t h e p a r t i c l e p e r p e n d i c u l a r to t h e film (P. Serwer,

278

P. SERWER

unpublished data). Because the envelopes of capsid I particles are flattened, it is not surprising t h a t some cores are also flattened. Flattening is presumably caused b y surface forces which occur during drying. Flattening of a capsid I particle allows the core axis to be both perpendicular to the capsid envelope, and roughly parallel to the electron beam, even though the core axis does not pass through the center of the particle. This probably accounts for the fact t h a t most cores seen in an end view do not appear at the center of the capsid envelope, even though the core axis is perpendicular to the envelope (see particles A and C in Fig. 3). Off-center cores should not be observed ff the envelopes are spherical. Alterations in the capsid I core apparently occur prior to electron microscopy. In one preparation of capsid I (not the one used for Figs 1 and 3) the proportion of capsid I cores with dimensions comparable to the cylindrical core in an end view increased from 21~ at one week after purification of capsid I to 5 5 ~ one and one-half months after purification (300 cores counted). At one and one-half months 6 2 ~ of the circular cores failed to elongate detectably when tilted through angles of ~ 4 5 ~ and --45 ~ (90 cores counted). However, in the preparation used for Fig. 3 only 10~o of the cores seen in an end view at 0 ~ failed to elongate when tilted (60 cores counted). Thus the core of capsid I appears to disintegrate during storage, releasing a ring-shaped structure. The remainder of the core either leaks out of capsid I or can no longer be seen inside the capsid. (c) Electron microscopy of the capsid I I core Electron micrographs of capsid I I reveal m a n y cores which resemble capsid I cores in a side view (Fig. l(b); Fig. l(c), particles 1 to 4). Some capsid I I cores have a circular profile with a central hole, an appearance which resembles the end view of capsid I cores (particle 7 in Fig. l(c)). I t is therefore likely that some of the capsid I I cores closely resemble cyhnders with a hole along the cylinder axis. Stereo electron microscopy has not been done with capsid II. The length of most capsid I I cores in a side view is between 200 and 250 A, suggesting that the capsid I I core is a little longer than the capsid I core. This could be because the part of the capsid I I core which is adjacent to the envelope is actually part of the envelope in the core of capsid I. An occasional capsid I particle appears to have a two-layered envelope at the point of core-envelope joining (wlfite arrow in Fig. 1 (a)). The inner envelope layer may appear to be part of the core when capsid I converts to capsid II. However, the core of capsid I I might also have a tendency to expand, perhaps as a prelude to disintegrating. A core which appears significantly longer than the average capsid I I core is shown in Figure l(c) (6). Capsid II cores have a variety of appearances which suggest that they are easily broken. In Figure l(c) 2 to 4 are capsid I I cores which are seen in a side view and which appear to be detached from the envelope. Capsid I cores of this type have never been observed. Other capsid I I cores appear to have broken into two pieces (Fig. l(c) ; 5, 8 to 10), a phenomenon which is also not observed with capsid I cores. Particle 9 has within it two ring-shaped pieces of the core. Presumably these rings are stacked together in the intact core with the ring axis coincident with the core axis. Other capsid I I cores have a smaller cross-section than cores seen in a side or end view, suggesting t h a t the core is disintegrating (Fig. l(c); 11, 12). Such cores are sometimes seen in capsid I particles. The capsid I I particles observed in Figure 1 were prepared for electron microscopy

PHAGE

T7 INTERNAL

PROTEINS

279

two weeks after purification. Two months later only 20~/o (300 counted) of the eapsid I I particles had visible cores. I t is not known whether the cores leak out of the capsid envelope or disintegrate and become invisible. (d) Fine structure of the capsid I and I I cores When seen in a side view capsid I cores sometimes appear to be composed of subunits which are concentrated in discs perpendicular to the core axis (particles D, --60~ E, --30~ F, ~ 4 5 ~ in Fig. 3). Gaps between these discs are often non-uniform in length (particles E, --30~ F, ~-45 ~ in Fig. 3). Capsid I I cores often have a similar appearance (particles 1 and 2 in Fig. 1(6)). Some cores seen in a side view have a more uniform distribution of matter. The uniform appearance is sometimes produced by superposition of comparatively non-uniform detail from the sides of the core proximal and distal to the electron beam. This is shown for cores such as D (Fig. 3), which has a fairly uniform appearance at --45 ~ but after tilting to --60 ~ there is a strong suggestion of substructure. Although electron micrographs of the T7 core have detail at the subunit level, a three-dimensional model at this level has not yet been developed. Difficulties in analyzing electron mierographs of the core include: (1) the possibility t h a t the cores do not all have the same structure; (2) distortions in the core produced during specimen preparation such as the flattening already discussed, and bending; (3) superposition of detail from the eapsid envelope on detail within the core. There are techniques that can be used to reconstruct the three-dimensional arrangement of subunits within the core from stereo electron mierographs (reviewed by Frank, 1973). I t should be possible to obtain the appropriate data for such a study because images of the core do not suffer major alteration during the making of a stereo series. The bottom row of capsid I particles in Figure 3 illustrates this point. These micrographs are of the same particles and at the same angle of tilt (--45 ~ as in the second row; however, the micrographs in the bottom row were taken after the mierographs in rows 2 to 7 were made. Much of the fine structure in the cores has not been altered during the taking of this series of micrographs. I t is possible, however, t h a t modification of substructure occurred before taking the first mierograph.

(e) Calasids from temperature-shocked T7 phage To determine the proteins present in T7 cores it is helpful to separate cores from the eapsid envelopes. In an a t t e m p t to do this, T7 phage particles were disrupted using temperature shock. When bacteriophage T7 (5 to 20 ~g/ml) is temperature-shocked in a low salt, EDTA-containing buffer DNA comes out of the phage capsid, but roughly a third of the emptied capsids remain bound to their DNA (Serwer, 1974b). To determine the fate of capsids not bound to DNA, T7 phage were temperature-shocked at a much higher concentration (7.8 mg/ml). This was done to provide adequate material for characterization by negative stain electron microscopy and polyacrylamide/ sodium dodeeyl sulfate gel eleetrophoresis. The shocked phage were sedimented through a cesium chloride step gradient. Two turbid bands were formed, a sharp non-viscous band at 1-27 g/cm 3 and a more diffuse, extremely viscous band at 1.4 to 1.5 g/era 3 (Fig. 4(a)). T7 phage have a density of 1.509 g/era 8. The complex of T7 DNA with a single capsid, mentioned above, is found in the broad lower band. The lower band is broad because it also contains complexes of more t h a n one capsid with 79

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FIG. 4. Isolation of capsids IVA a n d IVB. A preparation of T7 phage (1.5 ml a t 7-8 mg/ml) was dialyzed into 0-1 M-NaCI, 0.02 ai-Tris (pH 7.4), 0.002 M-EDTA, a n d was t h e n heated a t 50.0~ for 35 min. This caused D N A to be ejected from the oapsid envelope so t h a t the solution became very viscous. The burst phage were layered on a cesium chloride step gradient containing 1.3 ml of 1.74 g/ml; 0'8 ml of 1.51 g/ml; 1.0 ml of 1.31 g]ml; 0.7 ml of 1.23 g/ml, all in 0-01 ~i-Tris (pH 7.4), 0-001 M-EDTA. The step gradient was centrifuged a t 40,000 revs/min a t 10~ for 16.2 h in a n SW50.1 rotor. Material in the capsid b a n d from this step gradient was dialyzed into Tris/Mg buffer a n d was then layered on a n l l . 0 - m l 5 % to 25% sucrose gradient in T 7 G - buffer. A 0.8-ml layer of 62% sucrose in T 7 G - buffer was under the sucrose gradient. The oapsids were t h e n sedimented a t 27,000 revs/min a t 18~ for 120 m i , in the SW41 rotor. Capsids I V A a n d IVB were separately t a k e n from the sucrose gradient, dialyzed into Tris/Mg buffer a n d were made to 5-2 ml with cesium chloride solution in 0-01 M-Tris, 0.001 MMgC12, final density = 1.278 g/em 8. The capsids were centrifuged for 20 h a t 10~ a t 42,000 revs/min in the SW50.1 rotor. Capsid I V B is slightly more dense in cesium chloride (1.284 g/cm 3) t h a n capsid I V A (1.268 g/cma), so t h a t this final centrifugation was used to further separate capsid IVA from capsid IVB. The purified eapsids were dialyzed in Tris/Mg buffer. (a) Cesium chloride step gradient. (b) Sucrose gradient. s i n g l e T 7 D N A m o l e c u l e . T h e c a p s i d s g e n e r a l l y t e n d t o b i n d i n a ~oTape-like c l u s t e r a t o n e location on the DNA. However, in about 15% of the multi-capsid-DNA complexes, e a p s i d s a p p e a r t o b e b o u n d a t m o r e t h a n o n e l o c a t i o n o n t h e D N A (Fig. 5). A l l b u t one of the capsids on a given DNA molecule must have bound to the DNA after heat shock. Multi-capsid-DNA complexes were not detected in previous experiments ( S e r w e r , 1974b), p r o b a b l y b e c a u s e o f t h e l o w e r c o n c e n t r a t i o n o f p h a g e u s e d .

PHAGE

T7 I N T E R N A L

PROTEINS

281

FIG. 5. Complex of T7 DNA with several T7 capsids. Capsid-DNA complexes from the lower b a n d of an experiment similar to the experiment shown in Fig. 4(a) were purified by a second buoyant density banding in a CsCl density gradient. Material with an average density of 1.305 g/era a was dialyzed into Tris/EDTA buffer and was prepared for electron microscopy using the protein monolayer technique. The black arrow indicates a cluster of at least 10 eapsids. The white arrows indicate clusters which probably have 2 eapsids (an electron mierograph of a monomer eapsid-DNA complex is shown by Serwer (1974b)). The cluster at the b o t t o m appears to have been pulled loose from the DNA during preparation. Magnification 52,000 x .

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P. S E R W E R

284

Temperature shock Copsid~iZA ( Tail-.Core - ) Capsid I + DNA + Tail (Tail-. C~ llnv/vo CapsidII A, B ( Tail-,Core +)

/ ~ . . ~ Capsid I~B (Tail+,Core -) w'vo =- T7phage ~ Capsid~A-DNA (Tail+ Core+) ~"'.........~ complex (Tail+,Core -1 - CopsidVB-DNA

NN complex(Toil-,Core-) Toil

FIG. 6. Schematic illustration of T7 capsids. Capsid I is depicted as reacting "in viva" with D N A to form either capsid I I (during a n ab3rtive a t t e m p t to package DNA) or T7 phage. W h e n T7 phage are temperature-shocked the phage breaks down into capsids IV, the capsid V - D N A complexes a n d tails. The presence of a core or tail in each structure, as determined b y negative stain electron microscopy, is indicated.

The band at 1-27 g/era ~ (capsid band) in Figure 4(a) is non-viscons and presumably contains little or no DNA. Material from the capsid band was sedimented through a sucrose gradient. Two bands are seen near the middle of the sucrose gradient; the faster band is skewed toward the bottom of the sucrose gradient (Fig. 4(b)). Material in the band closer to the top of gradient will be referred to as capsid IVA and material in the lower band as capsid IVB. (A list of the T7 capsids which have been isolated is given in Table 1, and a schematic illustration is shown in Fig. 6.) Capsids IVA and IVB were further purified by buoyant density banding in a cesium chloride density gradient. Capsid IVA is slightly less dense in CsC1 than capsid IVB (see the legend to Fig. 4). (f) Electron microscopy of capsids I VA, I VB and T7 tails Capsids IVA and IVB were observed in the electron microscope using a negative staining technique for specimen preparation. Capsid IVA appears to be an empty phage envelope which has no tail (Fig. 7(d)). Capsid IVB appears to be an empty phage envelope with a tail attached (Fig. 7(c)). Cores were not observed in capsid IVA or capsid IVB. The tails released by capsid IVA form a faintly turbid band near the top of the sucrose gradient in Figure 4(b) (the print in Fig. 4(b) is too dark to observe the tail band). The tail seen end-on has a strong component of sixfold rotational symmetry and sometimes appears to have six bent fibers radiating fi'om it (black arrow in Fig. 7(b)). When seen in a side view (white arrow) the tail appears tapered at one end. At the other, blunt end, two discs of subunits are held together b y a narrow neck. Judging from the appearance of the tail in capsid IVB (Fig. 7(c)), and the tail of T7 phage (Fig. 7(a)), the tapered end of the tail projects outside the capsid envelope, while the blunt end is joined to the envelope. When capsid IVB is in a favorable orientation, the outer envelope of the capsid appears to pass between the two discs of subunits at the blunt end of the tail (arrow in Fig. 7(c)). (g) Protein composition of T7 capsids: likely core proteins Bacteriophage T7 capsids have been isolated with and without cores. Therefore, analysis of the proteins in the capsids should yield information about which T7 proteins are in the cores. The proteins in T7 capsids, phage and tails have been determined on polyacrylamide/sodium dodecyl sulfate gels stained with Coomassie brilliant blue. The material used for electrophoresis is the same as t h a t used for

PHAGE

T7 I N T E R N A L

PROTEINS

285

FzG. 7. Electron microscopy of capsid IVA, capsid IVB, tails a n d T7 phage. Capsids I V A a n d I V B were purified as described in the legend to Fig. 4. The tails were t a k e n from the sucrose gradient in Fig. 4(b) a n d were dialyzed into Tris/Mg buffer. Electron microscopy of capsid IVA, capsid I V B a n d the tails was done as in Fig. 1, F o r electron microscopy of phage T7 a sample (400 ~g/ml) was allowed to adsorb to the support film for 1 min. After adsorption the grid was washed w i t h 4 drops of distilled water a n d t h e n with 2 drops of 1% uranyl acetate. The grid was dried using filter paper. (a) T7 phage, magnification 224,000 x ; (bl tails, magnification 288,000• (e) capsid IVB, magnification 2 2 4 , 0 0 0 • (d) capsid IVA, maoonaification 224,000 •

286

P. S E R W E R

Fro. 8. Polyacrylamide]sodium dodseyl sulfate gel electrophoresis of T7 phage, capsids, tails, a n d eapsid-DNA complexes. Capsids I a n d I I were prepared as described in Materials and Methods. Capsids I~rA, IVB a n d the tails were prepared as described in the legend to Fig. 4. Material from the eapsid-Dl~A b a n d of Fig. 4(a) was treated with 1"5~o Sarkosyl NL30 (Geigy Chemical Co.) for 30 rain a t 37~ a n d t h e n was sedimented to a n equilibrium density in a cesium chloride density gradient (5.2 ml, initial density ~ 1.51 g]em 3, SW50.1 rotor, 40,000 revs]min, 20 h, 10~ The monomer capsid-DNA complexes were collected in a single fraction a n d were dialyzed into Tris]EDTA buffer. This material was t h e n mixed with sodium i e t h a l a m a t e solution a n d sedimented to a n equilibrium density (5.2 ml, initial density = 1.185 g/cm 3, SW50.1 rotor, 45,000 revs]min, 32 h, 10~ The capsid-DNA complexes, which b a n d a t 1.17 g]em ~ in sodium iothalamate (Serwer, 1975), were t h e n dialyzed into Tris]EDTA buffer a n d were r e b a n d e d in cesium chloride as described above. During the final banding the eapsid-DNA complex with capsid VA (density ~ 1.511 g]em s) was collected separately from the complex which has capsid VB (density = 1.522 g]cm3). B o t h eapsid-DNA complexes were dialyzed against Tris]EDTA buffer. The above structures were subjected to polyacrylamide]sodium dodeeyl sulfate gel electrophoresis as described in Materials a n d Methods. Slots A to G are from the same slab gel which h a d a strip of 17.0% separating gel a t its top. Slots H to J are from a second gel which h a d a 16-0~o top separating gel. W i t h the exception of slot C, a b o u t I0 ~g of protein was p u t in each slot. Electrophorcsis is from b o t t o m to top. The arrow indicates the border between the 10% gel and the 16~o or 17~o gel. Numbered proteins were identified b y comparison with the gels of

PHAGE

T7 I N T E R N A L

PROTEINS

287

e l e c t r o n m i c r o s c o p y . Capsid I V A , w h i c h has no core or tail, is m a d e p r e d o m i n a n t l y o f P10 ; p r o t e i n s Q a n d P 9 a r e also p r e s e n t in c a p s i d I V A (Fig. 8, slot A). T h e r e f o r e P10, Q a n d P9 a r e p r o b a b l y p a r t o f t h e e n v e l o p e of t h e phage. H o w e v e r , n o t all p r e p a r a t i o n s of T7 p h a g e c o n t a i n Q a n d P9 (P. Serwer, u n p u b l i s h e d d a t a ) . Therefore, p r o t e i n s P9 a n d Q a p p e a r n o t to be essential c o m p o n e n t s o f t h e envelope. Capsid I V B (Fig. 8, slot B) has t h e p r o t e i n s o f e a p s i d I V A a n d also has PS, P l l , P12, P17, which a r e t h e p r o t e i n s p r e s e n t in t h e tails (Fig. 8, slot C). This confirms t h e conclusion t h a t c a p s i d I V B is a p h a g e e n v e l o p e w i t h a t a i l a t t a c h e d . T h e i d e n t i f i c a t i o n o f P l l , P12 a n d P17 as t a i l p r o t e i n s has been m a d e b y S t u d i e r (1972). T h e m a j o r p r o t e i n o f capsids I a n d I I , b o t h o f w h i c h h a v e cores b u t n o t tails, is P10. Capsids I a n d I I are missing t a i l p r o t e i n s P l l a n d P 1 7 ; t h e y are also deficient in t a i l p r o t e i n P12~ (Fig. 8. slots F a n d E, respectively). Capsids I a n d I I c o n t a i n P14, P15, P 1 6 (slots F a n d E, r e s p e c t i v e l y ) which a r e p r o t e i n s also p r e s e n t in T7 p h a g e (slots D a n d G) b u t n o t in capsids I V A , I V B or t h e tails:~. Cores a r e t h e o n l y s t r u c t u r e s seen in capsids I, I I a n d T7 p h a g e which are n o t seen in e a p s i d s I V A a n d I V B . T h u s t h e cores are p r o b a b l y c o m p r i s e d o f a t l e a s t one a n d p o s s i b l y all o f t h e p r o t e i n s P14, P15 a n d P16. I t is possible, however, t h a t one or m o r e o f t h e s e p r o t e i n s is e i t h e r p a r t o f t h e e n v e l o p e of capsids I, I I a n d T7 phage, or is inside t h e e n v e l o p e in a form n o t visible in t h e e l e c t r o n microscope. P13, like P14, P15 a n d P16, is p r e s e n t in c a p s i d I a n d T7 phage, b u t n o t in capsids I V A a n d I V B w H o w e v e r , u n l i k e P14, P15 a n d P16, P13 is missing from c a p s i d I I , a c a p s i d which has a core. I f P13 is a core p r o t e i n , i t can a p p a r e n t l y be r e m o v e d f r o m t h e core w i t h o u t c o m p l e t e l y d e s t r o y i n g t h e core. Capsids I a n d I I c o n t a i n P8, a p r o t e i n in T7 phage, c a p s i d I V B a n d tails b u t n o t in c a p s i d I V A . This suggests t h a t P8 is b o t h a t a i l a n d a core p r o t e i n . E l e c t r o n microscope e v i d e n c e p r e s e n t e d in section (a) a b o v e i n d i c a t e s t h a t t h e core is j o i n e d t o t h e c a p s i d e n v e l o p e in c a p s i d I a n d T7 phage. Therefore, P8 is p r o b a b l y t h e p r o t e i n a t t h e j u n c t u r e o f t h e core, e n v e l o p e a n d t a i l in T7 p h a g e a n d a t t h e c o r e - e n v e l o p e t The P12 on capsid I may be in an appendage of non-uniform size s0metinaes seen on capsid I (black arrows in Fig. l(a)). These appendages are contiguous with the core and thus appear to be near the taft attachment site. When x4C-labeled capsid I is prepared without a concentration step prior to cesium chloride banding, no P12 was detected on autoradiographed gels of capsid I. Thus the P12 on capsid I in Fig. l(a} may have bound after cell lysis. :~There are small amounts of P15 and P16 in eapsid IVB; however, this P15 and P16 proabbly comes from contamination of capsid IVB with material that sediments faster than eapsid IVB (Fig. 4(b)}. This fast-sedimenting material has comparatively large amounts of P15 and P16. Thus, capsid IVB will be considered to be missing P15 and P16. wThe amount of P13 in capsid I is significantly smaller relative to P14, P15 and P16 than the amount in T7 phage. Studier (1972). (A) Capsid IVA; (B) capsid IVB; (C) tails; (D) T7 phage; (E) cupsid I I ; (F) capsid I; (G) T7 phage; (H) T7 phage; (I) capsid VA; (J) eapsid VB. Band D is present in T7 phage, but not in any DNA-free structure. Band D is also present in: ( 1) DNA from the pellet of the cesium chloride gradient of Fig. 4(a) ; (2) DNA obtained by extracting material in the capsid-DNA band of Fig. 4(a) with phenol and chloroform. DNAase treatment of (1) and (2} eliminates the D band. The D band could be DNA which has somehow trapped stain or it could be protein bound to DNA. The latter is thought to be the case, because some preparations of T7 phage do not have D band material when submitted to electrophoresis at the same concentration and on the same gel as phage which do have a D band. The D band has penetrated the separating gel through a region of the separating gel which is less than 10% (w/v) acrylamide. This region is present because of dilution of the acrylamide near the acrylamidewater interface during polymerization. I t is not known whether the D band will migrate into a 10% gel which does not have this diluted region.

288

P. SERWER

juncture in eapsid I. In purified tails P8 would be at the end of the taft which projects into the capsid envelope when the tails are joined to capsids, i.e. the blunt end. To be certain of the composition of cores it is necessary to isolate the cores intact, free of the capsid envelope. Attempts were made, therefore, to determine the fate of the possible core proteins, P13 to P16, after heat shock of T7 phage. (h) Fate of P13 to P16 after heat 8hock: aggregate8 of P15 A barely visible turbid band was observed above the tail band in the sucrose gradient of Figure 4(b) (1)15 band). When analyzed on polyacrylamide/sodium dodecyl sulfate gels this material was about 80% 1)15 and the remainder was 1)17. An electron micrograph of this material after glutaraldehyde fixation is shown in Figure 2(c). Two types of structure are observed: (1) a kinked fibre 2 9 0 ~ 2 0 / ~ in total length (arrows in Fig. 2(e)), and (2) a structure of irregular contour varying from 100 A to 190 A in cross-section. The fibers axe presumably the same fibers observed on tails and should be made of P17, because P15 is not found on tails. It has been shown t h a t gene 17 amber mutants of T7 do not have fibers on their tails when grown under non-permissive conditions (Studier, personal communication). This also suggests that the fiber is made of 1)17. The irregular structures seen in Figure 2(c) are presumably made of P15, but are too large to be single molecules of P15. A single molecule of P15 (M~. -~ 83,000) would have a cross-section of about 11 A if it were spherical. These aggregates of P15 were not observed if the sample was not fixed with glutaxaldehyde. However, the aggregates were probably present before fixation, because changing the concentration of sample by a factor of 10 during fixation does not affect the size of the aggregates. The maximum dimension of P15 aggregates is somewhat less than the length of the core of capsids I and I I and the morphology of the P15 aggregates is clearly not the same as the morphology of the cores. Therefore, the P15 aggregates are probably a component of the core, but it is also possible that these aggregates formed after ejection of 1)15 from the phage capsid. Proteins were also found in regions 1 and 2 of the cesium chloride step gradient in Figure 4(a). Region 1 contained electrophoretically pure P13. Region 2 contained P13 (35%) and P15 (65%). Preliminary amino acid composition data from hydrochloric acid hydrolysates of P13 from region 1 suggest that P13 is a basic protein (Argq-Lys, 19.0%; Asp~-Glu, 21.8%). This suggests that P13 may bind to DNA. I t is interesting to note that whenever DNA has entered and then left a T7 capsid, P13 is missing from the capsid envelope, even when P15 and P16 do not leave the envelope (Table 1). After sedimenting material in the capsid band (Fig. 4(a)) four times as far as in Figure 4(b), the top 1 ml of the sucrose gradient contains 1)15 which is about 95% pure. Preliminary amino acid composition data from acid hydrolysates of 1)15 indicate the P15 is not a basic protein and is high in glycine (at least 25%). Roughly 35% of the P15 originally in the heat-shocked phage of Figure 4 was recovered in the capsid band (Fig. 4(a)) and later at the top of the sucrose gradient of Figure 4(b). Of the P15 50% was recovered in region 2 of the gradient in Figure 4(a). Most of the remaining P15 was recovered in the capsid-DNA band (Fig. 4(a)) or in the capsids which sediment faster than capsid IVB (Fig. 4(b)). Of the P13 originally in the heat-shocked phage, 70% was recovered in region 1 and 30% in region 2 (Fig. 4(a)). The high concentrations of material used in the experiment in Figure 4 appear

PHAGE

T7 INTERNAL

PROTEINS

289

to be essential to a high recovery of P13 and P15. When T7 phage, labeled with 14C-labeled amino acids, was heat-shocked at 1 ~g/ml and was sedimented as described by Serwer (19745), no detectable P15 (~5~ was recovered. Losses of P13 occur when T7 is heat-shocked at 10 ~g/ml. The recovery of P14, even at the high concentrations of phage used in Figure 4, is only 10%. This P14 is found in the capsid-DNA band and in capsids which sediment more rapidly than capsid IVB. P16 is found associated with material in the capsid-DNA band and in capsids which sediment more rapidly than capsid IVB. (i) Proteins in T7 capsid-DNA complexes To help determine which protein is responsible for binding DNA to capsids in the capsid-DNA complexes discussed in section (e) above, the proteins in these complexes were analyzed on polyacrylamide/sodium dodecyl sulfate gels. Complexes of one eapsid and a T7 DNA molecule were obtained by incubating material from the capsid-DNA band of Figure 4(a) with 1 ~ Sarkosyl NL30 (Ciba-Geigy) at 37~ to help dissociate loosely aggregated capsids (the monomer capsid-DNA complex is insensitive to ionic detergents at 37~ This material was then (1) subjected to buoyant density banding in a cesium chloride density-gradient to complete the separation from DNA not bound to capsids; (2) subjected to buoyant density banding in a sodium iothalamate density-gradient to remove phage which were not burst by the heat shock (see Serwer, 1975) ; (3) subjected to a final banding in cesium chloride at which time the purified monomer capsid-DNA complex forms two bands (density 1.511 g/era 3 and 1.522 g/cm3). The capsid in the less dense capsid-DNA complex (capsid VA) has proteins Q, P8, P9, P10, P l l , P12, P17 in relative amounts comparable to those found in T7 phage (Fig. 8, slot I). This indicates that capsid VA is an envelope with a tail attached. Capsid VA also contains P14, P15 and P16, suggesting that a core may be present. Capsid VA was observed in the electron microscope after digestion of DNA bound to it. As expected, many capsid VA particles have tails (75%, 300 counted). None of the particles has internal structures with appearances resembling the end or side view of the cores in capsids I and II. However, amorphous material which usually does not appear to be bound to a tail is often observed inside capsid VA (Fig. 2(d)). Thus, eapsid VA appears to have cores which are disrupted. Capsid VA cores also appear disrupted when capsid VA is prepared without Sarkosyl treatment. That disrupted cores do not leave capsicl VA after heat shock and do leave capsid IVB, a DNA-free capsid which has a tail, suggests that DNA may be occluding a hole in the capsid envelope when it is bound to capsid VA. The capsid in the denser capsid-DNA complex (capsid VB) has proteins Q, P9 and P10 in relative amounts comparable to what is found in T7 phage and capsids IV (Fig. 8, slot J). Compared to capsid VA (slot I) and T7 phage (slot H), there are only small amounts of P8, P l l , P12, P14, P15, P16, P17 in capsid VB. These latter proteins are almost certainly in capsid VA particles which contaminate capsid VB preparations. It is difficult to prepare large amounts of purified capsid VA and VB for the following reasons: (1) DNA present in the eapsid V-DNA complexes causes capsid-DNA bands in cesium chloride density-gradients to be viscous and therefore difficult to collect without mixing; (2) about 50% of the capsid-DNA complexes are not recovered after a 15-hour banding in cesium chloride. (The lost material has presumably attached to the centrifuge tube wall.) (3) For unknown reasons the

290

P. S E R W E R

capsid-DNA complex bands broaden during purification. To reduce the amount of material needed for electrophoresis, T7 phage was labeled with 14C-labeled amino acids and the proteins in capsid V derived from these phage were detected on polyacrylamide/sodium dodecyl sulfate gels using autoradiography. Capsid VB was produced which contained only P10 and Q. This suggests t h a t capsid VB is a head envelope without any other structure, which has been confirmed by electron microscopy. The results in the previous paragraph indicate that at ]east one of the proteins Q, P9 or P10 is involved in binding DNA to capsid VB. As mentioned earlier, some preparations of T7 phage do not contain Q and P9. When such phage are heatshocked they yield normal amounts of the capsid-DNA complexes which contain capsid VA and VB~. Therefore, it appears that Q and P9 are not necessary f o r capsidDNA binding and t h a t P10 alone is capable of binding DNA. Capsid-DNA complexes are also found in lysates of T7-infected E.coli prepared with a non-ionic detergent (in vivo capsid-DNA complexes). Complexes of capsids with monomer, coneatemer, and replicating T7 DNA have been observed (Serwer, 1974a,b). The proteins in these capsids have been labeled with 140 and have been analyzed on polyacrylamide/sodium dodecyl sulfate gels. The results are summarized in Table 1; these data will be presented in more detail in a future communication. The capsids in the capsid-DNA complexes in vivo (capsids III) resemble capsid I I in that they contain P8, P14, P15, P16, but lack P9, P13 and are defective in the tail proteins, P l l and P12. However, the capsids I I I do contain normal amounts of P17, which comprises the T7 taft fiber. The protein composition of the capsids I I I suggests that they have a core. However, sufficient material for negative stain electron microscopy has not yet been prepared.

4. D i s c u s s i o n

Two forms of phage T7 capsid, isolated from lysates of wild-type T7-infected E. coli, have been shown to contain an internal core (capsids I and II). A similar core is present in T7 phage, but is not present in DNA-free capsids obtained from T7 phage using temperature shock (capsids IVA and IVB). Comparison of the protein compositions of capsids with and without cores suggests that P14, P15 and P16 are components of the core. Aggregates of P15 whose dimensions are comparable to the dimensions of cores have been isolated after heat shock of T7, further suggesting that P15 is a major core constituent. I t is likely that an early event in DNA packaging is the binding of capsid I to T7 DNA. The initial binding event presumably occurs on the exterior of capsid I and therefore may not involve the core. Evidence presented in Results suggests that P10, the major envelope protein, is capable of binding DNA. I t would not be surprising if DNA binds to P10 in capsid I during packaging. However, thus far there is no direct evidence indicating which protein binds DNA during packaging. The binding site presumably is located near a hole in capsid I through which DNA t Capsid-DNA complexes were detected after heat shock by banding 16/zg of heat-shocked phage in a sodium iothalamate density gradient (Serwer, 1975). The sodium iothalamate gradient contained 50 ~g ethidium bromide/ml which does not affect the buoyant density of DNA and which causes any DNA-eontaining structure in the gradient to fluoresce in the presence of ultraviolet light. The capsid-DNA complexes were therefore observed as fluorescent bands. The complex with eapsid VA is slightly more dense in sodium iothalamate gradients than the complex with capsid VB.

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will enter the capsid. The hole which runs along the axis of the core and m a y go through the outer envelope of capsid I is a good candidate for the hole through which DNA enters the capsid during packaging. One can imagine at least two roles for the core: (1) the core helps to package DNA in the proper configuration for its subsequent injection into the host; (2) the core helps in cutting mature T7 DNA, which is linear, terminally repetitious and nonpermuted (Thomas etal., 1968) from T7 concatemers, an apparent precursor to the mature DNA (Kelly & Thomas, 1969; Serwer, 1974a). This latter possibility receives support from the data which suggest that T7 DNA is packaged before it is cut to mature size (Serwer, 19745). I t would not be surprising to find that one of the core proteins is an endonuclease. The electron microscope data suggest that the capsid I envelope changes in thickness and diameter during its conversion to the capsid of mature phage. Because of flattening and possible dehydration effects which occur during negative staining, accurate T7 phage and capsid envelope dimensions cannot be reliably obtained from micrographs (see also Serwer, 1976). However, small-angle X-ray scattering studies of T7 phage and capsids confirm t h a t the diameter of the capsid I envelope is less than the diameter of capsids II, IVB and T7 phage, and that the capsid I envelope is thicker than the envelope of eapsids I I and IVB (Ruark, Stroud & Serwer, unpublished data). The major difference in composition between capsids I and I I is a difference in the amount of P9. The major difference in structure between capsids I and I I is in the outer envelope. Therefore it is likely that P9 is located in the outer envelope of capsid I and plays a role in maintaining the capsid I envelope smaller and thicker than the envelopes of the other capsids. For valuable criticism during the course of this work, I thank Drs W. B. Wood, F. W. Studier and J. P. Revel. I also thank Dr Studier for communicating to me unpublished results, and Patrick F. Koen for advice on technical matters concerning the electron microscope. Support was received from an American Cancer Society postdoctoral fellowship, a grant to Dr W. B. Wood from the United States Public Health Service (A102938), and grants from the National Science Foundation to Dr Robert M. Stroud (BMS 75-04105), and Dr J. P. Revel (06965). REFERENCES Bancroft, F. C. & Freifelder, D. (1970). J. Mol. Biol. 54, 537-546. Casjens, S. & King, J. (t975). Annu. Roy. B w c ~ m . 44, 555-611. Frank, J. (1973). In Advanced Techniques in Biological Elect/ton Microscopy (Koehler, J. K., ed.), pp. 215-274, Springer-Verlag, New York. Gordon, C. N. (1972). J. Ultrastruct. Res. 39, 173-185. Huxley, H. E. & Zubay, G. (1960). J. Mol. Biol. 2, 10-18. Ifft, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phys. Chem. 65, 1138-1145. Kellenberger, E. & Sechaud, J. (1957). Virology, 3, 256-274. Kelly, T. J. & Thomas, C. A. (1969}. J. Mol. Biol. 44, 459-475. Serwer, P. (1974a). Virology, 59, 70-88. Serwer, P. (1974b). Virology, 59, 89-107. Serwer, P. (1975). J. Mol. Biol. 92, 433-448. Serwer, P. (1976). J. Ul~ra~truct. Res. In the press. Studier, F. W. (1972). Science, 176, 367-376. Studier, F. W. (1973). J. Mol. Biol. 79, 237-248, Thomas, C. A., Kelly, T. J. & Rhoades, M. (1968}. Gold Spring Harbor Syrup. Quant. Biol. 33, 417-424. Yamamoto, K. R., Alber~s, B. M., Benzinger, R., Lawhorne, T.. & Treiber, G. (1970}. Virology, 40, 734-744.